G2Cdb::Gene report

Gene id
G00000030
Gene symbol
NOS1 (HGNC)
Species
Homo sapiens
Description
nitric oxide synthase 1 (neuronal)
Orthologue
G00000005 (Mus musculus)

Databases (8)

Gene
ENSG00000089250 (Ensembl human gene)
4842 (Entrez Gene)
76 (G2Cdb plasticity & disease)
NOS1 (GeneCards)
Literature
163731 (OMIM)
Marker Symbol
HGNC:7872 (HGNC)
Protein Expression
2167 (human protein atlas)
Protein Sequence
P29475 (UniProt)

Synonyms (1)

  • nNOS

Diseases (16)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000289: Infantile pyloric stenosis N Y (8571963) Unknown (?) Y
D00000231: Essential hypertension N Y (9298742) Repeat polymorphism (RP) N
D00000240: Asthma N Y (10641565) Unknown (?) Y
D00000240: Asthma N Y (10673365) Microsatellite variation (MSV) Y
D00000240: Asthma N Y (10833424) Dinucleotide polymorphism (DNP) Y
D00000270: End-stage renal disease N Y (11071967) Microsatellite variation (MSV) ?
D00000240: Asthma N Y (11112111) Dinucleotide polymorphism (DNP) Y
D00000240: Asthma N Y (11668616) Single nucleotide polymorphism (SNP) Y
D00000191: Parkinson's disease Y Y (11809160) Dinucleotide polymorphism (DNP) Y
D00000166: Schizophrenia Y Y (12140778) Single nucleotide polymorphism (SNP) Y
D00000193: Alzheimer's disease Y Y (12210288) Dinucleotide polymorphism (DNP) N
D00000191: Parkinson's disease Y Y (12490535) Dinucleotide polymorphism (DNP) Y
D00000276: Preeclampsia N Y (12699878) Microinsertion (MI) N
D00000171: Major depressive disorder Y Y (12759556) Single nucleotide polymorphism (SNP) N
D00000166: Schizophrenia Y Y (14623375) Repeat polymorphism (RP) N
D00000170: Bipolar disorder Y Y (14681919) Single nucleotide polymorphism (SNP) N
D00000195: Multiple sclerosis Y Y (14759629) Microsatellite polymorphism (MSP) N
D00000156: Cystic fibrosis N Y (14760158) Repeat polymorphism (RP) Y
D00000316: Immunoglobulin E-mediated allergic diseases N Y (15080837) Single nucleotide polymorphism (SNP) ?
D00000204: Migraine Y Y (16191396) Microsatellite repeat polymorphism (MSRP) N
D00000242: Achalasia N Y (16848803) Microsatellite repeat polymorphism (MSRP) N
D00000242: Achalasia N Y (16848803) Restriction fragment length polymorphism (RFLP) N
D00000181: Primary nocturnal enuresis N Y (17365914) Single nucleotide polymorphism (SNP) Y

References

  • Nitric oxide synthase gene polymorphisms in children with primary nocturnal enuresis: a preliminary study.

    Balat A, Alasehirli B, Oguzkan S and Gungor M

    Department of Pediatric Nephrology, Gaziantep University, Medical Faculty, Gaziantep, Turkey. aysebalat@hotmail.com

    Aims: Recent studies demonstrated some differences in urinary electrolytes of enuretic children. Intrarenal nitric oxide (NO) serves as a major regulator of renal sodium and water excretion like an endogenous diuretic. This study aimed to investigate endothelial (eNOS), and neuronal (nNOS) NO synthase gene polymorphisms in children with primary nocturnal enuresis (PNE).

    The eNOS gene polymorphism was investigated in 171 Turkish children (57 PNE cases and 114 healthy, non-enuretic controls), and nNOS gene polymorphism was determined in 158 Turkish children (83 PNE cases and 75 healthy, non-enuretic controls). The glu298asp (G/T) polymorphism of the eNOS and C276T (C/T) polymorphism of nNOS genes were genotyped using PCR.

    Results: The distribution of GG, TG, and TT genotypes for eNOS gene was 48%, 33%, and 19% in PNE, compared with 61%, 26%, and 13% in the controls (p > 0.05). The distribution of CC, TC, TT and genotypes for nNOS gene was 31%, 29%, and 40% in PNE compared with 10%, 43%, and 47% in the controls. CC genotype was found higher in enuretic children (p = 0.002). The eNOS and nNOS gene polymorphisms were not associated with positive family history, frequency of enuresis, and clinical response to desmopressin.

    Conclusions: This study is the first to search the NOS gene polymorphisms in children with PNE. It was determined that eNOS gene polymorphism may not be associated with PNE, while nNOS gene polymorphism, a predominantly CC genotype, may be associated with PNE in Turkish children. Further studies with larger samples together with the detection of enuresis gene may help determine the exact role of nNOS gene polymorphism in enuresis.

    Renal failure 2007;29;1;79-83

  • Association between achalasia and nitric oxide synthase gene polymorphisms.

    Mearin F, García-González MA, Strunk M, Zárate N, Malagelada JR and Lanas A

    Institute of Functional and Motor Digestive Disorders, Centro Médico Teknon, Barcelona, Spain.

    Background: Our group previously reported the absence of nitric oxide synthase (NOS) in the gastroesophageal junction of patients with achalasia. NOS exists in three distinct isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible isoform (iNOS). Some studies have shown that NO production is regulated by NOS polymorphisms.

    Aim: To assess whether some functional polymorphisms in the nNOS, iNOS, or eNOS genes are involved in susceptibility to suffer from achalasia.

    Methods: Genomic DNA from 80 unrelated Spanish Caucasian patients with sporadic achalasia and 144 healthy subjects matched for age (+/-5 yr) and gender was typed by PCR and RFLP methods for the 27-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 of the eNOS gene, a CA microsatellite repeat and a Nla III (C-->T) restriction fragment length polymorphism (RFLP) in exon 29 of the nNOS gene, and two nucleotide substitutions located in exon 16 (C-->T) and exon 22 (G-->A) of the iNOS gene.

    Results: No significant differences in carriage, genotype, and allele frequencies of the nNOS, iNOS, or eNOS gene polymorphisms were found between patients with achalasia and controls. Individuals homozygous for genotype iNOS22*A/A tended to be more frequent in achalasia (20%vs 11%, odds ratio [OR] 1.79, 95% confidence interval [CI] 0.89-3.59, p= 0.09) as were those homozygous for the rare eNOS*4a allele (6.2%vs 1.4%, OR 4.5, 95% CI 0.89-22.67, p= 0.1) although the difference did not reach statistical significance. No differences in genotype and allele distribution were found with respect to epidemiological and clinical characteristics of patients with achalasia.

    Conclusion: Our data suggest that NOS gene polymorphisms are not involved in the susceptibility to and nature of the clinical course of sporadic achalasia. However, studies in a greater number of patients are required to analyze the tendency toward a higher prevalence of genotypes iNOS22*A/A and eNOS*4a4a.

    The American journal of gastroenterology 2006;101;9;1979-84

  • A population genomics overview of the neuronal nitric oxide synthase (nNOS) gene and its relationship to migraine susceptibility.

    Johnson MP, Lea RA, Colson NJ, Macmillan JC and Griffiths LR

    Genomics Research Centre, School of Health Science, Griffith University, PMB 50 GCMC, Gold Coast 9726, Australia.

    The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5' and 3' regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5' region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5' repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5' and 3' genetic variants were investigated.

    Cellular and molecular biology (Noisy-le-Grand, France) 2005;51;3;285-92

  • Neuronal nitric oxide synthase gene polymorphism and IgE-mediated allergy in the Central European population.

    Hollá LI, Schüller M, Bucková D and Vácha J

    Department of Pathological Physiology, Masaryk University Brno, Brno, Czech Republic.

    Background: Several findings suggest that nitric oxide (NO) plays a significant role in the regulation of the Th1/Th2 balance and contributes to the development of allergic diseases. Our study investigates a possible association of C/T transition located 276-bp downstream from the translation termination site in exon 29 of the human nitric oxide synthase type 1 (NOS1) gene with immunoglobulin E (IgE)-mediated allergic diseases in the Czech population.

    Methods: The study included 688 subjects - 368 patients with clinically manifested allergic diseases and 320 unrelated controls with negative familial history of asthma/atopy. The NOS1 genotypes were determined by polymerase chain reaction (PCR) and restriction analysis by Eco72I.

    Results: No significant differences were found for allele or genotype frequencies of the 5266 C/T polymorphism in exon 29 of the NOS1 gene between IgE-mediated allergic diseases (or asthma alone) and healthy subjects. However, this common polymorphism showed a significant association with signs of atopy, especially with total serum IgE levels [log(e) IgE levels (mean +/- SD): CC genotype = 4.34 +/- 1.40; CT genotype = 4.58 +/- 1.53; TT genotype = 5.01 +/- 1.61; P < 0.05).

    Conclusions: Our findings suggest that NOS1 gene may participate in the pathogenesis of high total serum IgE levels in allergic diseases in our population. These findings provide support for NOS1 as a candidate gene for IgE-mediated allergy.

    Allergy 2004;59;5;548-52

  • Investigation of a neuronal nitric oxide synthase gene (NOS1) polymorphism in a multiple sclerosis population.

    Tajouri L, Ferreira L, Ovcaric M, Curtain R, Lea R, Csurhes P, Pender MP and Griffiths LR

    Genomics Research Centre, School of Health Science, Griffith University Gold Coast, Southport, Queensland, 4215 Australia.

    Multiple Sclerosis (MS) is a chronic neurological disease characterized by demyelination associated with infiltrating white blood cells in the central nervous system (CNS). Nitric oxide synthases (NOS) are a family of enzymes that control the production of nitric oxide. It is possible that neuronal NOS could be involved in MS pathophysiology and hence the nNOS gene is a potential candidate for involvement in disease susceptibility. The aim of this study was to determine whether allelic variation at the nNOS gene locus is associated with MS in an Australian cohort. DNA samples obtained from a Caucasian Australian population affected with MS and an unaffected control population, matched for gender, age and ethnicity, were genotyped for a microsatellite polymorphism in the promoter region of the nNOS gene. Allele frequencies were compared using chi-squared based statistical analyses with significance tested by Monte Carlo simulation. Allelic analysis of MS cases and controls produced a chi-squared value of 5.63 with simulated P = 0.96 (OR(max) = 1.41, 95% CI: 0.926-2.15). Similarly, a Mann-Whitney U analysis gave a non-significant P-value of 0.377 for allele distribution. No differences in allele frequencies were observed for gender or clinical course subtype (P > 0.05). Statistical analysis indicated that there is no association of this nNOS variant and MS and hence the gene does not appear to play a genetically significant role in disease susceptibility.

    Journal of the neurological sciences 2004;218;1-2;25-8

  • Nitric oxide synthase 1 as a potential modifier gene of decline in lung function in patients with cystic fibrosis.

    Texereau J, Marullo S, Hubert D, Coste J, Dusser DJ, Dall'Ava-Santucci J and Dinh-Xuan AT

    Service de Physiologie-Explorations Fonctionnelles, Hôpital Cochin, AP-HP, Université Paris 5, Paris, France.

    Background: The severity of lung disease varies widely in patients with cystic fibrosis (CF) who have the same type of mutations of the cystic fibrosis transmembrane regulator (CFTR) gene, suggesting involvement of "modifier" genes. The nitric oxide synthase 1 (NOS1) gene is a candidate for this role because exhaled nitric oxide (NO) is reduced in patients with CF and NOS1 activity contributes to transepithelial ionic transport, immune defence, and non-specific inflammation of the airways.

    Methods: Dinucleotide GT repeat polymorphism was studied in the 5' untranslated region of the NOS1 gene, immediately upstream from the transcription initiation site, in 59 patients with CF and 59 healthy controls.

    Results: Nineteen alleles of the NOS1 gene were identified according to the number of GT repeats (from 18 to 36) in the 5 untranslated region. Exhaled NO levels were significantly correlated with the number of GT repeats. Patients with CF who had the NOS1 genotype associated with high NO production had a slower decline in lung function during the 5 year follow up period. There was no confounding effect of age, chronic bacterial colonisation of the airway, or CFTR genotype.

    Conclusions: These data suggest a possible link between the NOS1 gene locus and the rate of decline in lung function in patients with CF.

    Thorax 2004;59;2;156-8

  • No association between a neuronal nitric oxide synthase (NOS1) gene polymorphism on chromosome 12q24 and bipolar disorder.

    Buttenschön HN, Mors O, Ewald H, McQuillin A, Kalsi G, Lawrence J, Gurling H and Kruse TA

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2004;124B;1;73-5

  • Association analysis for the CA repeat polymorphism of the neuronal nitric oxide synthase (NOS1) gene and schizophrenia.

    Liou YJ, Tsai SJ, Hong CJ and Liao DL

    Schizophrenia research 2003;65;1;57-9

  • Endothelial nitric oxide synthase polymorphism in preeclampsia.

    Häkli T, Romppanen EL, Hiltunen M, Helisalmi S, Punnonen K and Heinonen S

    Department of Obstetrics and Gynecology, Kuopio University and University Hospital, Kuopio, Finland.

    Objective: We wished to determine whether genetic variability in the gene encoding endothelial nitric oxide synthase (eNOS) modifies individual susceptibility to the development of preeclampsia.

    Methods: The study involved 132 preeclamptic and 113 healthy control pregnant women who were genotyped for the Glu298Asp polymorphism in the eNOS gene. Chi(2) analysis was used to assess genotype and allele frequency differences between preeclamptic women and controls.

    Results: A statistically similar allelic distribution of eNOS Glu298Asp polymorphism was observed in the two groups, with the frequency of the variant G allele being 74.6% in the preeclampsia group and 67.7% in the control group (P = .091; odds ratio 1.40, 95% confidence interval 0.95, 2.01). Accordingly, the genotype distribution of the NOS polymorphism in the preeclamptic and control groups was found to be similar (P = .233).

    Conclusion: These genotype data in subjects from eastern Finland were not suggestive of an important contribution of the Glu298Asp polymorphism in the NOS gene on preeclampsia across populations. However, the observed association between the G allele and disease risk, of borderline significance, may imply that other polymorphism(s) in the gene may modify disease risk.

    Journal of the Society for Gynecologic Investigation 2003;10;3;154-7

  • Association between Parkinson's disease and polymorphisms in the nNOS and iNOS genes in a community-based case-control study.

    Levecque C, Elbaz A, Clavel J, Richard F, Vidal JS, Amouyel P, Tzourio C, Alpérovitch A and Chartier-Harlin MC

    INSERM Unit 508 Institut Pasteur de Lille, 59019 Lille, France.

    Excess of nitric oxide (NO) has been shown to exert neurotoxic impacts in the brain. Moreover, inhibition of two NO-synthesizing enzymes, neuronal NOS (nNOS) and inducible NOS (iNOS), displays neuroprotective effects in the MPTP model of Parkinson's disease (PD). These data suggest a possible involvement of NOS as factors controlling the resistance of the nigral dopaminergic neurons to environmental insults. Therefore, we investigated whether polymorphisms present in these genes could contribute to the risk of developing PD. We carried out a community-based case-control study among subjects enrolled in the Mutualité Sociale Agricole, the French health insurance organization for workers connected to agriculture. Two-hundred and nine PD patients and 488 controls of European (mostly French) ancestry and matched for age, sex and region of residency were included in this study. Associations were observed with polymorphisms present in exon 22 of iNOS (OR for AA carriers=0.50, 95% CI=0.29-0.86, P=0.01) and in exon 29 of nNOS (OR for carriers of the T allele=1.53, 95% CI=1.08-2.16, P=0.02); no association was observed with a polymorphism in exon 18 of nNOS (OR for carriers of the T allele=1.20, 95% CI=0.85-1.69, P=0.30). Moreover, a significant interaction of the nNOS polymorphisms with current and ever cigarette smoking was found (nNOS 18, P=0.05; nNOS 29, P=0.04). All together, these data favour an involvement of these two genes as new modifier genes in PD.

    Human molecular genetics 2003;12;1;79-86

  • Association analysis for neuronal nitric oxide synthase gene polymorphism with major depression and fluoxetine response.

    Yu YW, Chen TJ, Wang YC, Liou YJ, Hong CJ and Tsai SJ

    Yu's Psychiatric Clinic, Kaohsiung, Taipei, Taiwan, ROC.

    Nitric oxide (NO) is produced from its precursor L-arginine by the enzyme NO synthase (NOS), which includes at least three distinct isoforms - neuronal (nNOS), endothelial, and inducible NOS. Recent studies have implicated NOS in the mechanism that underlies the therapeutic efficacy of antidepressant medication. In addition, major depressive disorder (MDD) patients were found to have significantly higher plasma nitrate concentrations than normal subjects, an index of NO production, in comparison to normal subjects. In a population-based association study, we tested the hypothesis that the nNOS C276T polymorphism confers susceptibility to MDD. We also examined the association between this polymorphism and therapeutic fluoxetine response in 114 MDD patients who underwent a 4-week fluoxetine treatment. The results demonstrate that the nNOS variants are found at similar frequencies in MDD patients and healthy control subjects. Further, we did not discover any genetic variants that influenced the fluoxetine response in MDD patients treated with fluoxetine. Our findings suggest that this nNOS C276T polymorphism does not play a major role in the susceptibility to, or fluoxetine response in, MDD. However, the association between other NOS variants and MDD or antidepressant response, including sexual dysfunction, may warrant further investigation.

    Neuropsychobiology 2003;47;3;137-40

  • No association between the neuronal nitric oxide synthase gene polymorphism and Alzheimer Disease.

    Liou YJ, Hong CJ, Liu HC, Liu CY, Liu TY, Chen IC and Tsai SJ

    Section of Psychiatry, Yu-Li Veterans Hospital, Hualien, Taiwan, Republic of China.

    Nitric oxide synthase (NOS) has been implicated in the pathogenesis of Alzheimer disease (AD). To examine the role of the neuronal NOS (nNOS) gene in AD, patients (n = 139) and control subjects (n = 101) were genotyped for the nNOS dinucleotide polymorphism. No association was demonstrated for AD and this particular nNOS polymorphism.

    American journal of medical genetics 2002;114;6;687-8

  • 5'-flanking region polymorphism of the neuronal nitric oxide synthase gene with Parkinson's disease in Taiwan.

    Lo HS, Hogan EL and Soong BW

    Department of Neurology, Taiwan Adventist Hospital, Taipei, Taiwan.

    Though the etiology of Parkinson's disease (PD) is unresolved and may be heterogeneous involving both environmental and genetic factors, there are indications that oxidative stress plays an important role in dopaminergic neuronal death. And, it has been reported that inhibition of nitric oxide synthase (NOS) can prevent the destruction of dopaminergic neurons in mammals. To determine if NOS gene polymorphism affects the 5' flanking region that is immediately upstream of the transcription start site lying between the TATA element and CAATT boxes in PD, and differs significantly between patients with PD and normal controls, we studied genetic polymorphism in that region of the neuronal NOS gene in Chinese patients with PD living in Taiwan. The results indicate that the allele size distribution in that region was statistically significantly different between patients with PD and normal.

    Journal of the neurological sciences 2002;194;1;11-3

  • Allelic association of the neuronal nitric oxide synthase (NOS1) gene with schizophrenia.

    Shinkai T, Ohmori O, Hori H and Nakamura J

    Department of Psychiatry, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, 807-8555, Japan.

    Nitric oxide (NO) has been identified as a widespread and multifunctional biological messenger molecule in the central nervous system (CNS), with possible roles in neurotransmission, neurosecretion, synaptic plasticity, and tissue injury in many neurological disorders, including schizophrenia. Neuronal NO is widely produced in the brain from L-arginine catalyzed by neuronal NO synthase (NOS1). We therefore hypothesized that the NOS1 gene may play a role in the pathophysiology of schizophrenia. In the present study, we examined the genetic association between a novel single nucleotide polymorphism (SNP: a C-->T transition located 276 base pairs downstream from the translation termination site) of the human NOS1 gene, which is located in chromosome 12q24, and schizophrenia (215 Japanese patients with schizophrenia and 182 healthy controls). The allele frequencies of the polymorphism in exon 29 of the NOS1 gene differed significantly between patients with schizophrenia and controls (chi(2) = 20.10, df = 1, P = 0.000007; relative risk = 1.92; 95% confidence interval = 1.44-2.55). Our results suggest that the NOS1 gene polymorphism may confer increased susceptibility to schizophrenia.

    Molecular psychiatry 2002;7;6;560-3

  • Fine mapping and single nucleotide polymorphism association results of candidate genes for asthma and related phenotypes.

    Immervoll T, Loesgen S, Dütsch G, Gohlke H, Herbon N, Klugbauer S, Dempfle A, Bickeböller H, Becker-Follmann J, Rüschendorf F, Saar K, Reis A, Wichmann HE and Wjst M

    GSF-National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany. immervoll@gsf.de

    Several genome-wide screens for asthma and related phenotypes have been published to date but data on fine-mapping are scarce. For higher resolution we performed a fine-mapping study with 2 cM average spacing in often discussed asthma candidate regions (2p, 5q, 6p, 7p, 9q, 11p, and 12q) to narrow down the regions of interest. All participants of a Caucasian family study (97 families with at least two affected sib pairs) were genotyped for 49 supplementary polymorphic dinucleotide markers. Our results indicate increased evidence for linkage on chromosome 6p, 9q, and 12q. These candidate regions were further analyzed with SNP polymorphisms in the endothelin 1 (EDN1), lymphotoxin alpha (LTA), and neuronal nitric oxide synthase (NOS1) genes. In addition, IL4 -590C>T and IL10 -592C>A, localized on chromosomes 5q and 1q, respectively, have been analyzed for SNP association. Of the six SNPs tested, four revealed weak association with the examined phenotypes. These are the IL10 -592C>A SNP in the interleukin 10 gene (p=0.036 for eosinophil cell counts), the 4124T>C SNP in EDN1 (p=0.044 for asthma), the 3391C>T SNP in NOS1 with eosinophil cell counts (p=0.0086), and the 5266C>T polymorphism, also in the NOS1 gene, for high IgE levels (p=0.022). In summary, fine mapping data enable us to confine asthma candidate regions, while variants of EDN1 and NOS1, or nearby genes, may play an important role in this context.

    Human mutation 2001;18;4;327-36

  • Exhaled nitric oxide in patients with asthma: association with NOS1 genotype.

    Wechsler ME, Grasemann H, Deykin A, Silverman EK, Yandava CN, Israel E, Wand M and Drazen JM

    Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    An increased concentration of nitric oxide (NO) in exhaled air (FENO) is now recognized as a critical component of the asthmatic phenotype. When we identified patients with asthma on the basis of a standard case definition alone, we found that they were remarkably heterogeneous with respect to their FENO. However, when we included genotype at a prominent asthma candidate gene (i.e., NOS1) in the case definition, and determined the number of AAT repeats in intron 20, we identified a remarkably homogeneous cohort of patients with respect to FENO. Both mean FENO (p = 0.00008) and variability around the mean (p = 0.000002) were significantly lower in asthmatic individuals with a high number (> or = 12) of AAT repeats at this locus than in those with fewer repeats. These data provide a biologically tenable link between genotype at a candidate gene in a region of linkage, NOS1, and an important component of the asthmatic phenotype, FENO. We show that addition of NOS1 genotype to the case definition of asthma allows the identification of a uniform cohort of patients, with respect to FENO, that would have been indistinguishable by other physiologic criteria. Our isolation of this homogeneous cohort of patients ties together the well-established associations among asthma, increased concentrations of NO in the exhaled air of asthmatic individuals, and variations of trinucleotide repeat sequences as identified in several neurologic conditions.

    Funded by: NHLBI NIH HHS: P50-HL-56383

    American journal of respiratory and critical care medicine 2000;162;6;2043-7

  • Genetic analysis of nitric oxide and endothelin in end-stage renal disease.

    Freedman BI, Yu H, Anderson PJ, Roh BH, Rich SS and Bowden DW

    Department of Internal Medicine/Nephrology, The Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1053, USA.

    Background: Genetic factors have been implicated in the development of the common aetiologies of end-stage renal disease (ESRD), including renal failure attributed to hypertension, diabetes mellitus, systemic lupus erythematosus and human immunodeficiency virus infection. Nitric oxide (NO) and endothelin are powerful vasoactive mediators involved in inflammation and regulation of vascular tone and blood pressure. We evaluated the role of the neuronal constitutive (NOS1) and endothelial constitutive (NOS3) nitric oxide synthase genes and the endothelin-1 (EDN-1) gene in predisposition to chronic renal failure in African-Americans.

    Methods: The study population for the linkage and association analyses in ESRD consisted of 361 individuals from 168 multiplex African-American families. These individuals comprised 207 unweighted sibling pairs concordant for all-cause ESRD. Microsatellite markers NOS1B (NOS1), D7S636 (NOS3) and CPHD1-1/2 (EDN-1) were genotyped in the sample. In addition, a mutation, Glu298Asp, in exon 7 of NOS3 and a 27 bp variable number tandem repeat (VNTR) marker in intron 4 of NOS3 were evaluated in the sibling pairs and in an additional 92 unrelated African-Americans with type 2 diabetes mellitus-associated ESRD (singletons). Association analyses utilized the relative predispositional effect method. Model independent linkage analyses were performed using GeneHunter-plus and MapMaker/SIBS (exclusion analysis) software.

    Results: Significant evidence for association with ESRD was detected for alleles 7 and 9 of the NOS1 gene (11.9 and 34.2%, respectively, in unrelated probands of ESRD families versus 6.5 and 27.5%, respectively, in race-matched controls, both P:<0.01). These associations were maintained when the unrelated first sibling from each family was used in a case-control comparison and was most pronounced in the non-diabetic ESRD cases. The NOS3 and EDN-1 markers failed to provide consistent evidence for association in the sibling pairs and the diabetic ESRD singletons, although we identified two novel endothelial constitutive NOS4 (ecNOS4) VNTR alleles in African-Americans. Significant evidence for linkage was not detected between the NOS genes or the EDN-1 gene in either all-cause ESRD or when the ESRD sibling pairs were stratified by aetiology (type 2 diabetic ESRD or non-diabetic aetiologies).

    Conclusion: Based upon the consistent allelic associations, we believe that further evaluation of the NOS1 gene in ESRD susceptibility in African-Americans is warranted.

    Funded by: NHLBI NIH HHS: R01 HL56266; NIDDK NIH HHS: R01 DK53591

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2000;15;11;1794-800

  • A neuronal NO synthase (NOS1) gene polymorphism is associated with asthma.

    Grasemann H, Yandava CN, Storm van's Gravesande K, Deykin A, Pillari A, Ma J, Sonna LA, Lilly C, Stampfer MJ, Israel E, Silverman EK and Drazen JM

    Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

    Recent family-based studies have revealed evidence for linkage of chromosomal region 12q to both asthma and high total serum immunoglobulin E (IgE) levels. Among the candidate genes in this region for asthma is neuronal nitric oxide synthase (NOS1). We sought a genetic association between a polymorphism in the NOS1 gene and the diagnosis of asthma, using a case-control design. Frequencies for allele 17 and 18 of a CA repeat in exon 29 of the NOS1 gene were significantly different between 490 asthmatic and 350 control subjects. Allele 17 was more common in the asthmatics (0.83 vs 0.76, or 1.49 [95% CI 1.17-1.90], P = 0.013) while allele 18 was less common in the asthmatics (0.06 vs 0.12, or 0.49 [95% CI 0.34-0. 69], P = 0.0004). To confirm these results we genotyped an additional 1131 control subjects and found the frequencies of alleles 17 and 18 to be virtually identical to those ascertained in our original control subjects. Total serum IgE was not associated with any allele of the polymorphism. These findings provide support, from case-control association analysis, for NOS1 as a candidate gene for asthma.

    Funded by: NHLBI NIH HHS: HL-56383

    Biochemical and biophysical research communications 2000;272;2;391-4

  • Variants of NOS1, NOS2, and NOS3 genes in asthmatics.

    Gao PS, Kawada H, Kasamatsu T, Mao XQ, Roberts MH, Miyamoto Y, Yoshimura M, Saitoh Y, Yasue H, Nakao K, Adra CN, Kun JF, Moro-oka S, Inoko H, Ho LP, Shirakawa T and Hopkin JM

    Experimental Medicine Unit, University of Wales Swansea, Swansea, United Kingdom.

    Nitric oxide (NO) gas concentrations are higher in expired air in asthmatics. NO is synthesized by three isoforms of NO synthase (NOS) encoded by three distinct genes, NOS1, NOS2, and NOS3. Genome-wide searches have identified linkages to asthma on chromosomes 7, 12, and 17 where these three genes are localized. No association study, however, has been reported to date. To test whether variants of NOS1, NOS2, and NOS3 relate to asthma, a genetic association study was conducted in a British population (n = 300). Intragenic microsatellite variants of NOS1 were significantly associated with asthma [odds ratio (OR) = 2.08, 95% CI: 1.20-3.57 (95% CI), P = 0.008 (Pc = 0.048)], but not with IgE levels. Neither NOS2 nor NOS3 variants showed any association with asthma nor IgE levels. These findings suggest that NOS1 variants may be a significant contributor to asthma in a British population.

    Biochemical and biophysical research communications 2000;267;3;761-3

  • Neuronal NO synthase (NOS1) is a major candidate gene for asthma.

    Grasemann H, Yandava CN and Drazen JM

    Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Asthma is a common, but heterogeneous disease, characterized by reversible airway obstruction, bronchial hyperresponsiveness (BHR); and is commonly associated with atopy. The messenger molecule nitric oxide (NO), that is formed by neuronal NO synthase (NOS1), is known to have a key role in bronchomotor control in animals. In humans the gene for NOS1 is located on chromosome 12q24, in a region that had been shown in family studies to be linked to the diagnosis of asthma. We identified variants of the NOS1 gene, and assessed whether there was a genetic association between these variants of NOS1 and the diagnosis asthma. A total of 410 Caucasian asthma patients and 228 Caucasian controls were screened for three bi-allelic polymorphisms in the NOS1 gene that had been detected by single-stranded conformational polymorphism (SSCP) analysis and confirmed by sequencing. Allele frequencies of a polymorphism in exon 29 of the NOS1 gene were significantly different between asthmatics and controls (P<0.05). These findings suggest that variants of the NOS1 gene may be one source of genetic risk for asthma.

    Funded by: NHLBI NIH HHS: HL-56383

    Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 1999;29 Suppl 4;39-41

  • Association analysis of TG repeat polymorphism of the neuronal nitric oxide synthase gene with essential hypertension.

    Takahashi Y, Nakayama T, Soma M, Uwabo J, Izumi Y and Kanmatsuse K

    Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.

    The nitric oxide synthase (NOS) gene is thought to be involved in essential hypertension because nitric oxide is implicated in endothelium-mediated or nitroxidergic neuron-mediated vasodilation. Using simple tandem repeat DNA polymorphism of the neuronal constitutive NOS (nNOS) gene, we carried out an association study in patients with essential hypertension. One hundred and thirty-one patients with essential hypertension and 147 subjects with normal blood pressure were studied. Polymerase chain reaction was applied to amplify the TG repeat site in the nNOS gene, and alleles based on the TG repeat number were determined. Eight alleles were identified in this study of Japanese subjects. Overall distributions of allele frequencies in the two groups were not significantly different. Thus, the genes detected by examination of this microsatellite polymorphism in the nNOS gene are not associated with essential hypertension.

    Clinical genetics 1997;52;2;83-5

  • Genetic evidence for the neuronal nitric oxide synthase gene (NOS1) as a susceptibility locus for infantile pyloric stenosis.

    Chung E, Curtis D, Chen G, Marsden PA, Twells R, Xu W and Gardiner M

    Department of Paediatrics, University College London Medical School, Rayne Institute, United Kingdom. echung@hgmp.mrc.ac.uk

    The etiological role of the gene for neuronal nitric oxide synthase (NOS1) in infantile pyloric stenosis (PS) was investigated by analysis of two intragenic polymorphisms (NOS1a and NOS1b) in 27 families. There was significant overall transmission disequilibrium between PS and NOS1a (P = .006). Consideration of each allele independently revealed a highly significant tendency for allele 7 (210 bp) to be preferentially transmitted to the affected offspring (P = .0006). These observations suggest that NOS1 is a susceptibility locus for PS.

    American journal of human genetics 1996;58;2;363-70

Literature (215)

Pubmed - human_disease

  • Nitric oxide synthase gene polymorphisms in children with primary nocturnal enuresis: a preliminary study.

    Balat A, Alasehirli B, Oguzkan S and Gungor M

    Department of Pediatric Nephrology, Gaziantep University, Medical Faculty, Gaziantep, Turkey. aysebalat@hotmail.com

    Aims: Recent studies demonstrated some differences in urinary electrolytes of enuretic children. Intrarenal nitric oxide (NO) serves as a major regulator of renal sodium and water excretion like an endogenous diuretic. This study aimed to investigate endothelial (eNOS), and neuronal (nNOS) NO synthase gene polymorphisms in children with primary nocturnal enuresis (PNE).

    The eNOS gene polymorphism was investigated in 171 Turkish children (57 PNE cases and 114 healthy, non-enuretic controls), and nNOS gene polymorphism was determined in 158 Turkish children (83 PNE cases and 75 healthy, non-enuretic controls). The glu298asp (G/T) polymorphism of the eNOS and C276T (C/T) polymorphism of nNOS genes were genotyped using PCR.

    Results: The distribution of GG, TG, and TT genotypes for eNOS gene was 48%, 33%, and 19% in PNE, compared with 61%, 26%, and 13% in the controls (p > 0.05). The distribution of CC, TC, TT and genotypes for nNOS gene was 31%, 29%, and 40% in PNE compared with 10%, 43%, and 47% in the controls. CC genotype was found higher in enuretic children (p = 0.002). The eNOS and nNOS gene polymorphisms were not associated with positive family history, frequency of enuresis, and clinical response to desmopressin.

    Conclusions: This study is the first to search the NOS gene polymorphisms in children with PNE. It was determined that eNOS gene polymorphism may not be associated with PNE, while nNOS gene polymorphism, a predominantly CC genotype, may be associated with PNE in Turkish children. Further studies with larger samples together with the detection of enuresis gene may help determine the exact role of nNOS gene polymorphism in enuresis.

    Renal failure 2007;29;1;79-83

  • Investigation of a neuronal nitric oxide synthase gene (NOS1) polymorphism in a multiple sclerosis population.

    Tajouri L, Ferreira L, Ovcaric M, Curtain R, Lea R, Csurhes P, Pender MP and Griffiths LR

    Genomics Research Centre, School of Health Science, Griffith University Gold Coast, Southport, Queensland, 4215 Australia.

    Multiple Sclerosis (MS) is a chronic neurological disease characterized by demyelination associated with infiltrating white blood cells in the central nervous system (CNS). Nitric oxide synthases (NOS) are a family of enzymes that control the production of nitric oxide. It is possible that neuronal NOS could be involved in MS pathophysiology and hence the nNOS gene is a potential candidate for involvement in disease susceptibility. The aim of this study was to determine whether allelic variation at the nNOS gene locus is associated with MS in an Australian cohort. DNA samples obtained from a Caucasian Australian population affected with MS and an unaffected control population, matched for gender, age and ethnicity, were genotyped for a microsatellite polymorphism in the promoter region of the nNOS gene. Allele frequencies were compared using chi-squared based statistical analyses with significance tested by Monte Carlo simulation. Allelic analysis of MS cases and controls produced a chi-squared value of 5.63 with simulated P = 0.96 (OR(max) = 1.41, 95% CI: 0.926-2.15). Similarly, a Mann-Whitney U analysis gave a non-significant P-value of 0.377 for allele distribution. No differences in allele frequencies were observed for gender or clinical course subtype (P > 0.05). Statistical analysis indicated that there is no association of this nNOS variant and MS and hence the gene does not appear to play a genetically significant role in disease susceptibility.

    Journal of the neurological sciences 2004;218;1-2;25-8

  • Nitric oxide synthase 1 as a potential modifier gene of decline in lung function in patients with cystic fibrosis.

    Texereau J, Marullo S, Hubert D, Coste J, Dusser DJ, Dall'Ava-Santucci J and Dinh-Xuan AT

    Service de Physiologie-Explorations Fonctionnelles, Hôpital Cochin, AP-HP, Université Paris 5, Paris, France.

    Background: The severity of lung disease varies widely in patients with cystic fibrosis (CF) who have the same type of mutations of the cystic fibrosis transmembrane regulator (CFTR) gene, suggesting involvement of "modifier" genes. The nitric oxide synthase 1 (NOS1) gene is a candidate for this role because exhaled nitric oxide (NO) is reduced in patients with CF and NOS1 activity contributes to transepithelial ionic transport, immune defence, and non-specific inflammation of the airways.

    Methods: Dinucleotide GT repeat polymorphism was studied in the 5' untranslated region of the NOS1 gene, immediately upstream from the transcription initiation site, in 59 patients with CF and 59 healthy controls.

    Results: Nineteen alleles of the NOS1 gene were identified according to the number of GT repeats (from 18 to 36) in the 5 untranslated region. Exhaled NO levels were significantly correlated with the number of GT repeats. Patients with CF who had the NOS1 genotype associated with high NO production had a slower decline in lung function during the 5 year follow up period. There was no confounding effect of age, chronic bacterial colonisation of the airway, or CFTR genotype.

    Conclusions: These data suggest a possible link between the NOS1 gene locus and the rate of decline in lung function in patients with CF.

    Thorax 2004;59;2;156-8

  • No association between a neuronal nitric oxide synthase (NOS1) gene polymorphism on chromosome 12q24 and bipolar disorder.

    Buttenschön HN, Mors O, Ewald H, McQuillin A, Kalsi G, Lawrence J, Gurling H and Kruse TA

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2004;124B;1;73-5

  • Association between Parkinson's disease and polymorphisms in the nNOS and iNOS genes in a community-based case-control study.

    Levecque C, Elbaz A, Clavel J, Richard F, Vidal JS, Amouyel P, Tzourio C, Alpérovitch A and Chartier-Harlin MC

    INSERM Unit 508 Institut Pasteur de Lille, 59019 Lille, France.

    Excess of nitric oxide (NO) has been shown to exert neurotoxic impacts in the brain. Moreover, inhibition of two NO-synthesizing enzymes, neuronal NOS (nNOS) and inducible NOS (iNOS), displays neuroprotective effects in the MPTP model of Parkinson's disease (PD). These data suggest a possible involvement of NOS as factors controlling the resistance of the nigral dopaminergic neurons to environmental insults. Therefore, we investigated whether polymorphisms present in these genes could contribute to the risk of developing PD. We carried out a community-based case-control study among subjects enrolled in the Mutualité Sociale Agricole, the French health insurance organization for workers connected to agriculture. Two-hundred and nine PD patients and 488 controls of European (mostly French) ancestry and matched for age, sex and region of residency were included in this study. Associations were observed with polymorphisms present in exon 22 of iNOS (OR for AA carriers=0.50, 95% CI=0.29-0.86, P=0.01) and in exon 29 of nNOS (OR for carriers of the T allele=1.53, 95% CI=1.08-2.16, P=0.02); no association was observed with a polymorphism in exon 18 of nNOS (OR for carriers of the T allele=1.20, 95% CI=0.85-1.69, P=0.30). Moreover, a significant interaction of the nNOS polymorphisms with current and ever cigarette smoking was found (nNOS 18, P=0.05; nNOS 29, P=0.04). All together, these data favour an involvement of these two genes as new modifier genes in PD.

    Human molecular genetics 2003;12;1;79-86

  • Association analysis for neuronal nitric oxide synthase gene polymorphism with major depression and fluoxetine response.

    Yu YW, Chen TJ, Wang YC, Liou YJ, Hong CJ and Tsai SJ

    Yu's Psychiatric Clinic, Kaohsiung, Taipei, Taiwan, ROC.

    Nitric oxide (NO) is produced from its precursor L-arginine by the enzyme NO synthase (NOS), which includes at least three distinct isoforms - neuronal (nNOS), endothelial, and inducible NOS. Recent studies have implicated NOS in the mechanism that underlies the therapeutic efficacy of antidepressant medication. In addition, major depressive disorder (MDD) patients were found to have significantly higher plasma nitrate concentrations than normal subjects, an index of NO production, in comparison to normal subjects. In a population-based association study, we tested the hypothesis that the nNOS C276T polymorphism confers susceptibility to MDD. We also examined the association between this polymorphism and therapeutic fluoxetine response in 114 MDD patients who underwent a 4-week fluoxetine treatment. The results demonstrate that the nNOS variants are found at similar frequencies in MDD patients and healthy control subjects. Further, we did not discover any genetic variants that influenced the fluoxetine response in MDD patients treated with fluoxetine. Our findings suggest that this nNOS C276T polymorphism does not play a major role in the susceptibility to, or fluoxetine response in, MDD. However, the association between other NOS variants and MDD or antidepressant response, including sexual dysfunction, may warrant further investigation.

    Neuropsychobiology 2003;47;3;137-40

  • No association between the neuronal nitric oxide synthase gene polymorphism and Alzheimer Disease.

    Liou YJ, Hong CJ, Liu HC, Liu CY, Liu TY, Chen IC and Tsai SJ

    Section of Psychiatry, Yu-Li Veterans Hospital, Hualien, Taiwan, Republic of China.

    Nitric oxide synthase (NOS) has been implicated in the pathogenesis of Alzheimer disease (AD). To examine the role of the neuronal NOS (nNOS) gene in AD, patients (n = 139) and control subjects (n = 101) were genotyped for the nNOS dinucleotide polymorphism. No association was demonstrated for AD and this particular nNOS polymorphism.

    American journal of medical genetics 2002;114;6;687-8

  • 5'-flanking region polymorphism of the neuronal nitric oxide synthase gene with Parkinson's disease in Taiwan.

    Lo HS, Hogan EL and Soong BW

    Department of Neurology, Taiwan Adventist Hospital, Taipei, Taiwan.

    Though the etiology of Parkinson's disease (PD) is unresolved and may be heterogeneous involving both environmental and genetic factors, there are indications that oxidative stress plays an important role in dopaminergic neuronal death. And, it has been reported that inhibition of nitric oxide synthase (NOS) can prevent the destruction of dopaminergic neurons in mammals. To determine if NOS gene polymorphism affects the 5' flanking region that is immediately upstream of the transcription start site lying between the TATA element and CAATT boxes in PD, and differs significantly between patients with PD and normal controls, we studied genetic polymorphism in that region of the neuronal NOS gene in Chinese patients with PD living in Taiwan. The results indicate that the allele size distribution in that region was statistically significantly different between patients with PD and normal.

    Journal of the neurological sciences 2002;194;1;11-3

  • Exhaled nitric oxide in patients with asthma: association with NOS1 genotype.

    Wechsler ME, Grasemann H, Deykin A, Silverman EK, Yandava CN, Israel E, Wand M and Drazen JM

    Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    An increased concentration of nitric oxide (NO) in exhaled air (FENO) is now recognized as a critical component of the asthmatic phenotype. When we identified patients with asthma on the basis of a standard case definition alone, we found that they were remarkably heterogeneous with respect to their FENO. However, when we included genotype at a prominent asthma candidate gene (i.e., NOS1) in the case definition, and determined the number of AAT repeats in intron 20, we identified a remarkably homogeneous cohort of patients with respect to FENO. Both mean FENO (p = 0.00008) and variability around the mean (p = 0.000002) were significantly lower in asthmatic individuals with a high number (> or = 12) of AAT repeats at this locus than in those with fewer repeats. These data provide a biologically tenable link between genotype at a candidate gene in a region of linkage, NOS1, and an important component of the asthmatic phenotype, FENO. We show that addition of NOS1 genotype to the case definition of asthma allows the identification of a uniform cohort of patients, with respect to FENO, that would have been indistinguishable by other physiologic criteria. Our isolation of this homogeneous cohort of patients ties together the well-established associations among asthma, increased concentrations of NO in the exhaled air of asthmatic individuals, and variations of trinucleotide repeat sequences as identified in several neurologic conditions.

    Funded by: NHLBI NIH HHS: P50-HL-56383

    American journal of respiratory and critical care medicine 2000;162;6;2043-7

  • Genetic analysis of nitric oxide and endothelin in end-stage renal disease.

    Freedman BI, Yu H, Anderson PJ, Roh BH, Rich SS and Bowden DW

    Department of Internal Medicine/Nephrology, The Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1053, USA.

    Background: Genetic factors have been implicated in the development of the common aetiologies of end-stage renal disease (ESRD), including renal failure attributed to hypertension, diabetes mellitus, systemic lupus erythematosus and human immunodeficiency virus infection. Nitric oxide (NO) and endothelin are powerful vasoactive mediators involved in inflammation and regulation of vascular tone and blood pressure. We evaluated the role of the neuronal constitutive (NOS1) and endothelial constitutive (NOS3) nitric oxide synthase genes and the endothelin-1 (EDN-1) gene in predisposition to chronic renal failure in African-Americans.

    Methods: The study population for the linkage and association analyses in ESRD consisted of 361 individuals from 168 multiplex African-American families. These individuals comprised 207 unweighted sibling pairs concordant for all-cause ESRD. Microsatellite markers NOS1B (NOS1), D7S636 (NOS3) and CPHD1-1/2 (EDN-1) were genotyped in the sample. In addition, a mutation, Glu298Asp, in exon 7 of NOS3 and a 27 bp variable number tandem repeat (VNTR) marker in intron 4 of NOS3 were evaluated in the sibling pairs and in an additional 92 unrelated African-Americans with type 2 diabetes mellitus-associated ESRD (singletons). Association analyses utilized the relative predispositional effect method. Model independent linkage analyses were performed using GeneHunter-plus and MapMaker/SIBS (exclusion analysis) software.

    Results: Significant evidence for association with ESRD was detected for alleles 7 and 9 of the NOS1 gene (11.9 and 34.2%, respectively, in unrelated probands of ESRD families versus 6.5 and 27.5%, respectively, in race-matched controls, both P:<0.01). These associations were maintained when the unrelated first sibling from each family was used in a case-control comparison and was most pronounced in the non-diabetic ESRD cases. The NOS3 and EDN-1 markers failed to provide consistent evidence for association in the sibling pairs and the diabetic ESRD singletons, although we identified two novel endothelial constitutive NOS4 (ecNOS4) VNTR alleles in African-Americans. Significant evidence for linkage was not detected between the NOS genes or the EDN-1 gene in either all-cause ESRD or when the ESRD sibling pairs were stratified by aetiology (type 2 diabetic ESRD or non-diabetic aetiologies).

    Conclusion: Based upon the consistent allelic associations, we believe that further evaluation of the NOS1 gene in ESRD susceptibility in African-Americans is warranted.

    Funded by: NHLBI NIH HHS: R01 HL56266; NIDDK NIH HHS: R01 DK53591

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2000;15;11;1794-800

  • A neuronal NO synthase (NOS1) gene polymorphism is associated with asthma.

    Grasemann H, Yandava CN, Storm van's Gravesande K, Deykin A, Pillari A, Ma J, Sonna LA, Lilly C, Stampfer MJ, Israel E, Silverman EK and Drazen JM

    Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.

    Recent family-based studies have revealed evidence for linkage of chromosomal region 12q to both asthma and high total serum immunoglobulin E (IgE) levels. Among the candidate genes in this region for asthma is neuronal nitric oxide synthase (NOS1). We sought a genetic association between a polymorphism in the NOS1 gene and the diagnosis of asthma, using a case-control design. Frequencies for allele 17 and 18 of a CA repeat in exon 29 of the NOS1 gene were significantly different between 490 asthmatic and 350 control subjects. Allele 17 was more common in the asthmatics (0.83 vs 0.76, or 1.49 [95% CI 1.17-1.90], P = 0.013) while allele 18 was less common in the asthmatics (0.06 vs 0.12, or 0.49 [95% CI 0.34-0. 69], P = 0.0004). To confirm these results we genotyped an additional 1131 control subjects and found the frequencies of alleles 17 and 18 to be virtually identical to those ascertained in our original control subjects. Total serum IgE was not associated with any allele of the polymorphism. These findings provide support, from case-control association analysis, for NOS1 as a candidate gene for asthma.

    Funded by: NHLBI NIH HHS: HL-56383

    Biochemical and biophysical research communications 2000;272;2;391-4

  • Variants of NOS1, NOS2, and NOS3 genes in asthmatics.

    Gao PS, Kawada H, Kasamatsu T, Mao XQ, Roberts MH, Miyamoto Y, Yoshimura M, Saitoh Y, Yasue H, Nakao K, Adra CN, Kun JF, Moro-oka S, Inoko H, Ho LP, Shirakawa T and Hopkin JM

    Experimental Medicine Unit, University of Wales Swansea, Swansea, United Kingdom.

    Nitric oxide (NO) gas concentrations are higher in expired air in asthmatics. NO is synthesized by three isoforms of NO synthase (NOS) encoded by three distinct genes, NOS1, NOS2, and NOS3. Genome-wide searches have identified linkages to asthma on chromosomes 7, 12, and 17 where these three genes are localized. No association study, however, has been reported to date. To test whether variants of NOS1, NOS2, and NOS3 relate to asthma, a genetic association study was conducted in a British population (n = 300). Intragenic microsatellite variants of NOS1 were significantly associated with asthma [odds ratio (OR) = 2.08, 95% CI: 1.20-3.57 (95% CI), P = 0.008 (Pc = 0.048)], but not with IgE levels. Neither NOS2 nor NOS3 variants showed any association with asthma nor IgE levels. These findings suggest that NOS1 variants may be a significant contributor to asthma in a British population.

    Biochemical and biophysical research communications 2000;267;3;761-3

  • Neuronal NO synthase (NOS1) is a major candidate gene for asthma.

    Grasemann H, Yandava CN and Drazen JM

    Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Asthma is a common, but heterogeneous disease, characterized by reversible airway obstruction, bronchial hyperresponsiveness (BHR); and is commonly associated with atopy. The messenger molecule nitric oxide (NO), that is formed by neuronal NO synthase (NOS1), is known to have a key role in bronchomotor control in animals. In humans the gene for NOS1 is located on chromosome 12q24, in a region that had been shown in family studies to be linked to the diagnosis of asthma. We identified variants of the NOS1 gene, and assessed whether there was a genetic association between these variants of NOS1 and the diagnosis asthma. A total of 410 Caucasian asthma patients and 228 Caucasian controls were screened for three bi-allelic polymorphisms in the NOS1 gene that had been detected by single-stranded conformational polymorphism (SSCP) analysis and confirmed by sequencing. Allele frequencies of a polymorphism in exon 29 of the NOS1 gene were significantly different between asthmatics and controls (P<0.05). These findings suggest that variants of the NOS1 gene may be one source of genetic risk for asthma.

    Funded by: NHLBI NIH HHS: HL-56383

    Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 1999;29 Suppl 4;39-41

  • Association analysis of TG repeat polymorphism of the neuronal nitric oxide synthase gene with essential hypertension.

    Takahashi Y, Nakayama T, Soma M, Uwabo J, Izumi Y and Kanmatsuse K

    Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.

    The nitric oxide synthase (NOS) gene is thought to be involved in essential hypertension because nitric oxide is implicated in endothelium-mediated or nitroxidergic neuron-mediated vasodilation. Using simple tandem repeat DNA polymorphism of the neuronal constitutive NOS (nNOS) gene, we carried out an association study in patients with essential hypertension. One hundred and thirty-one patients with essential hypertension and 147 subjects with normal blood pressure were studied. Polymerase chain reaction was applied to amplify the TG repeat site in the nNOS gene, and alleles based on the TG repeat number were determined. Eight alleles were identified in this study of Japanese subjects. Overall distributions of allele frequencies in the two groups were not significantly different. Thus, the genes detected by examination of this microsatellite polymorphism in the nNOS gene are not associated with essential hypertension.

    Clinical genetics 1997;52;2;83-5

  • Genetic evidence for the neuronal nitric oxide synthase gene (NOS1) as a susceptibility locus for infantile pyloric stenosis.

    Chung E, Curtis D, Chen G, Marsden PA, Twells R, Xu W and Gardiner M

    Department of Paediatrics, University College London Medical School, Rayne Institute, United Kingdom. echung@hgmp.mrc.ac.uk

    The etiological role of the gene for neuronal nitric oxide synthase (NOS1) in infantile pyloric stenosis (PS) was investigated by analysis of two intragenic polymorphisms (NOS1a and NOS1b) in 27 families. There was significant overall transmission disequilibrium between PS and NOS1a (P = .006). Consideration of each allele independently revealed a highly significant tendency for allele 7 (210 bp) to be preferentially transmitted to the affected offspring (P = .0006). These observations suggest that NOS1 is a susceptibility locus for PS.

    American journal of human genetics 1996;58;2;363-70

Pubmed - other

  • EDNRA variants associate with smooth muscle mRNA levels, cell proliferation rates, and cystic fibrosis pulmonary disease severity.

    Darrah R, McKone E, O'Connor C, Rodgers C, Genatossio A, McNamara S, Gibson R, Stuart Elborn J, Ennis M, Gallagher CG, Kalsheker N, Aitken M, Wiese D, Dunn J, Smith P, Pace R, Londono D, Goddard KA, Knowles MR and Drumm ML

    Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106, USA.

    Airway inflammation and pulmonary disease are heterogeneous phenotypes in cystic fibrosis (CF) patients, even among patients with the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Endothelin, a proinflammatory peptide and smooth muscle agonist, is increased in CF airways, potentially contributing to the pulmonary phenotype. Four cohorts of CF patients were screened for variants in endothelin pathway genes to determine whether any of these variants associated with pulmonary function. An initial cohort of 808 CF patients homozygous for the common CF mutation, DeltaF508, showed significant association for polymorphisms in the endothelin receptor A gene, EDNRA (P = 0.04), but not in the related endothelin genes (EDN1, EDN2, EDN3, or EDNRB) or NOS1, NOS2A, or NOS3. Variants within EDNRA were examined in three additional cohorts of CF patients, 238 patients from Seattle, WA, 303 from Ireland and the U.K., and 228 from Cleveland, OH, for a total of 1,577 CF patients. The three additional groups each demonstrated a significant association between EDNRA 3'-untranslated region (UTR) variant rs5335 and pulmonary function (P = 0.002). At the molecular level, single nucleotide primer extension assays suggest that the effect of the variants is quantitative. EDNRA mRNA levels from cultured primary tracheal smooth muscle cells are greater for the allele that appears to be deleterious to lung function than for the protective allele, suggesting a mechanism by which increased receptor function is harmful to the CF airway. Finally, cell proliferation studies using human airway smooth muscle cells demonstrated that cells homozygous for the deleterious allele proliferate at a faster rate than those homozygous for the protective allele.

    Funded by: NHLBI NIH HHS: HL-68890, K23-HL-70849-01, T32-HL-07415

    Physiological genomics 2010;41;1;71-7

  • Association of a functional variant of neuronal nitric oxide synthase gene with self-reported impulsiveness, venturesomeness and empathy in male offenders.

    Retz W, Reif A, Freitag CM, Retz-Junginger P and Rösler M

    Neurocentre, Institute for Forensic Psychology and Psychiatry, Saarland University Hospital, Homburg, Saar, Germany. wolfgang.retz@uks.eu

    It has been shown that a functional promoter dinucleotide repeat length variation of the neuronal nitric oxide synthase gene (NOS1 Ex1f-VNTR) is associated with impulsivity-related behavioral phenotypes. In this study, the Eysenck Impulsivity Questionnaire (IVE-7) was administered to 182 male offenders to prove the hypothesis that NOS1 Ex1f-VNTR is associated with self-reported impulsiveness, venturesomeness and empathy. Multivariate analysis of variance (MANCOVA) revealed a significant multivariate effect of NOS1 Ex1f-VNTR genotype on IVE-7 measures (P = 0.0006). The effect was more pronounced regarding impulsiveness and empathy (P = 0.0052 and P = 0.0036, respectively) as compared with venturesomeness, which was only of borderline significance. The findings give additional evidence that NOS-I is involved in the regulation of impulsive personality traits and support the notion that the NOS1 gene takes part in the regulation of social behavior.

    Journal of neural transmission (Vienna, Austria : 1996) 2010;117;3;321-4

  • Lys842 in neuronal nitric-oxide synthase enables the autoinhibitory insert to antagonize calmodulin binding, increase FMN shielding, and suppress interflavin electron transfer.

    Guan ZW, Haque MM, Wei CC, Garcin ED, Getzoff ED and Stuehr DJ

    Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.

    Neuronal nitric-oxide synthase (nNOS) contains a unique autoinhibitory insert (AI) in its FMN subdomain that represses nNOS reductase activities and controls the calcium sensitivity of calmodulin (CaM) binding to nNOS. How the AI does this is unclear. A conserved charged residue (Lys(842)) lies within a putative CaM binding helix in the middle of the AI. We investigated its role by substituting residues that neutralize (Ala) or reverse (Glu) the charge at Lys(842). Compared with wild type nNOS, the mutant enzymes had greater cytochrome c reductase and NADPH oxidase activities in the CaM-free state, were able to bind CaM at lower calcium concentration, and had lower rates of heme reduction and NO synthesis in one case (K842A). Moreover, stopped-flow spectrophotometric experiments with the nNOS reductase domain indicate that the CaM-free mutants had faster flavin reduction kinetics and had less shielding of their FMN subdomains compared with wild type and no longer increased their level of FMN shielding in response to NADPH binding. Thus, Lys(842) is critical for the known functions of the AI and also enables two additional functions of the AI as newly identified here: suppression of electron transfer to FMN and control of the conformational equilibrium of the nNOS reductase domain. Its effect on the conformational equilibrium probably explains suppression of catalysis by the AI.

    Funded by: NCI NIH HHS: CA53914, R01 CA053914; NHLBI NIH HHS: HL58883, HL76491, P01 HL076491, R01 HL058883; NIGMS NIH HHS: GM51491, R01 GM051491

    The Journal of biological chemistry 2010;285;5;3064-75

  • Replication of association between schizophrenia and ZNF804A in the Irish Case-Control Study of Schizophrenia sample.

    Riley B, Thiselton D, Maher BS, Bigdeli T, Wormley B, McMichael GO, Fanous AH, Vladimirov V, O'Neill FA, Walsh D and Kendler KS

    Department of Psychiatry, Virginia Commonwealth University, Richmond, VA 23298-0424, USA. bpriley@vcu.edu

    A recent genome-wide association study reported association between schizophrenia and the ZNF804A gene on chromosome 2q32.1. We attempted to replicate these findings in our Irish Case-Control Study of Schizophrenia (ICCSS) sample (N=1021 cases, 626 controls). Following consultation with the original investigators, we genotyped three of the most promising single-nucleotide polymorphisms (SNPs) from the Cardiff study. We replicate association with rs1344706 (trend test one-tailed P=0.0113 with the previously associated A allele) in ZNF804A. We detect no evidence of association with rs6490121 in NOS1 (one-tailed P=0.21), and only a trend with rs9922369 in RGRIP1L (one-tailed P=0.0515). On the basis of these results, we completed genotyping of 11 additional linkage disequilibrium-tagging SNPs in ZNF804A. Of 12 SNPs genotyped, 11 pass quality control criteria and 4 are nominally associated, with our most significant evidence of association at rs7597593 (P=0.0013) followed by rs1344706. We observe no evidence of differential association in ZNF804A on the basis of family history or sex of case. The associated SNP rs1344706 lies in approximately 30 bp of conserved mammalian sequence, and the associated A allele is predicted to maintain binding sites for the brain-expressed transcription factors MYT1l and POU3F1/OCT-6. In controls, expression is significantly increased from the A allele of rs1344706 compared with the C allele. Expression is increased in schizophrenic cases compared with controls, but this difference does not achieve statistical significance. This study replicates the original reported association of ZNF804A with schizophrenia and suggests that there is a consistent link between the A allele of rs1344706, increased expression of ZNF804A and risk for schizophrenia.

    Funded by: NIMH NIH HHS: R01 MH041953, R01 MH041953-08S1, R01 MH041953-09A2, R01 MH041953-10, R01 MH041953-11, R01 MH041953-12, R01 MH041953-13, R01 MH041953-14, R01 MH041953-15, R01 MH041953-16, R01 MH041953-17, R01 MH083094, R01-MH41953

    Molecular psychiatry 2010;15;1;29-37

  • Genetic association analysis of functional polymorphisms in neuronal nitric oxide synthase 1 gene (NOS1) and mood disorders and fluvoxamine response in major depressive disorder in the Japanese population.

    Okumura T, Kishi T, Okochi T, Ikeda M, Kitajima T, Yamanouchi Y, Kinoshita Y, Kawashima K, Tsunoka T, Inada T, Ozaki N and Iwata N

    Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Japan.

    Nitric oxide has been reported to play a role in neural transmitter release and N-methyl-D-aspartate receptor activation, as well as to be related to oxidative stress. Abnormalities in both of these mechanisms are thought to be involved in the pathophysiology of mood disorders including major depressive disorder (MDD) and bipolar disorder (BP). In addition, several lines of evidence support an association between abnormalities in neuronal nitric oxide synthases (nNOS) and mood disorders. Therefore, we studied the association of nNOS gene (NOS1) and mood disorders and the efficacy of fluvoxamine treatment in Japanese MDD patients.

    Using a single nucleotide polymorphism (SNP; rs41279104, also called ex1c), we conducted a genetic association analysis of case-control samples (325 MDD patients, 154 BP patients and 807 controls) in the Japanese population. In addition, we performed an association analysis between NOS1 and the efficacy of fluvoxamine treatment in 117 MDD patients. We defined a clinical response as a decrease of more than 50% in baseline SIGH-D (Structured Interview Guide for the Hamilton Rating Scale for Depression) score within 8 weeks, and clinical remission as an SIGH-D score of less than 7 at 8 weeks.

    Results: We did not detect a significant association between NOS1 and MDD, BP or the fluvoxamine therapeutic response in MDD in allele/genotype-wise analysis.

    Conclusions: We did not detect an association between only one marker (rs41279104) in NOS1 and Japanese mood disorder patients and fluvoxamine response, but sample sizes were probably too small to allow a meaningful test. Moreover, because we did not perform an association analysis based on linkage disequilibrium and a mutation scan of NOS1, a replication of the study using a larger sample and based on linkage disequilibrium may be required for conclusive results.

    Neuropsychobiology 2010;61;2;57-63

  • No association between a promoter NOS1 polymorphism (rs41279104) and Infantile Hypertrophic Pyloric Stenosis.

    Lagerstedt-Robinson K, Svenningsson A and Nordenskjöld A

    Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. kristina.lagerstedt@ki.se

    Infantile hypertrophic pyloric stenosis (IHPS) is a condition affecting infants in the first few months of life. The condition is manifested by persistent vomiting and is caused by a hypertrophied muscle obstructing the gastric outlet. The condition is treated by pyloromyotomy. The incidence is 1-8/1000 births and varies among different populations. The etiology of IHPS is unknown, but both genetic and environmental factors are thought to contribute to the disease. Genetic linkage analysis has so far localized five loci that could harbor genes contributing to IHPS. The only gene implicated in IHPS is the nitric oxide synthase gene (NOS1), in which a single nucleotide polymorphism (rs41279104) in the promoter region has been associated with the disease in 16 patients. In this study, we examined an association of this SNP in 54 familial and 28 sporadic cases with IHPS, and compared the results with normal controls using univariate and multiple logistic regression analysis. We could not confirm any association between the analyzed SNP and infantile hypertrophic pyloric stenosis.

    Journal of human genetics 2009;54;12;706-8

  • Retinoic acid-induced nNOS expression depends on a novel PI3K/Akt/DAX1 pathway in human TGW-nu-I neuroblastoma cells.

    Nagl F, Schönhofer K, Seidler B, Mages J, Allescher HD, Schmid RM, Schneider G and Saur D

    II. Medizinische Klinik, Technische Universität München, 81675 Munich, Germany.

    Neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) acts as a neurotransmitter and intracellular signaling molecule in the central and peripheral nervous system. NO regulates multiple processes like neuronal development, plasticity, and differentiation and is a mediator of neurotoxicity. The nNOS gene is highly complex with 12 alternative first exons, exon 1a-1l, transcribed from distinct promoters, leading to nNOS variants with different 5'-untranslated regions. Transcriptional control of the nNOS gene is not understood in detail. To investigate regulation of nNOS gene expression by retinoic acid (RA), we used the human neuroblastoma cell line TGW-nu-I as a model system. We show that RA induces nNOS transcription in a protein synthesis-dependent fashion. We identify the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and the atypical orphan nuclear receptor DAX1 (NR0B1) as critical mediators involved in RA-induced nNOS gene transcription. RA treatment increases DAX1 expression via PI3K/Akt signaling. Upregulation of DAX1 expression in turn induces nNOS transcription in response to RA. These results identify nNOS as a target gene of a novel RA/PI3K/Akt/DAX1-dependent pathway in human neuroblastoma cells and stress the functional importance of the transcriptional regulator DAX1 for nNOS gene expression in response to RA treatment.

    American journal of physiology. Cell physiology 2009;297;5;C1146-56

  • ChAT and NOS in human myenteric neurons: co-existence and co-absence.

    Beck M, Schlabrakowski A, Schrödl F, Neuhuber W and Brehmer A

    Institute of Anatomy I, University of Erlangen-Nuremberg, Krankenhausstrasse 9, 91054, Erlangen, Germany.

    Most myenteric neurons contain one of the two generating enzymes for major excitatory and inhibitory neurotransmitters: choline acetyltransferase (ChAT) or neuronal nitric oxide synthase (NOS). Two minor groups of myenteric neurons contain either both enzymes or neither. Our study had two aims: (1) to compare the proportions of neurons stained for ChAT and/or NOS in human small and large intestinal whole-mounts by co-staining with an antibody against the human neuronal protein Hu C/D (HU); (2) to characterize these neurons morphologically by co-staining with a neurofilament (NF) antibody. In small intestinal whole-mounts co-stained with HU, we counted more ChAT-positive (ChAT+) than NOS+ neurons (52% vs. 38%), whereas the large intestine exhibited fewer ChAT+ than NOS+ neurons (38% vs. 50%). Neurons co-reactive for both ChAT and NOS accounted for about 3% in both regions, whereas neurons negative for both enzymes accounted for 7% in the small intestine and 8% in the large intestine. Co-staining with NF revealed that, in both small and large intestine, ChAT+/NOS+ neurons were either spiny (type I) neurons or displayed smaller perikarya that were weakly or not NF-stained. Of all spiny neurons, almost one third was co-reactive for ChAT and NOS, whereas nearly two thirds were positive only for NOS. Neurons negative for both ChAT and NOS were heterogeneous in size and NF reactivity. Thus, neither the co-existence nor the co-absence of ChAT and NOS in human myenteric neurons is indicative for particular neuron types, with several qualitative and quantitative parameters showing a wide range of interindividual variability.

    Cell and tissue research 2009;338;1;37-51

  • Evidence for increased methylglyoxal in the vasculature of women with preeclampsia: role in upregulation of LOX-1 and arginase.

    Sankaralingam S, Xu H, Jiang Y, Sawamura T and Davidge ST

    Department of Physiology, University of Alberta, Edmonton, Alberta, Canada.

    Preeclampsia is characterized by vascular endothelial dysfunction partly attributed to oxidative stress. In the vasculature of preeclamptic women, we have shown increased lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and arginase expression, which can contribute to vascular oxidative stress. However, the mechanisms of such upregulation are unknown. Methylglyoxal (MG) that plays a role in the vascular complications of diabetes mellitus and the development of hypertension can be one potential factor that can affect LOX-1 and arginase through its ability to induce oxidative stress in vascular cells. MG also reacts with lysine residues in proteins to generate advanced glycation end product, N(epsilon)-carboxy ethyl lysine, which also serves as a marker of MG. We hypothesized that markers of MG formation will be increased in the vasculature of preeclamptic women and that exogenous MG will induce oxidative stress by the upregulation of LOX-1 via arginase. We observed increased N(epsilon)-carboxy ethyl lysine expression in the vasculature of women with preeclampsia in comparison with normotensive pregnant women. Moreover, glyoxalase I and II, enzymes that detoxify MG, and glutathione reductase, which generates reduced glutathione, a cofactor for glyoxalase, are also reduced in preeclampsia. In cultured endothelial cells, MG increased arginase expression by 6 hours and LOX-1 expression by 24 hours. Inhibition of arginase or NO synthase significantly reduced MG-induced LOX-1 expression, superoxide levels, and nitrotyrosine staining. In conclusion, MG-induced LOX-1 expression is mediated via arginase upregulation likely because of uncoupling of NO synthase, which may have implications in preeclampsia.

    Hypertension (Dallas, Tex. : 1979) 2009;54;4;897-904

  • Influence of NOS1 on verbal intelligence and working memory in both patients with schizophrenia and healthy control subjects.

    Donohoe G, Walters J, Morris DW, Quinn EM, Judge R, Norton N, Giegling I, Hartmann AM, Möller HJ, Muglia P, Williams H, Moskvina V, Peel R, O'Donoghue T, Owen MJ, O'Donovan MC, Gill M, Rujescu D and Corvin A

    Neuropsychiatric Genetics Research Group, Department of Psychiatry, Trinity Health Sciences Bldg, St James's Hospital, Dublin 8, Ireland. gary.donohoe@tcd.ie

    Context: Human and animal studies have implicated the gene NOS1 in both cognition and schizophrenia susceptibility.

    Objective: To investigate whether a potential schizophrenia risk single-nucleotide polymorphism (rs6490121) identified in a recent genome-wide association study negatively influences cognition in patients with schizophrenia and healthy control subjects.

    Design: A comparison of both cases and controls grouped according to NOS1 genotype (GG vs AG vs AA) on selected measures of cognition in 2 independent samples. We tested for association between NOS1 rs6490121 and cognitive functions known to be impaired in schizophrenia (IQ, episodic memory, working memory, and attentional control) in an Irish sample. We then sought to replicate the significant results in a German sample.

    Setting: Unrelated patients from general adult psychiatric inpatient and outpatient services and unrelated healthy volunteers from the general population were ascertained.

    Participants: Patients with DSM-IV-diagnosed schizophrenia and healthy control subjects from independent samples of Irish (cases, n = 349; controls, n = 230) and German (cases, n = 232; controls, n = 1344) nationality.

    Results: A main effect of NOS1 genotype on verbal IQ and working memory was observed in the Irish sample where the homozygous carriers of the schizophrenia risk G allele performed poorly compared with the other genotype groups. These findings were replicated in the German sample, again with the GG genotype carriers performing below other genotype groups. Post hoc analysis of additional IQ measures (full-scale and performance IQ) in the German sample revealed that NOS1 GG carriers underperformed on these measures also.

    Conclusions: NOS1 is associated with clinically significant variation in cognition. Whether this is a mechanism by which schizophrenia risk is increased (eg, via an influence on cognitive reserve) is yet to be confirmed.

    Funded by: Medical Research Council: G0601635, G0800509; Wellcome Trust

    Archives of general psychiatry 2009;66;10;1045-54

  • Identification of new putative susceptibility genes for several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment.

    Gratacòs M, Costas J, de Cid R, Bayés M, González JR, Baca-García E, de Diego Y, Fernández-Aranda F, Fernández-Piqueras J, Guitart M, Martín-Santos R, Martorell L, Menchón JM, Roca M, Sáiz-Ruiz J, Sanjuán J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X, Carracedo A and Psychiatric Genetics Network Group

    CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.

    A fundamental difficulty in human genetics research is the identification of the spectrum of genetic variants that contribute to the susceptibility to common/complex disorders. We tested here the hypothesis that functional genetic variants may confer susceptibility to several related common disorders. We analyzed five main psychiatric diagnostic categories (substance-abuse, anxiety, eating, psychotic, and mood disorders) and two different control groups, representing a total of 3,214 samples, for 748 promoter and non-synonymous single nucleotide polymorphisms (SNPs) at 306 genes involved in neurotransmission and/or neurodevelopment. We identified strong associations to individual disorders, such as growth hormone releasing hormone (GHRH) with anxiety disorders, prolactin regulatory element (PREB) with eating disorders, ionotropic kainate glutamate receptor 5 (GRIK5) with bipolar disorder and several SNPs associated to several disorders, that may represent individual and related disease susceptibility factors. Remarkably, a functional SNP, rs945032, located in the promoter region of the bradykinin receptor B2 gene (BDKRB2) was associated to three disorders (panic disorder, substance abuse, and bipolar disorder), and two additional BDKRB2 SNPs to obsessive-compulsive disorder and major depression, providing evidence for common variants of susceptibility to several related psychiatric disorders. The association of BDKRB2 (odd ratios between 1.65 and 3.06) to several psychiatric disorders supports the view that a common genetic variant could confer susceptibility to clinically related phenotypes, and defines a new functional hint in the pathophysiology of psychiatric diseases.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;6;808-16

  • Candidate gene approach evaluates association between innate immunity genes and breast cancer risk in Korean women.

    Lee JY, Park AK, Lee KM, Park SK, Han S, Han W, Noh DY, Yoo KY, Kim H, Chanock SJ, Rothman N and Kang D

    Department of Preventive Medicine, Seoul National University College of Medicine, 103 Daehak-Ro, Jongno-Gu, Seoul 110-799, Korea.

    Objectives: This study was conducted to investigate the role of common variation in innate immunity-related genes as susceptibility factors to breast cancer risk in Korean women.

    Methods: Total 1536 single-nucleotide polymorphisms (SNPs) in 203 genes were analyzed by Illumina GoldenGate assay in 209 cases and the same numbers of controls. Both SNP and gene-based tests were used to evaluate the association with breast cancer risk. The robustness of results was further evaluated with permutation method, false discovery rate and haplotype analyses.

    Results: Both SNP and gene-based analyses showed promising associations with breast cancer risk for 17 genes: OR10J3, FCER1A, NCF4, CNTNAP1, CTNNB1, KLKB1, ITGB2, ALOX12B, KLK2, IRAK3, KLK4, STAT6, NCF2, CCL1, C1QR1, MBP and NOS1. The most significant association with breast cancer risk was observed for the OR10J3 SNP (rs2494251, P-value = 1.2 x 10(-4)) and FCER1A SNP (rs7548864, P-value = 7.7 x 10(-4)). Gene-based permutation and false discovery rate P-values for OR10J3 SNP (rs2494251) with breast cancer risk were also significant (P = 4 x 10(-5) and 0.008, respectively). Haplotype analyses supported these findings that OR10J3 and FCER1A were most significantly associated with risk for breast cancer (P = 2 x 10(-4) and 0.004, respectively).

    Conclusion: This study suggests that common genetic variants in the OR10J3 and FCER1A be strongly associated with breast cancer risk among Korean women.

    Carcinogenesis 2009;30;9;1528-31

  • Case-control association study of 65 candidate genes revealed a possible association of a SNP of HTR5A to be a factor susceptible to bipolar disease in Bulgarian population.

    Yosifova A, Mushiroda T, Stoianov D, Vazharova R, Dimova I, Karachanak S, Zaharieva I, Milanova V, Madjirova N, Gerdjikov I, Tolev T, Velkova S, Kirov G, Owen MJ, O'Donovan MC, Toncheva D and Nakamura Y

    Laboratory for International Alliance, RIKEN Center for Genomic Medicine, Tsurumi-ku, Yokohama, Japan.

    Background: Bipolar affective disorder (BAD) is a psychiatric illness characterized by episodes of mania and depression. Although the etiology is not clear, epidemiological studies suggest it is a result of an interaction of genetic and environmental factors. Despite of enormous efforts and abundant studies conducted, none has yet been identified definitively a gene susceptible to bipolar disorder.

    Methods: Ninety-four Bulgarian patients diagnosed with bipolar disorder and 184 Bulgarian healthy individuals, were used for genotyping of 191 single nucleotide polymorphisms (SNPs) by TaqMan and/or Invader assays. Seventeen SNPs that revealed P value less than 0.05 in the first screening were genotyped using an additional independent set of samples, consisting of 78 BAD cases and 372 controls.

    Results: After applying the Bonferonni correction on genotyping results of 172 cases and 556 controls, only one SNP, rs1800883, in the HTR5A gene revealed a significant level of P value (P=0.000097; odds ratio=1.80 (95%CI, 1.27-2.54); corrected P=0.017).

    Conclusions: Our findings suggest that HTR5A gene could play an important role in the pathogenesis of bipolar disorder in our population. However these findings should be viewed with caution and replication studies in other populations are necessary in support of these findings.

    Funded by: Medical Research Council: G0800509

    Journal of affective disorders 2009;117;1-2;87-97

  • The effect of a promoter polymorphism on the transcription of nitric oxide synthase 1 and its relevance to Parkinson's disease.

    Rife T, Rasoul B, Pullen N, Mitchell D, Grathwol K and Kurth J

    James Madison University Biology Department, Harrisonburg, VA 22807, USA. rifetk@jmu.edu

    Transcriptional changes of the enzyme nitric oxide synthase I (NOS1) are believed to play a role in the development of many diseases. The gene for NOS1 has 12 alternative first exons (1A-1L). The 1F exon is one of the most highly utilized first exons in the brain and has a polymorphism ((TG)(m)TA(TG)(n)) located in its promoter region. The polymorphism's length has been suggested to affect NOS1 transcription and play a role in Parkinson's disease (PD); however, the actual influence of the polymorphism on NOS1 transcription has not been studied. To better characterize the links of the polymorphism with PD, a genotyping study was done comparing polymorphism length among 170 PD patients and 150 age-matched controls. The pattern of changes between the two group's allele frequencies shows statistical significance (P = 0.0359). The smallest polymorphism sizes are more predominant among PD patients than controls. To study the effects of this polymorphism on NOS1 gene transcription, reporter gene constructs were made by cloning the NOS1 1F promoter with polymorphism lengths of either 42, 54, or 62 bp in front of the luciferase gene and transfecting them into HeLa or Sk-N-MC cells. NOS1-directed reporter gene constructs with the 62-bp polymorphism increased transcription of luciferase 2.2-fold in HeLa and 1.8-fold in Sk-N-MC cells compared with reporter gene constructs with the 42-bp polymorphism. These data suggest that if smaller polymorphism size contributes to the higher NOS1 levels in PD patients, an as yet unknown transcriptional mechanism is required.

    Journal of neuroscience research 2009;87;10;2319-25

  • Association of five single nucleotide polymorphism loci with asthma in children of Chinese Han nationality.

    Lv J, Liu Q, Hua L, Dong X and Bao Y

    Department of Pediatrics, Xin Hua Hospital, Xin Hua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China.

    Background: Increasing research is available verifying that asthma is a familial and hereditary disorder of a complex of factors and genes. Objective. To investigate the single and combined associations of five single nucleotide polymorphism loci in three genes with the development of asthma in children of Chinese Han Nationality.

    Methods: A total of 192 children with asthma and an equal number of control subjects were recruited in this study. Asthma was diagnosed in accordance with American Thoracic Society criteria. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the genotypes of the single nucleotide polymorphism loci.

    Results: No statistic differences (p > 0.05) were found between experimental and control group in genotype distribution among three loci (NOS1 C5266T, TGF-beta1 L10P, and TGF-beta1 R25P). However, a significant diversity was observed among IL4-RA Q551R (p = 0.004) and TGF-beta1 C-509T (p = 0.000). Furthermore, the frequency of IL4-RA Q551R A/G and TGF-beta1 C-509T C/T in the asthma group were significantly higher than those in the control group (OR = 1.91, P = 0.002; OR = 2.90, P = 0.000, respectively). The carriers of both IL4-RA Q551R A/G and TGF-beta1 C-509T C/T did not have a more significant risk of having asthma than those who covered either.

    Conclusion: The three loci (NOS1 C5266T, TGF-beta1 L10P and TGF-beta1 R25P) have little contribution to the development of childhood asthma in Chinese Han Nationality. IL4-RA Q551R and TGF-beta1 C-509T are single nucleotide polymorphism loci with significant association to childhood asthma, but they do not have synergistic effects.

    The Journal of asthma : official journal of the Association for the Care of Asthma 2009;46;6;582-5

  • Environmental exposures, nitric oxide synthase genes, and exhaled nitric oxide in asthmatic children.

    Spanier AJ, Kahn RS, Hornung RW, Wang N, Sun G, Lierl MB and Lanphear BP

    Cincinnati Children's Environmental Health Center, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA. adam.spanier@cchmc.org

    Exhaled nitric oxide (FeNO), a measure of airway inflammation, is being explored as a tool to guide asthma management in children. Investigators have identified associations of genetic polymorphisms in nitric oxide synthase genes (NOS1 and NOS3) with FeNO levels; however, none have explored whether these polymorphisms modify the relationship of environmental exposures with FeNO. The objective of this project was to evaluate the association of NOS polymorphisms and environmental exposures with FeNO levels among children with asthma. We conducted a 12-month prospective cohort study of 225 tobacco-smoke exposed children (6-12 years) with doctor-diagnosed asthma. We assessed environmental exposures (tobacco, indoor allergens, & airborne particulates), polymorphisms in NOS1 (an intronic AAT tandem repeat) and NOS3 (G894T), and FeNO levels. There was no association of NOS1 or NOS3 polymorphisms with FeNO levels. There were no significant interactions of environmental exposures and the NOS1 polymorphism with FeNO levels. In contrast, there was an interaction of the NOS3 polymorphism and airborne nicotine concentration with FeNO levels (P = 0.01). Among GG genotype individuals, nicotine exposure did not affect FeNO levels; however, among individuals with at least one T allele, higher nicotine exposure was associated with lower FeNO levels (approximately 5 ppb decrease from the lowest to the highest quartile). We conclude that genetic differences may explain some of the conflicting results in studies of the effects of tobacco smoke exposure on FeNO levels and may make FeNO interpretation difficult for a subset of children with asthma.

    Funded by: NHLBI NIH HHS: 1R21HL083145-01A1, R01 HL065731, R01-HL65731-01, R21 HL083145, R21 HL083145-02

    Pediatric pulmonology 2009;44;8;812-9

  • [Expression of nNOS, Pax3 and Cx43 proteins in early developing posterior horn of embryonic and fetal human spinal cord].

    Zhang Y and Liu XH

    School of Medicine, Shaoxing University, Shaoxing 312000, China. hhren6588@sina.com

    Objective: To investigate the distribution pattern of the expressions neuronal nitric oxide synthase (nNOS), Pax3 and connexin 43 (Cx43) proteins in the early developing posterior horn of embryonic and fetal human spinal cord.

    Methods: Immunohistochemistry was used to detect the expressions of nNOS, Pax3 and Cx43 proteins in the posterior horn of the spinal cord during the second, third and fourth month of human embryonic and fetal development.

    Results: In the second to fourth month of gestation, the expressions of nNOS and Pax3 proteins increased gradually from weak expression to strong expression in the posterior horn of the spinal cord. In the second to third month of development, Cx43 protein expression was negative in the posterior horn of the spinal cord, but positive in the myelin sheath. In the fourth month, positive Cx43 expression was detected in some of the cells in the posterior horn of the spinal cord.

    Conclusion: nNOS, Pax3 and Cx43 proteins are closely related to the growth and development of the spinal cord in human embryos and fetuses.

    Nan fang yi ke da xue xue bao = Journal of Southern Medical University 2009;29;8;1651-3

  • Calpain-mediated activation of NO synthase in human neuroblastoma SK-N-BE cells.

    Averna M, Stifanese R, De Tullio R, Beccaria F, Salamino F, Pontremoli S and Melloni E

    Department of Experimental Medicine (DIMES), Biochemistry Section, and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Genoa, Italy.

    In resting human neuronal cells, nitric oxide synthase (nNOS) is present in its native 160 kDa form in a quiescent state predominantly co-localized on the plasma membrane, via its PDZ (Psd-95/Discs-large/Zona Occludens) domain, with NMDA receptor (NMDA-R) and in tight association with heat shock protein 90 (HSP90). Following exposure of the cells to Ca(2+)-ionophore or to NMDA, nNOS undergoes proteolytic removal of the PDZ domain being converted into a fully active 130 kDa form. The newly generated nNO synthase form dissociates from NMDA-R and extensively diffuses into the cytosol in direct correlation with NO production. Intracellular redistribution and activation of nNOS are completely prevented in cells preloaded with calpain inhibitor-1, indicating that these processes are triggered by a concomitant activation of calpain. The role of calpain has been confirmed by immunoprecipitation experiments revealing that also mu-calpain is specifically recruited into the NMDA-R-nNOS-HSP90 complex following calcium loading. Thus, the formation of clusters containing HSP90, mu-calpain, nNOS and NMDA-R represents the limiting step for the operation of the mechanism that links an efficient synthesis of NO to a local increase in Ca(2+) influx.

    Journal of neurochemistry 2009;110;1;412-21

  • Microarray analysis of multiple candidate genes and associated plasma proteins for nephropathy secondary to type 2 diabetes among Chinese individuals.

    Lim SC, Liu JJ, Low HQ, Morgenthaler NG, Li Y, Yeoh LY, Wu YS, Goh SK, Chionh CY, Tan SH, Kon YC, Soon PC, Bee YM, Subramaniam T, Sum CF and Chia KS

    Department of Medicine, Alexandra Hospital, 378 Alexandra Road, Singapore, Republic of Singapore. Su_chi_lim@alexhosp.com.sg

    Evolving research suggests that common and rare alleles jointly constitute the genetic landscape of complex disease. We studied the association between 43 pathway-related candidate genes with 'intermediate phenotype' (i.e. corresponding plasma protein) and diabetic nephropathy in a customised microarray of 1,536 SNPs.

    Methods: In this case-control study of type 2 diabetic Chinese individuals with and without diabetic nephropathy, cases (n = 545) were defined on the basis of a spot urinary albumin/creatinine ratio (ACR) > 113 mg/mmol; the value for controls (n = 503) was ACR < 3.3 mg/mmol. Genotyping was performed using Illumina GoldenGate assay.

    Results: No single nucleotide polymorphism (SNP) remained significant in single locus analysis after correction for multiple testing. Therefore, we explored the best approximately 1% SNPs. Of these 13 SNPs, four clustered to a 5' end NADPH oxidase homologue 4 (NOX4) haplotype (GGCC frequency = 0.776) with estimated OR for diabetic nephropathy of 2.05 (95% CI 1.04-4.06) (heterozygous) and 2.48 (1.27-4.83) (homozygous) (p = 0.0055). The haplotype was correlated with plasma Cu/Zn superoxide dismutase (SOD) concentration, suggesting increased oxidative burden. Endothelin-1 SNP (rs1476046G>A, frequency = 0.252) was correlated with plasma C-terminal pro-endothelin-1 concentrations with an estimated OR for diabetic nephropathy of (heterozygous) 1.26 (0.96-1.66) and (homozygous) 1.87 (1.13-3.12) (p = 0.0072). Nitric oxide synthase 1 (NOS1) 5' haplotype (TGTC frequency = 0.38) also revealed a suggestive association with diabetic nephropathy: heterozygous 1.26 (0.95-1.67), homozygous 1.57 (1.04-2.35) (p = 0.0073). A rare NADPH oxidase homologue 1 (NOX1)-coding non-synonymous SNP (Arg315His, frequency = 0.006) was found exclusively among cases.

    Our preliminary observations suggest that common haplotypes from NOX4 and endothelin-1 SNP correlated with plasma Cu/Zn SOD and C-terminal pro-endothelin-1 concentrations, respectively, and might have conferred diabetic nephropathy susceptibility. Common NOS1 and rare NOX1 variants also revealed a suggestive association with diabetic nephropathy. Future studies to validate our observation are needed.

    Diabetologia 2009;52;7;1343-51

  • Lead exposure, polymorphisms in genes related to oxidative stress, and risk of adult brain tumors.

    Bhatti P, Stewart PA, Hutchinson A, Rothman N, Linet MS, Inskip PD and Rajaraman P

    Department of Health ad Human Services, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland, USA. pbhatti@fhcrc.org

    There is some evidence that oxidative stress plays a role in lead-induced toxicity. Mechanisms for dealing with oxidative stress may be of particular relevance in the brain given the high rate of oxygen metabolism. Using a hospital-based case-control study, we investigated the role of oxidative stress in the potential carcinogenicity of lead through examination of effect modification of the association between occupational lead exposure and brain tumors by single nucleotide polymorphisms in genes with functions related to oxidative stress. The study included 362 patients with glioma (176 of which had glioblastoma multiforme), 134 patients with meningioma, and 494 controls. Lead exposure was estimated by expert review of detailed job history data for each participant. We evaluated effect modification with 142 single nucleotide polymorphisms using likelihood ratio tests that compared nested unconditional logistic regression models that did and did not include a cross-product term for cumulative lead exposure and genotype. When the analyses were restricted to cases with glioblastoma multiforme, RAC2 rs2239774 and two highly correlated GPX1 polymorphisms (rs1050450 and rs18006688) were found to significantly modify the association with lead exposure (P <or= 0.05) after adjustment for multiple comparisons. Furthermore, the same GPX1 polymorphisms and XDH rs7574920 were found to significantly modify the association between cumulative lead exposure and meningioma. Although the results of this study provide some evidence that lead may cause glioblastoma multiforme and meningioma through mechanisms related to oxidative damage, the results must be confirmed in other populations.

    Funded by: Intramural NIH HHS: NIH0011946573, Z01 CP010135-12; NCI NIH HHS: N01-CO-12400, N01CO12400

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2009;18;6;1841-8

  • Effects of neuronal nitric oxide synthase on human coronary artery diameter and blood flow in vivo.

    Seddon M, Melikian N, Dworakowski R, Shabeeh H, Jiang B, Byrne J, Casadei B, Chowienczyk P and Shah AM

    King's College London British Heart Foundation Centre, Cardiovascular Division, Department of Cardiology, London, UK.

    Background: Nitric oxide (NO)-mediated local regulation of vascular tone is considered to involve endothelial NO synthase (eNOS). However, we recently reported that human forearm basal microvascular tone in vivo is tonically regulated by neuronal NO synthase (nNOS), in contrast to an acetylcholine-stimulated reduction in tone, which is eNOS dependent. Here, we investigated the in vivo effects of an nNOS-selective inhibitor, S-methyl-L-thiocitrulline (SMTC), on the human coronary circulation and on flow-mediated dilatation in the forearm.

    In patients with angiographically normal coronary arteries, intracoronary infusion of SMTC (0.625 micromol/min) reduced basal coronary blood flow by 34.1+/-5.2% (n=10; P<0.01) and epicardial coronary diameter by 3.6+/-1.2% (P=0.02) but had no effect on increases in flow evoked by intracoronary substance P (20 pmol/min). The nonselective NOS inhibitor N(G)-monomethyl-L-arginine (25 micromol/min) also reduced basal coronary flow (by 22.3+/-5.3%; n=8; P<0.01) but, in contrast to SMTC, inhibited substance P-induced increases in flow (P<0.01). In healthy volunteers, local infusion of SMTC (0.2 micromol/min) reduced radial artery blood flow by 36.0+/-6.4% (n=10; P=0.03) but did not affect flow-mediated dilatation (P=0.55). In contrast, N(G)-monomethyl-L-arginine (2 micromol/min) infusion reduced radial blood flow to a similar degree (by 39.7+/-11.8%; P=0.02) but also inhibited flow-mediated dilatation by approximately 80% (P<0.01).

    Conclusions: These data indicate that local nNOS-derived NO regulates basal blood flow in the human coronary vascular bed, whereas substance P-stimulated vasodilatation is eNOS mediated. Thus, nNOS and eNOS have distinct local roles in the physiological regulation of human coronary vascular tone in vivo.

    Funded by: British Heart Foundation: CVH/99001, FS/09/062/27958, PG/05/138/20014; Department of Health

    Circulation 2009;119;20;2656-62

  • Analyses of associations with asthma in four asthma population samples from Canada and Australia.

    Daley D, Lemire M, Akhabir L, Chan-Yeung M, He JQ, McDonald T, Sandford A, Stefanowicz D, Tripp B, Zamar D, Bosse Y, Ferretti V, Montpetit A, Tessier MC, Becker A, Kozyrskyj AL, Beilby J, McCaskie PA, Musk B, Warrington N, James A, Laprise C, Palmer LJ, Paré PD and Hudson TJ

    James Hogg iCAPTURE Center, University of British Columbia (UBC), 1081 Burrard Street, Vancouver, BC, V6Z 1Y6, Canada.

    Asthma, atopy, and related phenotypes are heterogeneous complex traits, with both genetic and environmental risk factors. Extensive research has been conducted and over hundred genes have been associated with asthma and atopy phenotypes, but many of these findings have failed to replicate in subsequent studies. To separate true associations from false positives, candidate genes need to be examined in large well-characterized samples, using standardized designs (genotyping, phenotyping and analysis). In an attempt to replicate previous associations we amalgamated the power and resources of four studies and genotyped 5,565 individuals to conduct a genetic association study of 93 previously associated candidate genes for asthma and related phenotypes using the same set of 861 single-nucleotide polymorphisms (SNPs), a common genotyping platform, and relatively harmonized phenotypes. We tested for association between SNPs and the dichotomous outcomes of asthma, atopy, atopic asthma, and airway hyperresponsiveness using a general allelic likelihood ratio test. No SNP in any gene reached significance levels that survived correction for all tested SNPs, phenotypes, and genes. Even after relaxing the usual stringent multiple testing corrections by performing a gene-based analysis (one gene at a time as if no other genes were typed) and by stratifying SNPs based on their prior evidence of association, no genes gave strong evidence of replication. There was weak evidence to implicate the following: IL13, IFNGR2, EDN1, and VDR in asthma; IL18, TBXA2R, IFNGR2, and VDR in atopy; TLR9, TBXA2R, VDR, NOD2, and STAT6 in airway hyperresponsiveness; TLR10, IFNGR2, STAT6, VDR, and NPSR1 in atopic asthma. Additionally we found an excess of SNPs with small effect sizes (OR < 1.4). The low rate of replication may be due to small effect size, differences in phenotypic definition, differential environmental effects, and/or genetic heterogeneity. To aid in future replication studies of asthma genes a comprehensive database was compiled and is available to the scientific community at http://genapha.icapture.ubc.ca/.

    Human genetics 2009;125;4;445-59

  • Genetic polymorphisms in nitric oxide synthase genes modify the relationship between vegetable and fruit intake and risk of non-Hodgkin lymphoma.

    Han X, Zheng T, Lan Q, Zhang Y, Kilfoy BA, Qin Q, Rothman N, Zahm SH, Holford TR, Leaderer B and Zhang Y

    Yale University School of Public Health, New Haven, CT 06520-8034, USA.

    Oxidative damage caused by reactive oxygen species and other free radicals is involved in carcinogenesis. It has been suggested that high vegetable and fruit intake may reduce the risk of non-Hodgkin lymphoma (NHL) as vegetables and fruit are rich in antioxidants. The aim of this study is to evaluate the interaction of vegetable and fruit intake with genetic polymorphisms in oxidative stress pathway genes and NHL risk. This hypothesis was investigated in a population-based case-control study of NHL and NHL histologic subtypes in women from Connecticut, including 513 histologically confirmed incident cases and 591 randomly selected controls. Gene-vegetable/fruit joint effects were estimated using unconditional logistic regression model. The false discovery rate method was applied to adjust for multiple comparisons. Significant interactions with vegetable and fruit intake were mainly found for genetic polymorphisms on nitric oxide synthase (NOS) genes among those with diffuse large B-cell lymphoma and follicular lymphoma. Two single nucleotide polymorphisms in the NOS1 gene were found to significantly modify the association between total vegetable and fruit intake and risk of NHL overall, as well as the risk of follicular lymphoma. When vegetables, bean vegetables, cruciferous vegetables, green leafy vegetables, red vegetables, yellow/orange vegetables, fruit, and citrus fruits were examined separately, strong interaction effects were narrowed to vegetable intake among patients with diffuse large B-cell lymphoma. Our results suggest that genetic polymorphisms in oxidative stress pathway genes, especially in the NOS genes, modify the association between vegetable and fruit intake and risk of NHL.

    Funded by: FIC NIH HHS: 1D43TW007864-01, D43 TW007864; Intramural NIH HHS: Z01 CP010123-12; NCI NIH HHS: R01 CA062006; NCRR NIH HHS: UL1 RR024139

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2009;18;5;1429-38

  • Genetic susceptibility to respiratory syncytial virus bronchiolitis in preterm children is associated with airway remodeling genes and innate immune genes.

    Siezen CL, Bont L, Hodemaekers HM, Ermers MJ, Doornbos G, Van't Slot R, Wijmenga C, Houwelingen HC, Kimpen JL, Kimman TG, Hoebee B and Janssen R

    Laboratory for Health Protection Research, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.

    Prematurity is a risk factor for severe respiratory syncytial virus bronchiolitis. We show that genetic factors in innate immune genes (IFNA13, IFNAR2, STAT2, IL27, NFKBIA, C3, IL1RN, TLR5), in innate and adaptive immunity (IFNG), and in airway remodeling genes (ADAM33 and TGFBR1), affect disease susceptibility to a different extent in preterm children, born with underdeveloped lungs, than in term children.

    The Pediatric infectious disease journal 2009;28;4;333-5

  • High frequencies of short alleles of NOS1 (CA)n polymorphism in beta(0)39 carriers from Corsica Island (France).

    Piras IS, Falchi A, Melis A, De Cian MC, Calò CM, Vona G and Varesi L

    Department of Experimental Biology, University of Cagliari, Monserrato (CA), Italy. is.piras@tiscali.it

    In this work we investigated about the presence of a correlation between a (CA)n repeat located in exon 29 of NOS1 gene and the beta-thalassemia trait in Corsica Island (France). We genotyped a sample of individuals with beta-thalassemia minor (N=110) and an ethnically matched control (N=113) from Balagna, a region of Corsica Island (France). Results highlighted the high frequencies of allele with 16 and 17 repeats in the thalassemic sample. From these results we suggest, that high frequencies of alleles with 16 and 17 repeats, could be a consequence of past malarial endemicity.

    Experimental and molecular pathology 2009;86;2;136-7

  • Family-based association study between NOS-I and -IIA polymorphisms and autism spectrum disorders in Korean trios.

    Kim HW, Cho SC, Kim JW, Cho IH, Kim SA, Park M, Cho EJ and Yoo HJ

    Department of Child and Adolescent Psychiatry, Seoul National University, Korea.

    Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with a strong genetic component and environmental risk factors. Nitric oxide (NO), which is produced by nitric oxide synthase (NOS), may play a role in the development of ASD. We genotyped nine single nucleotide polymorphisms (SNPs) in the NOS-I gene and nine SNPs in the NOS-IIA gene and carried out the transmission disequilibrium test (TDT) and haplotype analysis in 151 Korean ASD trios. We found preferential transmission of the A allele of rs8068149 (P = 0.039) and G allele of rs1060826 (P = 0.035) of NOS-IIA in ASD and the haplotype analysis revealed that the two haplotypes had significant associations (P = 0.014 and 0.031, respectively). The behavioral subdomain score of failure to use nonverbal behaviors to regulate social interaction in Autism Diagnostic Interview-Revised (ADI-R) was significantly higher in subjects with the GG or AG allele in rs1060826 of NOS-IIA compared to those who had the AA allele (P = 0.027). These results provide significant but weak evidence for an association between NOS-IIA and ASD in the Korean population.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;2;300-6

  • An association study of 45 folate-related genes in spina bifida: Involvement of cubilin (CUBN) and tRNA aspartic acid methyltransferase 1 (TRDMT1).

    Franke B, Vermeulen SH, Steegers-Theunissen RP, Coenen MJ, Schijvenaars MM, Scheffer H, den Heijer M and Blom HJ

    Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands. b.franke@antrg.umcn.nl

    Background: Spina bifida is a class of neural tube defects, which are congenital malformations of the central nervous system with a prevalence of 0.5 to 12 per 1000 births globally. In this article we attempt to identify genes related to folate and its metabolic pathways that are involved in the etiology of spina bifida.

    Methods: We selected 50 folate metabolism-related genes and genotyped polymorphisms in those genes. Eighty-seven polymorphisms in 45 genes passed quality controls. Associations with spina bifida were investigated in 180 patients and 190 controls. For those polymorphisms that were nominally associated with spina bifida risk, the relation with serum and red blood cell folate, vitamin B(12), and homocysteine was evaluated in controls.

    Results: A polymorphism in CUBN was significantly associated with decreased spina bifida risk, after correction for multiple testing, and was related to increased vitamin B(12) (p = 0.039) and red blood cell folate (p = 0.001). The CUBN gene encodes the intrinsic factor-cobalamin receptor (or cubilin), a peripheral membrane protein that acts as a receptor for intrinsic factor-vitamin B(12) complexes. Vitamin B(12) is an important cofactor in the folate metabolism, and low B(12) status in mothers has been linked to neural tube defects in children. Other interesting findings include nominally significant associations with polymorphisms in TRDMT1, ALDH1L1, SARDH, and SLCA19A1 (RFC1).

    Conclusion: Our study indicates interesting new candidate genes and functional pathways for further study and confirms earlier findings. None of the genes CUBN, TRDMT1, ALDH1L1, or SARDH have been investigated previously for association with spina bifida.

    Birth defects research. Part A, Clinical and molecular teratology 2009;85;3;216-26

  • Nitric oxide synthase gene polymorphisms in children with minimal change nephrotic syndrome.

    Alasehirli B, Balat A, Barlas O and Kont A

    Department of Pharmacology, Medical Faculty, Gaziantep University, Gaziantep, Turkey.

    Aims: Nitric oxide (NO) attenuates many functions within the kidney, and all NO synthase (NOS) isoforms are constitutively expressed in the kidney. But the exact role of NO in renal diseases is still debatable. The aim of the present study was to investigate endothelial (eNOS), and neuronal (nNOS) NOS gene polymorphisms in children with minimal change nephrotic syndrome (MCNS).

    Eighty-six Turkish children with clinical MCNS, ranging in age from 2 to 10 years, were compared with 114 healthy age- and sex-matched controls. The glu 298 Asp (G/T) polymorphism of the eNOS, and C276T (C/T) polymorphism of nNOS genes were genotyped using polymerase chain reaction.

    Results: The distribution of GG, TG, and TT genotypes for eNOS was 52%, 33% and 15% in MCNS compared with 61%, 26% and 13% in the controls (P > 0.05). The distribution of CC, TC, and TT genotypes for nNOS was 16%, 66% and 18% in MCNS compared with 10%, 43% and 47% in the controls. TT genotype distribution of nNOS was found to be lower in patients (P = 0.003). The eNOS and nNOS gene polymorphisms were not associated with gender, positive family history, frequency of relapses, or response to steroid.

    Conclusions: The present study is the first to investigate eNOS and nNOS gene polymorphisms in children with MCNS. The nNOS gene polymorphism may be associated with MCNS in children, but further studies in a larger population with different glomerular diseases are needed to confirm the results.

    Pediatrics international : official journal of the Japan Pediatric Society 2009;51;1;75-8

  • The NOS3 G894T (Glu298Asp) polymorphism is a risk factor for frontotemporal lobar degeneration.

    Venturelli E, Villa C, Fenoglio C, Clerici F, Marcone A, Ghidoni R, Cortini F, Scalabrini D, Gallone S, Rainero I, Mandelli A, Restelli I, Binetti G, Cappa S, Mariani C, Giordana MT, Bresolin N, Scarpini E and Galimberti D

    Department of Neurological Sciences, 'Dino Ferrari' Center, University of Milan, IRCCS Fondazione Ospedale Maggiore Policlinico, Milan, Italy.

    Neuronal nitric oxide synthase (NOS)1 C276T polymorphism was shown to increase the risk for frontotemporal lobar degeneration (FTLD). In the brain, both NOS1 and NOS3 (endothelial isoform) have been detected. The distribution of NOS3 G894T (Glu298Asp) and T-786C single nucleotide polymorphisms (SNPs) was analyzed in a population of 222 patients with FTLD compared with 218 age-matched controls to determine whether they could influence the susceptibility to develop the disease.

    Results: A statistically significant increased frequency of the NOS3 G894T SNP was observed in patients as compared with controls (40.0 vs. 31.4%, P = 0.011, OR: 1.65, CI: 1.13-2.42). Conversely, the distribution of the T-786C SNP was similar in patients and controls. No differences were observed stratifying according to gender.

    Discussion: The NOS3 G894T polymorphism likely acts as risk factor for sporadic FTLD, but studies in larger populations are needed to confirm these preliminary findings.

    European journal of neurology 2009;16;1;37-42

  • Diminished NO generation by injured endothelium and loss of macula densa nNOS may contribute to sustained acute kidney injury after ischemia-reperfusion.

    Kwon O, Hong SM and Ramesh G

    Dept. of Medicine, Div. of Nephrology, Penn State Milton S. Hershey Medical Ctr., Penn State College of Medicine, 500 University Dr., Hershey, PA 17033-0850, USA. okwon@psu.edu

    In postischemic acute kidney injury (AKI) or acute renal failure, a dissipation of glomerular filtration pressure is associated with an altered renal vascular tone and reactivity, as well as a loss of vascular autoregulation. To test the hypothesis that renal nitric oxide (NO) generation reflects endothelial damage in the kidney after ischemia-reperfusion, we quantified the urinary NO levels and identified the site of its generation in postischemic AKI. Subjects were 50 recipients of cadaveric renal allografts: 15 with sustained AKI and 35 with recovering renal function. Urine and blood samples were obtained after transplant, and intraoperative allograft biopsies were performed to examine NO synthases (NOSs) in the kidney. In the sustained AKI group, urinary nitrite and nitrate excretion (in mumol/g urine creatinine) was lower (12.3 +/- 1.8 and 10.0 +/- 1.4 on postoperative days 0 and 3) than in the recovery group [20.0 +/- 3.6 and 35.1 +/- 5.3 (P < 0.005 vs. sustained AKI on days 0 and 3) on postoperative days 0 and 3]. Endothelial NOS expression diminished from the peritubular capillaries of 6 of 7 subjects in the sustained AKI group but from only 6 of 16 subjects in the recovery group. No differences were observed in the inducible NOS staining pattern between the two groups. Neuronal NOS staining was rarely observed in the macula densae of subjects but was prominent in control tissues. These findings suggest that a diminished NO generation by injured endothelium and loss of macula densa neuronal NOS could impair the vasodilatory ability of the renal vasculature and contribute to the reduction in the glomerular filtration rate in postischemic AKI.

    American journal of physiology. Renal physiology 2009;296;1;F25-33

  • Influence of functional variant of neuronal nitric oxide synthase on impulsive behaviors in humans.

    Reif A, Jacob CP, Rujescu D, Herterich S, Lang S, Gutknecht L, Baehne CG, Strobel A, Freitag CM, Giegling I, Romanos M, Hartmann A, Rösler M, Renner TJ, Fallgatter AJ, Retz W, Ehlis AC and Lesch KP

    Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Füchsleinstr 15, D-97080 Würzburg, Germany. reif_a@klinik.uni-wuerzburg.de

    Context: Human personality is characterized by substantial heritability but few functional gene variants have been identified. Although rodent data suggest that the neuronal isoform of nitric oxide synthase (NOS-I) modifies diverse behaviors including aggression, this has not been translated to human studies.

    Objectives: To investigate the functionality of an NOS1 promoter repeat length variation (NOS1 Ex1f variable number tandem repeat [VNTR]) and to test whether it is associated with phenotypes relevant to impulsivity.

    Design: Molecular biological studies assessed the cellular consequences of NOS1 Ex1f VNTR; association studies were conducted to investigate the impact of this genetic variant on impulsivity; imaging genetics was applied to determine whether the polymorphism is functional on a neurobiological level.

    Setting: Three psychiatric university clinics in Germany.

    Participants: More than 3200 subjects were included in the association study: 1954 controls, 403 patients with personality disorder, 383 patients with adult attention-deficit/hyperactivity disorder (ADHD), 151 with familial ADHD, 189 suicide attempters, and 182 criminal offenders.

    For the association studies, the major outcome criteria were phenotypes relevant to impulsivity, namely, the dimensional phenotype conscientiousness and the categorical phenotypes adult ADHD, aggression, and cluster B personality disorder.

    Results: A novel functional promoter polymorphism in NOS1 was associated with traits related to impulsivity, including hyperactive and aggressive behaviors. Specifically, the short repeat variant was more frequent in adult ADHD, cluster B personality disorder, and autoaggressive and heteroaggressive behavior. This short variant came along with decreased transcriptional activity of the NOS1 exon 1f promoter and alterations in the neuronal transcriptome including RGS4 and GRIN1. On a systems level, it was associated with hypoactivation of the anterior cingulate cortex, which is involved in the processing of emotion and reward in behavioral control.

    Conclusion: These findings implicate deficits in neuronal signaling via nitric oxide in moderation of prefrontal circuits underlying impulsivity-related behavior in humans.

    Archives of general psychiatry 2009;66;1;41-50

  • No association between polymorphisms of neuronal oxide synthase 1 gene (NOS1) and schizophrenia in a Japanese population.

    Okumura T, Okochi T, Kishi T, Ikeda M, Kitajima T, Yamanouchi Y, Kinoshita Y, Kawashima K, Tsunoka T, Ujike H, Inada T, Ozaki N and Iwata N

    Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Aichi, 470-1192, Japan.

    The neuronal nitric oxide synthase gene (NOS1) is located on 12q24, in a susceptibility region for schizophrenia, and produces nitric oxide (NO) in the brain. NO plays a role in neurotransmitter release and is the second messenger of the N-methyl-D-aspartate (NMDA) receptor. Furthermore, it is connected to the dopaminergic and serotonergic neural transmission systems. Therefore, abnormalities in the NO pathway are thought to be involved in the pathophysiology of schizophrenia. Several genetic studies showed an association of NOS1 with schizophrenia. However, results of replication studies have been inconsistent. Therefore, we conducted a replication study of NOS1 with schizophrenia in a Japanese sample. We selected seven SNPs (rs41279104, rs3782221, rs3782219, rs561712, rs3782206, rs2682826, and rs6490121) in NOS1 that were positively associated with schizophrenia in previous studies. Two SNPs showed an association with Japanese schizophrenic patients (542 cases and 519 controls, rs3782219: P allele = 0.0291 and rs3782206: P allele = 0.0124, P genotype = 0.0490), and almost these significances remained with an increased sample size (1154 cases and 1260 controls, rs3782219: P allele = 0.0197 and rs3782206: P allele = 0.0480). However, these associations also might have resulted from type I error on account of multiple testing (rs3782219: P allele = 0.133 and rs3782206: P allele = 0.168). In conclusion, we could not replicate the association between seven SNPs in NOS1 and schizophrenia found in several earlier studies, using larger Japanese schizophrenia and control samples.

    Neuromolecular medicine 2009;11;2;123-7

  • Genome-wide association scan of quantitative traits for attention deficit hyperactivity disorder identifies novel associations and confirms candidate gene associations.

    Lasky-Su J, Neale BM, Franke B, Anney RJ, Zhou K, Maller JB, Vasquez AA, Chen W, Asherson P, Buitelaar J, Banaschewski T, Ebstein R, Gill M, Miranda A, Mulas F, Oades RD, Roeyers H, Rothenberger A, Sergeant J, Sonuga-Barke E, Steinhausen HC, Taylor E, Daly M, Laird N, Lange C and Faraone SV

    Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    Attention deficit hyperactivity disorder (ADHD) is a complex condition with environmental and genetic etiologies. Up to this point, research has identified genetic associations with candidate genes from known biological pathways. In order to identify novel ADHD susceptibility genes, 600,000 SNPs were genotyped in 958 ADHD proband-parent trios. After applying data cleaning procedures we examined 429,981 autosomal SNPs in 909 family trios. We generated six quantitative phenotypes from 18 ADHD symptoms to be used in genome-wide association analyses. With the PBAT screening algorithm, we identified 2 SNPs, rs6565113 and rs552655 that met the criteria for significance within a specified phenotype. These SNPs are located in intronic regions of genes CDH13 and GFOD1, respectively. CDH13 has been implicated previously in substance use disorders. We also evaluated the association of SNPs from a list of 37 ADHD candidate genes that was specified a priori. These findings, along with association P-values with a magnitude less than 10(-5), are discussed in this manuscript. Seventeen of these candidate genes had association P-values lower then 0.01: SLC6A1, SLC9A9, HES1, ADRB2, HTR1E, DDC, ADRA1A, DBH, DRD2, BDNF, TPH2, HTR2A, SLC6A2, PER1, CHRNA4, SNAP25, and COMT. Among the candidate genes, SLC9A9 had the strongest overall associations with 58 association test P-values lower than 0.01 and multiple association P-values at a magnitude of 10(-5) in this gene. In sum, these findings identify novel genetic associations at viable ADHD candidate genes and provide confirmatory evidence for associations at previous candidate genes. Replication of these results is necessary in order to confirm the proposed genetic variants for ADHD.

    Funded by: NHLBI NIH HHS: R00 HL096840; NIMH NIH HHS: R01 MH062873, R01 MH081803, R01MH081803, R01MH62873

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;8;1345-54

  • Evidence for association between the 5' flank of the NOS1 gene and schizophrenia in the Chinese population.

    Tang W, Huang K, Tang R, Zhou G, Fang C, Zhang J, Du L, Feng G, He L and Shi Y

    Bio-X Center, Shanghai Jiao Tong University, Shanghai, PR China.

    Nitric oxide (NO) plays an important role in the dopaminergic and serotonergic system as the second messenger of the NMDA receptor and has possible roles in neurotransmission, neurosecretion, synaptic plasticity, and tissue injury in many neurological disorders, including schizophrenia. There is also genetic evidence to support the human NOS1 (neuronal nitric oxide synthase 1) gene as a promising candidate gene associated with schizophrenia. In this paper we conducted a case-control association study involving 1705 Chinese subjects and 12 genetic markers [11 single nucleotide polymorphisms (SNPs) and 1 microsatellite] mainly in the 5' flank region of the gene by direct sequencing and capillary electrophoresis. We identified SNP rs3782206 and several haplotypes derived from it as being significantly associated with schizophrenia and, specifically, in a paranoid subgroup. Our results strongly support a previous hypothesis that NOS1 contributes to the genetic risk of schizophrenia and suggest that further research on more NOS1 variants and its regular elements are warranted.

    The international journal of neuropsychopharmacology 2008;11;8;1063-71

  • Oxidative stress, telomere length and biomarkers of physical aging in a cohort aged 79 years from the 1932 Scottish Mental Survey.

    Starr JM, Shiels PG, Harris SE, Pattie A, Pearce MS, Relton CL and Deary IJ

    MRC Centre for Cognitive Ageing and Cognitive Epidemiology, University of Edinburgh, Royal Victoria Hospital, Edinburgh EH4 2DN, UK. jstarr@staffmail.ed.ac.uk

    Telomere shortening is a biomarker of cellular senescence and is associated with a wide range of age-related disease. Oxidative stress is also associated with physiological aging and several age-related diseases. Non-human studies suggest that variants in oxidative stress genes may contribute to both telomere shortening and biological aging. We sought to test whether oxidative stress-related gene polymorphisms contribute to variance in both telomere length and physical biomarkers of aging in humans. Telomere lengths were calculated for 190 (82 men, 108 women) participants aged 79 years and associations with 384 SNPs, from 141 oxidative stress genes, identified 9 significant SNPS, of which those from 5 genes (GSTZ1, MSRA, NDUFA3, NDUFA8, VIM) had robust associations with physical aging biomarkers, respiratory function or grip strength. Replication of associations in a sample of 318 (120 males, 198 females) participants aged 50 years confirmed significant associations for two of the five SNPs (MSRA rs4841322, p=0.008; NDUFA8 rs6822, p=0.048) on telomere length. These data indicate that oxidative stress genes may be involved in pathways that lead to both telomere shortening and physiological aging in humans. Oxidative stress may explain, at least in part, associations between telomere shortening and physiological aging.

    Funded by: Biotechnology and Biological Sciences Research Council: S18386; Chief Scientist Office: CZB/4/505, ETM/55; Medical Research Council; Wellcome Trust

    Mechanisms of ageing and development 2008;129;12;745-51

  • Sarcolemma-localized nNOS is required to maintain activity after mild exercise.

    Kobayashi YM, Rader EP, Crawford RW, Iyengar NK, Thedens DR, Faulkner JA, Parikh SV, Weiss RM, Chamberlain JS, Moore SA and Campbell KP

    Howard Hughes Medical Institute, University of Iowa, Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, Iowa 52242-1101, USA.

    Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response that is disproportionate to activity level. This fatigue is not necessarily correlated with greater central or peripheral fatigue in patients, and some patients experience severe fatigue without any demonstrable somatic disease. Except in myopathies that are due to specific metabolic defects, the mechanism underlying this type of fatigue remains unknown. With no treatment available, this form of inactivity is a major determinant of disability. Here we show, using mouse models, that this exaggerated fatigue response is distinct from a loss in specific force production by muscle, and that sarcolemma-localized signalling by neuronal nitric oxide synthase (nNOS) in skeletal muscle is required to maintain activity after mild exercise. We show that nNOS-null mice do not have muscle pathology and have no loss of muscle-specific force after exercise but do display this exaggerated fatigue response to mild exercise. In mouse models of nNOS mislocalization from the sarcolemma, prolonged inactivity was only relieved by pharmacologically enhancing the cGMP signal that results from muscle nNOS activation during the nitric oxide signalling response to mild exercise. Our findings suggest that the mechanism underlying the exaggerated fatigue response to mild exercise is a lack of contraction-induced signalling from sarcolemma-localized nNOS, which decreases cGMP-mediated vasomodulation in the vessels that supply active muscle after mild exercise. Sarcolemmal nNOS staining was decreased in patient biopsies from a large number of distinct myopathies, suggesting a common mechanism of fatigue. Our results suggest that patients with an exaggerated fatigue response to mild exercise would show clinical improvement in response to treatment strategies aimed at improving exercise-induced signalling.

    Funded by: Howard Hughes Medical Institute; NCRR NIH HHS: K26 RR017369, K26 RR017369-01A1, K26 RR017369-02, K26 RR017369-03, K26 RR017369-04, K26 RR017369-05, UL1 RR024979; NHLBI NIH HHS: T32 HL007121, T32 HL007121-26, T32 HL007121-27; NIA NIH HHS: R01 AG033610; NIAMS NIH HHS: F32 AR048742-01, F32 AR048742-02, R01 AR051199, R01 AR051199-01; NINDS NIH HHS: U54 NS053672, U54 NS053672-01, U54 NS053672-02, U54 NS053672-02S1, U54 NS053672-03, U54 NS053672-04

    Nature 2008;456;7221;511-5

  • Metabolic profile and nitric oxide synthase expression of skeletal muscle fibers are altered in patients with type 1 diabetes.

    Fritzsche K, Blüher M, Schering S, Buchwalow IB, Kern M, Linke A, Oberbach A, Adams V and Punkt K

    Institute of Anatomy, University of Leipzig, Germany.

    We investigate muscle fiber composition, fiber-specific glycolytic and oxidative enzyme capacity and nitric oxide synthase (NOS) expression in skeletal muscle of patients with type 1 diabetes (T1D) compared to individuals with normal glucose tolerance (NGT). Vastus lateralis muscle was obtained by percutaneous biopsy from 7 T1D patients and 10 healthy controls with similar characteristics. Using cytophotometry, muscle fiber composition and fiber type-specific glycolytic and oxidative enzyme activities were measured in slow oxidative (SO), fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers. In addition, NOS 1-3 protein expression was mea-sured. The glycolytic fiber fraction was 1.4 fold higher, whereas FOG and SO fiber fractions were significantly reduced by 13.5% and 6.2% in skeletal muscle from T1D patients. Glycolytic enzyme activities and fiber-specific ratio of glycolytic relative to oxidative enzyme activity were significantly higher in all fiber types of T1D patients and correlated with HbA (1c). Expression of NOS1-3 isoforms was reduced in skeletal muscle of T1D subjects. Increased glycolytic enzyme activity in muscle of T1D patients is most likely due to both a higher number of fast glycolytic fibers and a shift towards increased glycolytic metabolism in all fiber types. Alterations in muscle fiber distribution and enzyme activities seem to be due to impaired long-term glycemic control.

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2008;116;10;606-13

  • Neuronal nitric oxide synthase expression in glial tumors: correlation with malignancy and tumor proliferation.

    Tanriover N, Ulu MO, Isler C, Durak H, Oz B, Uzan M and Akar Z

    Department of Neurosurgery, Cerrahpasa Medical Faculty, Istanbul University, Istanbul, Turkey. dronurulu@gmail.com

    Introduction: Increased vascular permeability, vasodilatation, neovascularization and free radical injury in malignant tumors and adjacent normal tissues are believed to be mediated by nitric oxide (NO). High levels of neuronal nitric oxide synthase (nNOS) have been demonstrated in cultured and intracerebral cells. Our aim was to investigate nNOS expression in human glial tumors and to assess its correlation with the histologic grade and proliferative potential.

    Methods: Tissue specimens were obtained from 29 patients with supratentorial astrocytomas [15 glioblastoma multiforme (GBM), six anaplastic astrocytomas (AA) and eight low grade astrocytomas (LGA)] diagnosed and classified according to the current WHO classification of nervous system tumors. Immunohistochemical staining was performed in paraffin embedded specimens with polyclonal anti-nNOS antibody, and the levels of nNOS expression was evaluated as slight, moderate or dense on the basis of intensity and the extent of distribution of nNOS immunoreactivity. Proliferative potential was evaluated with immunostaining for Ki-67.

    Results: There was a significant positive correlation between the histologic grade and nNOS expression in terms of intensity and the extent of distribution of nNOS immunoreactivity (p<0.05). Greater values of Ki-67 indices were demonstrated in tumors with higher nNOS expression, indicating a positive correlation between proliferative potentials and expression of nNOS immunoreactivity.

    Conclusion: Our study suggests that nNOS expression is increased in glial tumors, which was significantly correlated with histologic grade and proliferative potential. NO overproduction due to overexpression of nNOS activity, seems to have significant correlation with malignancy in glial tumors, and may provide another target for anti-proliferative therapy in the future.

    Neurological research 2008;30;9;940-4

  • Nitric oxide synthase isoforms and NF-kappaB activity in normal and osteoarthritic human chondrocytes: regulation by inducible nitric oxide.

    Rosa SC, Judas F, Lopes MC and Mendes AF

    Center for Neurosciences and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal.

    To elucidate the role of endogenous inducible nitric oxide (NO) on the regulation of NF-kappaB activity in human chondrocytes, we evaluated (i) the pattern of expression of the neuronal (nNOS) and inducible (iNOS) NO synthase isoforms and the basal NF-kappaB activity in normal and osteoarthritic (OA) human chondrocytes, (ii) the role of cytokines and growth factors in modulating the protein levels of the two NOS isoforms, and (iii) the effect of inhibiting endogenous inducible NO production on the ability of interleukin-1beta (IL-1) to induce NF-kappaB activation. nNOS was more frequently expressed in normal than in OA chondrocytes, whereas the opposite was found for iNOS. IL-1 induced the degradation of both enzymes, but iNOS disappeared more rapidly. Although IkappaB-alpha was present in all the normal samples and in the majority of the OA samples, NF-kappaB-DNA binding activity in OA chondrocytes was increased approximately twofold relatively to normal cells. Addition of a NOS inhibitor, after induction of iNOS expression, induced IkappaB-alpha degradation and potenciated the effect of IL-1, indicating that endogenous inducible NO inhibits NF-kappaB activation. Taken together, these findings favor an inhibitory role of high NO levels on the regulation of NF-kappaB activation in chondrocytes, indicating that NF-kappaB activity is regulated, at least in part, by the balanced production of NO resulting from a dynamic process that, at any given moment, determines the availability of the constitutive and inducible NOS isoforms. Moreover, the down-regulatory role of NO on NF-kappaB activation warrants caution as to the possible utilization of NO inhibitors in the therapy of OA.

    Nitric oxide : biology and chemistry 2008;19;3;276-83

  • Association of a NOS1 promoter repeat with Alzheimer's disease.

    Galimberti D, Scarpini E, Venturelli E, Strobel A, Herterich S, Fenoglio C, Guidi I, Scalabrini D, Cortini F, Bresolin N, Lesch KP and Reif A

    Department of Neurological Sciences, Dino Ferrari Center, University of Milan, Fondazione Ospedale Maggiore Policlinico IRCCS, Milan, Italy. daniela.galimberti@unimi.it

    The gene encoding NOS-I (NOS1) displays a complex transcriptional regulation, with nine alternative first exons. Exon 1c and 1f are the most abundant forms in the brain. A functional single nucleotide polymorphism (SNP) in exon 1c and a polymorphism in exon 1f, consisting of a variable number of tandem repeats (VNTR) originating short (S) and long (L) alleles, were studied in 184 patients with Alzheimer's disease (AD) and 144 gender- and age-matched controls. No differences were found for the Ex1c G-84A. The Ex1f-VNTR S allele was significantly more common in AD (55% versus 44%, P=0.009, OR=1.52) as was the S/S genotype (28% versus 14%, P=0.008; OR=2.37). The S allele showed a highly significant interaction with the ApoE epsilon 4 allele (OR: 10.83). Therefore, short alleles of the NOS1 exon 1f-VNTR are likely to be susceptibility factors for AD, and interact with the epsilon 4 allele to markedly increase the AD risk.

    Neurobiology of aging 2008;29;9;1359-65

  • Acute exercise does not cause sustained elevations in AMPK signaling or expression.

    Lee-Young RS, Koufogiannis G, Canny BJ and McConell GK

    Department of Physiology, The University of Melbourne, Parkville, Victoria, Australia. robert.s.lee-young@vanderbilt.edu

    Purpose: No study has examined the response of skeletal muscle AMP-activated protein kinase (AMPK) signaling beyond the first 3 h after an acute exercise bout in humans. The purpose of this study was to assess AMPK signaling in human skeletal muscle immediately after a single bout of moderate-intensity endurance exercise and 3 and 24 h after the exercise bout.

    Methods: We examined AMPK signaling, and protein expression of AMPK alpha, ACC-beta, and nNOS mu in untrained individuals (four females and four males) during the 24-h period after a 60-min bout of moderate-intensity (63 +/- 1% VO2peak) cycling endurance exercise.

    Results: AMPK alpha2 activity, AMPK alpha2 Thr172 phosphorylation, and ACC-beta Ser222 phosphorylation were increased immediately after exercise. These increases had all returned to basal levels at 3 and 24 h after exercise. Furthermore, an acute bout of exercise did not alter AMPK alpha1, AMPK alpha2, ACC-beta, or nNOS mu protein expression during the 24-h period after exercise.

    Conclusion: Although an acute bout of exercise elicits increases in AMPK signaling, this alone is not sufficient to induce sustained increases in either AMPK signaling or protein expression during the postexercise period.

    Medicine and science in sports and exercise 2008;40;8;1490-4

  • Syntrophin mutation associated with long QT syndrome through activation of the nNOS-SCN5A macromolecular complex.

    Ueda K, Valdivia C, Medeiros-Domingo A, Tester DJ, Vatta M, Farrugia G, Ackerman MJ and Makielski JC

    Department of Medicine, University of Wisconsin, Madison, WI 53792, USA.

    Mutations in 11 genes that encode ion channels or their associated proteins cause inherited long QT syndrome (LQTS) and account for approximately 75-80% of cases (LQT1-11). Direct sequencing of SNTA1, the gene encoding alpha1-syntrophin, was performed in a cohort of LQTS patients that were negative for mutations in the 11 known LQTS-susceptibility genes. A missense mutation (A390V-SNTA1) was found in a patient with recurrent syncope and markedly prolonged QT interval (QTc, 530 ms). SNTA1 links neuronal nitric oxide synthase (nNOS) to the nNOS inhibitor plasma membrane Ca-ATPase subtype 4b (PMCA4b); SNTA1 also is known to associate with the cardiac sodium channel SCN5A. By using a GST-fusion protein of the C terminus of SCN5A, we showed that WT-SNTA1 interacted with SCN5A, nNOS, and PMCA4b. In contrast, A390V-SNTA1 selectively disrupted association of PMCA4b with this complex and increased direct nitrosylation of SCN5A. A390V-SNTA1 expressed with SCN5A, nNOS, and PMCA4b in heterologous cells increased peak and late sodium current compared with WT-SNTA1, and the increase was partially inhibited by NOS blockers. Expression of A390V-SNTA1 in cardiac myocytes also increased late sodium current. We conclude that the A390V mutation disrupted binding with PMCA4b, released inhibition of nNOS, caused S-nitrosylation of SCN5A, and was associated with increased late sodium current, which is the characteristic biophysical dysfunction for sodium-channel-mediated LQTS (LQT3). These results establish an SNTA1-based nNOS complex attached to SCN5A as a key regulator of sodium current and suggest that SNTA1 be considered a rare LQTS-susceptibility gene.

    Funded by: NHLBI NIH HHS: HL71092, R01 HL071092, R56 HL071092; NICHD NIH HHS: HD42569, R01 HD042569; NIDDK NIH HHS: DK17238, R01 DK017238

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;27;9355-60

  • Systematic analysis of glutamatergic neurotransmission genes in alcohol dependence and adolescent risky drinking behavior.

    Schumann G, Johann M, Frank J, Preuss U, Dahmen N, Laucht M, Rietschel M, Rujescu D, Lourdusamy A, Clarke TK, Krause K, Dyer A, Depner M, Wellek S, Treutlein J, Szegedi A, Giegling I, Cichon S, Blomeyer D, Heinz A, Heath S, Lathrop M, Wodarz N, Soyka M, Spanagel R and Mann K

    Interdisciplinary Research Group Addiction, MRC-SGDP Center, Institute of Psychiatry at King's College, POB 080, London SE5 8AF, England. g.schumann@iop.kcl.ac.uk

    Context: Glutamatergic neurotransmission is implicated in alcohol-drinking behavior in animal models.

    Objective: To investigate whether genetic variations in glutamatergic neurotransmission genes, which are known to alter alcohol effects in rodents, contribute to the genetic basis of alcoholism in humans.

    Design: Association analysis of alcohol dependence and haplotype-tagging single nucleotide polymorphisms (SNPs) covering 10 glutamatergic genes. Resequencing of functional domains of these genes identified 204 SNPs. Haplotypes with a frequency of 5% or greater could be discriminated by 21 haplotype-tagging SNPs analyzed for association in 2 independent samples of alcohol-dependent adult patients and controls as well as adolescent trios.

    Setting: Four university medical centers in the south of Germany.

    Participants: One thousand three hundred thirty-seven patients and 1555 controls (study 1: 544 patients, 553 controls; study 2: 793 patients, 1002 controls). One hundred forty-four trios of 15-year-old adolescents assessed for risky drinking behavior.

    Genotype profiles for GLAST; N-methyl-d-aspartate-receptor subunits NR1, NR2A, and NR2B; MGLUR5; NNOS; PRKG2; CAMK4; the regulatory subunit of PI3K; and CREB were analyzed for association with alcohol dependence using multivariate statistical analysis. Risky adolescent drinking was tested using the transmission disequilibrium test.

    Results: Analysis of study 1 revealed that NR2A and MGLUR5 have the greatest relevance for human alcohol dependence among the genes selected with odds ratios of 2.35 and 1.69, respectively. Replication analysis in study 2 confirmed an association of alcohol dependence with NR2A (odds ratio, 2.01) but showed no association with MGLUR5. Combined analysis of study 1 and study 2 exhibited a more significant association on the Cochran-Mantel-Haenszel test (P < .001) for NR2A; NR2A was associated with positive family history, early onset of alcoholism, and maximum number of drinks in adults as well as risky drinking patterns in adolescents.

    Conclusion: Genetic variations in NR2A have the greatest relevance for human alcohol dependence among the glutamatergic genes selected for their known alteration of alcohol effects in animal models.

    Funded by: Department of Health

    Archives of general psychiatry 2008;65;7;826-38

  • Cardiomyocyte overexpression of neuronal nitric oxide synthase delays transition toward heart failure in response to pressure overload by preserving calcium cycling.

    Loyer X, Gómez AM, Milliez P, Fernandez-Velasco M, Vangheluwe P, Vinet L, Charue D, Vaudin E, Zhang W, Sainte-Marie Y, Robidel E, Marty I, Mayer B, Jaisser F, Mercadier JJ, Richard S, Shah AM, Bénitah JP, Samuel JL and Heymes C

    Institut National de Santé et de Recherché Médicale (INSERM), Unit 689, Centre de Recherché Cardiovasculaire Lariboisière, Paris, France.

    Background: Defects in cardiomyocyte Ca(2+) cycling are a signature feature of heart failure (HF) that occurs in response to sustained hemodynamic overload, and they largely account for contractile dysfunction. Neuronal nitric oxide synthase (NOS1) influences myocyte excitation-contraction coupling through modulation of Ca(2+) cycling, but the potential relevance of this in HF is unknown.

    We generated a transgenic mouse with conditional, cardiomyocyte-specific NOS1 overexpression (double-transgenic [DT]) and studied cardiac remodeling, myocardial Ca(2+) handling, and contractility in DT and control mice subjected to transverse aortic constriction (TAC). After TAC, control mice developed eccentric hypertrophy with evolution toward HF as revealed by a significantly reduced fractional shortening. In contrast, DT mice developed a greater increase in wall thickness (P<0.0001 versus control+TAC) and less left ventricular dilatation than control+TAC mice (P<0.0001 for both end-systolic and end-diastolic dimensions). Thus, DT mice displayed concentric hypertrophy with fully preserved fractional shortening (43.7+/-0.6% versus 30.3+/-2.6% in control+TAC mice, P<0.05). Isolated cardiomyocytes from DT+TAC mice had greater shortening, intracellular Ca(2+) transients, and sarcoplasmic reticulum Ca(2+) load (P<0.05 versus control+TAC for all parameters). These effects could be explained, at least in part, through modulation of phospholamban phosphorylation status.

    Conclusions: Cardiomyocyte NOS1 may be a useful target against cardiac deterioration during chronic pressure-overload-induced HF through modulation of calcium cycling.

    Funded by: British Heart Foundation: RG/08/011/25922

    Circulation 2008;117;25;3187-98

  • Association of functional polymorphisms in NOS1 and NOS3 with loudness dependence of auditory evoked potentials.

    Kawohl W, Giegling I, Mavrogiorgou P, Pogarell O, Mulert C, Möller HJ, Hegerl U, Rujescu D and Juckel G

    Research Group Clinical and Experimental Psychopathology, Psychiatric University Hospital Zurich, Zurich, Switzerland.

    Nitric oxide (NO) is a gaseous molecule with neurotransmitter properties that is involved in numerous functions in the central nervous system (CNS), the vascular system and also in macrophages. Haplotypes of NOS1 and NOS3 genes have been shown to be associated with different psychiatric disorders such as schizophrenia and bipolar disorder. Therefore, the detection of other characteristics of nitrinergic transmission is desirable. Because nitrinergic functioning influences serotonergic transmission, a functional marker of the serotonergic transmission, the loudness dependence of auditory evoked potentials (LDAEP), can be assumed to be influenced by nitrinergic changes as well. In order to clarify the relationship between nitrinergic transmission and LDAEP, 95 healthy subjects (41 males, 54 females) underwent electrophysiological recording and blood drawing for genotyping of single nucleotide polymorphisms (SNPs) and haplotypes of the NOS1 and NOS3 genes. Interestingly, two functional SNPs in both NOS1 (G-84A_exon 1c promoter polymorphism) and NOS3 (Glu298Asp) were associated with lower LDAEP. Further studies are needed to fully clarify the relationship between nitrinergic transmission, LDAEP and complex disorders such as schizophrenia and affective disorders.

    The international journal of neuropsychopharmacology 2008;11;4;477-83

  • Neuronal nitric oxide synthase regulates basal microvascular tone in humans in vivo.

    Seddon MD, Chowienczyk PJ, Brett SE, Casadei B and Shah AM

    Department of Cardiology, King's College London School of Medicine, 125 Coldharbour Ln, London SE5 9NU, United Kingdom.

    Background: Nitric oxide (NO) has a pivotal role in the regulation of vascular tone and blood flow, with dysfunctional release contributing to disease pathophysiology. These effects have been attributed to NO production by the endothelial NO synthase (eNOS); however, recent evidence suggests that a neuronal NO synthase (nNOS) may also be expressed in arterial vessels.

    We undertook a first-in-humans investigation of the role of nNOS in the local regulation of vascular blood flow in healthy subjects. Brachial artery infusion of the nNOS-specific inhibitor S-methyl-L-thiocitrulline (SMTC, 0.025 micromol/min to 0.2 micromol/min) caused a dose-dependent reduction in basal flow, with a 30.1+/-3.8% decrease at the highest dose (n=10; mean+/-SE; P<0.01). The effect of SMTC was abolished by coinfusion of the NO synthase substrate L-arginine but was unaffected by D-arginine. A similar reduction in basal flow with the nonselective NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 37.4+/-3.1%, n=10) required a 20-fold higher dose of 4 micromol/min. At doses that produced comparable reductions in basal flow, only L-NMMA (4 micromol/min) and not SMTC (0.2 micromol/min) inhibited acetylcholine-induced vasodilation; however, both SMTC and L-NMMA inhibited the forearm vasodilator response to mental stress.

    Conclusions: Basal forearm blood flow in humans is regulated by nNOS-derived NO, in contrast to the acetylcholine-stimulated increase in blood flow, which, as shown previously, is mediated primarily by eNOS. These data indicate that vascular nNOS has a distinct local role in the physiological regulation of human microvascular tone in vivo.

    Funded by: British Heart Foundation: CVH/99001, PG/05/138/20014; Department of Health

    Circulation 2008;117;15;1991-6

  • Genetic profiling of genes from the oxidative stress pathway among North and South Indians.

    Tiwari AK, Punia S, Juyal RC and Thelma BK

    Department of Genetics, University of Delhi South Campus, New Delhi 110021, India.

    The case-control association study design has been extensively used for elucidating the genetic basis of complex traits. Considerable variation in frequencies of various gene polymorphisms has been reported across different populations and ethnic groups. Thus before beginning such studies, one must know the gene variants that exist in the population. Such information is not available for the ethnically distinct Indian population, which, on the basis of the languages spoken, can be further subdivided into Indo-Europeans (North Indians) and Dravidians (South Indians). In this study we provide information on allele and genotype frequencies, pairwise linkage disequilibrium, and predominant haplotypes in two populations (North India, n=96; South India, n=96) for several of the commonly investigated polymorphisms in the oxidative stress pathway genes. Of the 33 polymorphisms in 19 genes tested, significant differences in allele and genotype frequencies between the two populations were observed for SOD3 Ala58Thr, UCP1-3826 C/T, NOS3-786 T/C, and TNFA-308 G/A polymorphisms.

    Human biology 2008;80;2;161-79

  • Variants in the neuronal nitric oxide synthase (nNOS, NOS1) gene are associated with restless legs syndrome.

    Winkelmann J, Lichtner P, Schormair B, Uhr M, Hauk S, Stiasny-Kolster K, Trenkwalder C, Paulus W, Peglau I, Eisensehr I, Illig T, Wichmann HE, Pfister H, Golic J, Bettecken T, Pütz B, Holsboer F, Meitinger T and Müller-Myhsok B

    Institute of Human Genetics, GSF-National Research Center for Environment and Health, Munich, Germany. julianewinkelmann@t-online.de

    Sixty percent of the patients with restless legs syndrome (RLS) report a positive family history. To date five loci have been mapped on chromosome 12q, 14q, 9p, 2q, and 20p (RLS1-5) but no gene has been identified so far. To identify genes related to RLS, we performed a three-stage association study (explorative study, replication study, high-density mapping) in two Caucasian RLS case-control samples of altogether 918 independent cases and controls. In the explorative study (367 cases and controls, respectively), we screened 1536 SNPs in 366 genes in a 21 Mb region encompassing the RLS1 critical region on chromosome 12. Armitage trend test revealed three genomic regions that were significant (P < 0.05). In the replication study (551 cases and controls, respectively) we genotyped the most significant SNPs of Stage 1. After correction for multiple testing, association was observed with SNP rs7977109 (P(nominal) = 0.00175, P(Westfall-Young) = 0.04895, OR = 0.76228, 95% CI = 0.64310-0.90355), which is in the neuronal nitric oxide synthase (NOS1) gene. High-density mapping using altogether 34 tagging and coding SNPs of the NOS1 gene in both case-control samples further confirmed the significant association results to NOS1. Ten more SNPs revealed significance with nominal P-values from 0.0001 to 0.0482 (genotypic test and Armitage test). Altogether, this study provides evidence for an association of variants in the NOS1 gene and RLS, and suggests the involvement of the NO/arginine pathway in the pathogenesis of RLS. Potential usage of NO modulating agents as new treatment options for RLS have become a challenging aspect for future research of this disorder.

    Movement disorders : official journal of the Movement Disorder Society 2008;23;3;350-8

  • Neuronal nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo.

    Kellogg DL, Zhao JL and Wu Y

    Division of Geriatrics and Gerontology, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr, San Antonio, TX 78229, USA. kelloggd@uthscsa.edu

    The physiological roles of constitutively expressed nitric oxide synthase (NOS) isoforms in humans, in vivo, are unknown. Cutaneous vasodilatation during both central nervous system-mediated, thermoregulatory reflex responses to whole-body heat stress and during peripheral axon reflex-mediated, local responses to skin warming in humans depend on nitric oxide (NO) generation by constitutively expressed NOS of uncertain isoform. We hypothesized that neuronal NOS (nNOS, NOS I) effects cutaneous vasodilatation during whole-body heat stress, but not during local skin warming. We examined the effects of the nNOS inhibitor 7-nitroindazole (7-NI) administered by intradermal microdialysis on vasodilatation induced by whole-body heat stress or local skin warming. Skin blood flow (SkBF) was monitored by laser-Doppler flowmetry (LDF). Blood pressure (MAP) was monitored and cutaneous vascular conductance calculated (CVC = LDF/MAP). In protocol 1, whole-body heat stress was induced with water-perfused suits. In protocol 2, local skin warming was induced through local warming units at LDF sites. At the end of each protocol, 56 mm sodium nitroprusside was perfused at microdialysis sites to raise SkBF to maximal levels for data normalization. 7-NI significantly attenuated CVC increases during whole-body heat stress (P < 0.05), but had no effect on CVC increases induced by local skin warming (P > 0.05). These diametrically opposite effects of 7-NI on two NO-dependent processes verify selective nNOS antagonism, thus proving that the nNOS isoform affects NO increases and hence vasodilatation during centrally mediated, reflex responses to whole-body heat stress, but not during locally mediated, axon reflex responses to local skin warming. We conclude that the constitutively expressed nNOS isoform has distinct physiological roles in cardiovascular control mechanisms in humans, in vivo.

    The Journal of physiology 2008;586;3;847-57

  • Constitutive nitric oxide synthase gene polymorphisms and house dust mite respiratory allergy in an Algerian patient group.

    Djidjik R, Ghaffor M, Brun M, Gharnaout M, Salah SS, Boukouaci W, Djidjik H, Benyounes A, Koumaravelou K, Krishnamoorthy R, Abbadi MC, Charron D and Tamouza R

    Laboratoire de Biologie, Unité d'Immunologie, CHU Beni-Messous, Algiers, Algeria.

    Genetic polymorphisms in neuronal nitric oxide synthase (NOS1) and calmodulin-dependent endothelial NOS (NOS3) genes are known to influence the course of allergic respiratory disorders. We investigated the role of NOS1 -84 G-->A and NOS3 -786 T-->C, 894 G-->T and 27 base pair (bp) repeat polymorphisms in 125 patients suffering from asthma and/or rhinitis and monosensitized against Dermatophagoides pteronyssinus (Dpter) and 111 controls from Algeria. We found a higher frequency of the -786 C NOS3 allele in patients than in controls [corrected P value (Pc) = 0.04], especially in female cases (Pc = 0.02) and that the 'ab' genotype of the 27-bp polymorphism was significantly associated with specific immunoglobulin E production against Dpter (P = 0.006). This study brings further support for the participation of NOS3 gene polymorphism in the pathogenesis of respiratory allergic disorders.

    Tissue antigens 2008;71;2;160-4

  • Influence of salt on subcellular localization of nitric oxide synthase activity and expression in the renal inner medulla.

    Sullivan JC, Smart EJ, Pollock DM and Pollock JS

    Vascular Biology Center, Medical College of Georgia, Augusta, Georgia 30912, USA.

    1. The aims of this study were: (i) to characterize the subcellular localization of nitric oxide synthase (NOS) 1 and NOS3 activity and expression within the cytosolic, plasma membrane and intracellular membrane subcellular fractions of the renal inner medulla of rats; and (ii) to determine whether NOS1 and NOS3 activity and expression in subcellular fractions of the renal inner medulla are regulated by dietary salt intake. Although the NOS system is important in maintaining Na(+) and water homeostasis, the identity of the NOS isoform that is sensitive to dietary Na(+) remains unclear. In addition, subcellular localization of both NOS1 and NOS3 has been shown to regulate enzymatic activity and influence the ability of NOS to produce nitric oxide (NO). 2. Renal inner medullae were dissected from male Sprague-Dawley rats and separated into cytosolic, plasma membrane and intracellular membrane fractions for measurement of NOS activity and western blot analysis. 3. On a normal-salt diet, NOS activity and NOS1 and NOS3 protein expression were present in all three subcellular fractions, although total NOS activity was enriched in the intracellular membrane fraction. In response to a high-salt diet, urinary nitrate/nitrite (NO(x)) increased. Despite an increase in NO(x) excretion, total NOS activity in the renal inner medullary homogenate was decreased. There were no detectable differences in NOS activity in the subcellular fractions. Expression of NOS1 protein was decreased in the cytoplasmic and plasma membrane fractions, although maintained in the intracellular membrane fraction, in response to high salt. Expression of NOS3 protein was unaffected by high salt. 4. In conclusion, we hypothesize that NOS1 localization in the intracellular membrane is important in increasing NO production to aid Na(+) and water homeostasis.

    Funded by: NHLBI NIH HHS: HL60653, HL64776, P01 HL095499, R01 HL060653; NIDDK NIH HHS: DK69392

    Clinical and experimental pharmacology & physiology 2008;35;2;120-5

  • Possible role for nitric oxide dysregulation in critical illness myopathy.

    Capasso M, Di Muzio A, Pandolfi A, Pace M, Di Tomo P, Ragno M and Uncini A

    Neuromuscular Diseases Unit, Center for Excellence on Aging, G. d'Annunzio University Foundation, Clinica Neurologica, Ospedale SS Annunziata, Via dei Vestini, 66013 Chieti, Italy.

    Muscle fiber inexcitability and myosin loss underlie weakness in critical illness myopathy (CIM). Nitric oxide (NO) takes part in the maintenance of muscle fiber resting potential and, in pathological conditions accompanied by oxidative stress, may damage proteins through peroxynitrite generation. Sepsis and other conditions associated with CIM may differentially affect expression of NO synthases (NOSs), so that both downregulation and upregulation with excessive peroxynitrite production can be hypothesized. In six patients with CIM we studied NOS1, NOS2, and NOS3 protein expression by immunohistochemistry and Western blot. To investigate peroxynitrite production, we performed immunohistochemistry for nitrotyrosine and measured nitrotyrosine levels by enzyme-linked immunosorbent assay. In three patients, sarcolemmal staining for NOS1 was selectively absent. In the others, it was absent in atrophic fibers and absent or reduced in non-atrophic fibers. Total NOS1 protein content was reduced by 41% in patients compared to controls, whereas no significant changes were found in levels and localization of NOS2, NOS3, and nitrotyrosine. Further studies are warranted to determine whether NOS1 loss plays a role in the pathophysiology of CIM, possibly reducing the release of NO at the sarcolemma and affecting muscle fiber excitability.

    Muscle & nerve 2008;37;2;196-202

  • NOS-I and -III gene variants are differentially associated with facets of suicidal behavior and aggression-related traits.

    Rujescu D, Giegling I, Mandelli L, Schneider B, Hartmann AM, Schnabel A, Maurer K, Möller HJ and Serretti A

    Department of Psychiatry, Ludwig Maximilians University, Munich, Germany. Dan.Rujescu@med.uni-muenchen.de

    Nitric oxide (NO) is a neurotransmitter which modulates depressive and aggressive behaviors. We studied gene variants of NOS-I (rs2682826;rs1353939;rs2293049;rs693534) and NOS-III (rs2070744;rs1799983;rs891512) in a total of 571 cases (167 German suicide attempters, 92 Caucasian suicide completers and 312 German healthy subjects). A NOS-I risk haplotype C-G-G (rs2682826-rs1353939-rs693534) was associated with suicidal behavior (P = 0.01), and more specifically with suicide attempts (P = 0.01). Sliding windows analysis showed similar results for the haplotype G-G (rs1353939-rs693534) being a risk factor for suicidal behavior (P = 0.01) again especially in suicide attempters (P = 0.004). Additionally, the G-allele of rs693534 was associated with suicidal behavior (P = 0.005) and more specifically with suicide attempts (P = 0.003). Interestingly, the same haplotype (G-G) as well as the rs693534 G-allele were also associated with increased aggression. Regarding NOS-III, a protective haplotype C-T-A (rs2070744-rs1799983-rs891512) was observed (P = 0.01) with a pronounced effect against suicide completion (P = 0.005). Sliding window analysis showed the same effect of haplotype T-A (rs1799983-rs891512) (P = 0.01) which was again protective against suicide completion (P = 0.006). Single marker analysis showed the same protective effect of the rs891512 A-allele (P = 0.009) again especially against suicide completion (P = 0.007). Additionally, a second haplotype (T-T-G) was associated with increased aggression (P = 0.0002; sliding haplotype T-G, P = 0.002). In conclusion, our study suggests a possible involvement of NOS-I and NOS-III gene variants in suicidal behavior and related intermediate phenotypes.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2008;147B;1;42-8

  • IL10 polymorphisms are associated with airflow obstruction in severe alpha1-antitrypsin deficiency.

    Demeo DL, Campbell EJ, Barker AF, Brantly ML, Eden E, McElvaney NG, Rennard SI, Sandhaus RA, Stocks JM, Stoller JK, Strange C, Turino G and Silverman EK

    Brigham and Women's Hospital, Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115, USA. dawn.demeo@channing.harvard.edu

    Severe alpha(1)-antitrypsin (AAT) deficiency is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD), especially in individuals who smoke. There is marked variability in the development of lung disease in individuals homozygous (PI ZZ) for this autosomal recessive condition, suggesting that modifier genes could be important. We hypothesized that genetic determinants of obstructive lung disease may be modifiers of airflow obstruction in individuals with severe AAT deficiency. To identify modifier genes, we performed family-based association analyses for 10 genes previously associated with asthma and/or COPD, including IL10, TNF, GSTP1, NOS1, NOS3, SERPINA3, SERPINE2, SFTPB, TGFB1, and EPHX1. All analyses were performed in a cohort of 378 PI ZZ individuals from 167 families. Quantitative spirometric phenotypes included forced expiratory volume in one second (FEV(1)) and the ratio of FEV(1)/forced vital capacity (FVC). A qualitative phenotype of moderate-to-severe COPD was defined for individuals with FEV(1) </= 50 percent predicted. Six of 11 single-nucleotide polymorphisms (SNPs) in IL10 (P = 0.0005-0.05) and 3 of 5 SNPs in TNF (P = 0.01-0.05) were associated with FEV(1) and/or FEV(1)/FVC. IL10 SNPs also demonstrated association with the qualitative COPD phenotype. When phenotypes of individuals with a physician's diagnosis of asthma were excluded, IL10 SNPs remained significantly associated, suggesting that the association with airflow obstruction was independent of an association with asthma. Haplotype analysis of IL10 SNPs suggested the strongest association with IL10 promoter SNPs. IL10 is likely an important modifier gene for the development of COPD in individuals with severe AAT deficiency.

    Funded by: NHLBI NIH HHS: K08 HL072918, R01 HL68926

    American journal of respiratory cell and molecular biology 2008;38;1;114-20

  • Neuronal nitric oxide synthase C276T polymorphism increases the risk for frontotemporal lobar degeneration.

    Venturelli E, Villa C, Scarpini E, Fenoglio C, Guidi I, Lovati C, Marcone A, Cortini F, Scalabrini D, Clerici F, Bresolin N, Mariani C, Cappa S and Galimberti D

    Department of Neurological Sciences, Dino Ferrari Center, University of Milan, IRCCS Ospedale Maggiore Policlinico, Milan, Italy.

    The neuronal nitric oxide synthase (nNOS) is abundantly expressed in the brain and its transcripts have been found in the frontal cerebral cortex. Eighty-nine patients with different neurodegenerative tau-related disorders, including 71 patients with frontotemporal lobar degeneration (FTLD), 12 with progressive supranuclear palsy (PSP) and 6 with corticobasal degeneration (CBD), were genotyped for the C276T single nucleotide polymorphism (SNP) in exon 29 of the nNOS gene and compared with 190 age-matched controls (CON). A significantly increased allelic frequency of the T allele was observed in patients compared with CON (40.4% vs. 29.7%, P = 0.014, OR: 1.94, CI: 1.15-3.27). Considering each disorder separately, significance was reached for FTLD only (39.4%, P = 0.0248 versus controls, OR: 1.96, CI: 1.11-3.47). However, the frequency of the T allele was elevated also in patients with PSP (45.8%) and CBD (41.7%). No differences were observed stratifying according to gender or apolipoprotein E status. The C276T SNP acts as risk factor for sporadic FTLD, possibly influencing NOS1 transcription. Studies in larger populations are needed to confirm its role in PSP and CBD.

    European journal of neurology 2008;15;1;77-81

  • Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90.

    Averna M, Stifanese R, De Tullio R, Salamino F, Bertuccio M, Pontremoli S and Melloni E

    Department of Experimental Medicine (DIMES)-Biochemistry Section and Centre of Excellence for Biomedical Research (CEBR), University of Genoa, Italy.

    Ca2+ loading of Jurkat and bovine aorta endothelium cells induces the degradation of the neuronal and endothelial nitric oxide synthases that are selectively expressed in these cell lines. For neuronal nitric oxide synthase, this process involves a conservative limited proteolysis without appreciable loss of catalytic activity. By contrast, endothelial nitic oxide synthase digestion proceeds through a parallel loss of protein and catalytic activity. The chaperone heat shock protein 90 (HSP90) is present in a large amount in Jurkat cells and at significantly lower levels in bovine aorta endothelium cells. The differing ratios of HSP90/nitric oxide synthase (NOS) occurring in the two cell types are responsible for the conservative or nonconservative digestion of NOS isozymes. Consistently, we demonstrate that, in the absence of Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain. When Ca2+ is present, a ternary complex containing the three proteins is produced. In this associated state, HSP90 and NOS forms are almost completely resistant to calpain digestion, probably due to a structural hindrance and a reduction in the catalytic efficiency of the protease. Thus, the recruitment of calpain in the HSP90-NOS complexes reduces the extent of the proteolysis of these two proteins. We have also observed that calpastatin competes with HSP90 for the binding of calpain in reconstructed systems. Digestion of the proteins present in the complexes can occur only when free active calpain is present in the system. This process can be visualized as a novel mechanism involving the association of NOS with HSP90 and the concomitant recruitment of active calpain in ternary complexes in which the proteolysis of both NOS isozymes and HSP90 is significantly reduced.

    The FEBS journal 2007;274;23;6116-27

  • Different expression of NOS isoforms in early endothelial progenitor cells derived from peripheral and cord blood.

    Muscari C, Gamberini C, Carboni M, Basile I, Farruggia G, Bonafè F, Giordano E, Caldarera CM and Guarnieri C

    Department of Biochemistry G. Moruzzi, University of Bologna, Italy. claudio.muscari@unibo.it

    Cord blood and peripheral-adult blood were compared as different sources of early endothelial precursor cells (eEPCs). Total mononuclear cells (MNCs) were obtained from both blood types and committed to eEPCs by exposure to fibronectin, VEGF, IGF-I, and bFGF. Under this condition, MNCs seeded at the density of 3 x 10(5) cells/cm(2) assumed a spindle shape, which was indicative of developing eEPCs, and expanded in a similar manner irrespective to the blood sources. Ulex europaeus agglutinin (UEA-1) and acetylated low density lipoprotein (acLDL) double staining was present in 90% in both peripheral- and cord-blood eEPCs after 2-week expansion. Also, the ability of eEPCs to form tubule-like structures in Matrigel was independent of their blood source, but dependent on the presence of human umbilical vein endothelial cells (HUVECs). eNOS and nNOS were not detectable by Western blotting in both peripheral and cord-blood eEPCs upon 3 weeks and their mRNA levels were lower than 2% relative to those present in HUVECs. On the contrary, iNOS protein was detectable in peripheral-blood eEPCs, but not in cord-blood eEPCs and HUVECs, as well as iNOS mRNA was more concentrated in peripheral-blood eEPCs than in cord-blood eEPCs and HUVECs. These data suggest that: (a) peripheral and cord blood can be considered comparable sources of eEPCs when they are expanded and differentiated in a short-term period; (b) the extremely low expression of constitutive NOS isoforms in the eEPCs of both blood types should markedly reduce their ability to regulate NO-dependent vasorelaxation; (c) the presence of iNOS in peripheral-blood eEPCs could improve the process of vasculogenesis.

    Journal of cellular biochemistry 2007;102;4;992-1001

  • Alterations in nitric oxide synthase isoforms in acute lower limb ischemia and reperfusion.

    Tsui JC, Baker DM, Shaw SG and Dashwood MR

    Department of Surgery, Royal Free Hospital, London, UK. jcstsui@hotmail.com

    Alterations in nitric oxide synthase (NOS) are implicated in ischemia and ischemia-reperfusion injury. Changes in the 3 NOS isoforms in human skeletal muscle subjected to acute ischemia and reperfusion were studied. Muscle biopsies were taken from patients undergoing total knee replacement. Distribution of the specific NOS isoforms within muscle sections was studied using immunohistochemistry. NOS mRNA levels were measured using real-time reverse transcription-polymerase chain reaction and protein levels studied using Western blotting. NOS activity was also assessed using the citrulline assay. All 3 NOS isoforms were found in muscle sections associated with muscle fibers and microvessels. In muscle subjected to acute ischemia and reperfusion, NOS I/neuronal NOS mRNA and protein were elevated during reperfusion. NOS III/endothelial NOS was also upregulated at the protein level during reperfusion. No changes in NOS II/inducible NOS expression or NOS activity occurred. In conclusion, alterations in NOS I and III (neuronal NOS and endothelial NOS) at different levels occurred after acute ischemia and reperfusion in human skeletal muscle; however, this did not result in increased NOS activity. In the development of therapeutic agents based on manipulation of the NO pathway, targeting the appropriate NOS isoenzymes may be important.

    Angiology 2007;58;5;586-92

  • Cutaneous neuronal nitric oxide is specifically decreased in postural tachycardia syndrome.

    Stewart JM, Medow MS, Minson CT and Taneja I

    The Center for Pediatric Hypotension, New York Medical College, Ste. 3050, 19 Bradhurst Ave., Hawthorne, NY 10532, USA. stewart@nymc.edu

    Low flow postural tachycardia syndrome (POTS), is associated with reduced nitric oxide (NO) activity assumed to be of endothelial origin. We tested the hypothesis that cutaneous microvascular neuronal NO (nNO) is impaired, rather than endothelial NO (eNO), in POTS. We performed three sets of experiments on subjects aged 22.5 +/- 2 yr. We used laser-Doppler flowmetry response to sequentially increase acetylcholine (ACh) doses and the local cutaneous heating response of the calf as bioassays for NO. During local heating we showed that when the selective neuronal nNO synthase (nNOS) inhibitor N(omega)-nitro-L-arginine-2,4-L-diaminobutyric amide (N(omega), 10 mM) was delivered by intradermal microdialysis, cutaneous vascular conductance (CVC) decreased by an amount equivalent to the largest reduction produced by the nonselective NO synthase (NOS) inhibitor nitro-L-arginine (NLA, 10 mM). We demonstrated that the response to ACh was minimally attenuated by nNOS blockade using N(omega) but markedly attenuated by NLA, indicating that eNO largely comprises the receptor-mediated NO release by ACh. We further demonstrated that the ACh dose response was minimally reduced, whereas local heat-mediated NO-dependent responses were markedly reduced in POTS compared with control subjects. This is consistent with intact endothelial function and reduced NO of neuronal origin in POTS. The local heating response was highly attenuated in POTS [60 +/- 6 percent maximum CVC(%CVC(max))] compared with control (90 +/- 4 %CVC(max)), but the plateau response decreased to the same level with nNOS inhibition (50 +/- 3 %CVC(max) in POTS compared with 47 +/- 2 %CVC(max)), indicating reduced nNO bioavailability in POTS patients. The data suggest that nNO activity but not NO of endothelial NOS origin is reduced in low-flow POTS.

    Funded by: NHLBI NIH HHS: 5R01-HL-74873, R01 HL074873, R01 HL087803; NIDDK NIH HHS: 1R21-DK-071647, R21 DK071647

    American journal of physiology. Heart and circulatory physiology 2007;293;4;H2161-7

  • Genetic susceptibility to respiratory syncytial virus bronchiolitis is predominantly associated with innate immune genes.

    Janssen R, Bont L, Siezen CL, Hodemaekers HM, Ermers MJ, Doornbos G, van 't Slot R, Wijmenga C, Goeman JJ, Kimpen JL, van Houwelingen HC, Kimman TG and Hoebee B

    Laboratory for Toxicology, Pathology, and Genetics, National Institute for Public Health and the Environment, Bilthoven, The Netherlands. riny.janssen@rivm.nl

    Background: Respiratory syncytial virus (RSV) is a common cause of severe lower respiratory tract infection in infants. Only a proportion of children infected with RSV require hospitalization. Because known risk factors for severe disease, such as premature birth, cannot fully explain differences in disease severity, genetic factors have been implicated.

    Methods: To study the complexity of RSV susceptibility and to identify the genes and biological pathways involved in its development, we performed a genetic association study involving 470 children hospitalized for RSV bronchiolitis, their parents, and 1008 random, population controls. We analyzed 384 single-nucleotide polymorphisms (SNPs) in 220 candidate genes involved in airway mucosal responses, innate immunity, chemotaxis, adaptive immunity, and allergic asthma.

    Results: SNPs in the innate immune genes VDR (rs10735810; P=.0017), JUN (rs11688; P=.0093), IFNA5 (rs10757212; P=.0093), and NOS2 (rs1060826; P=.0031) demonstrated the strongest association with bronchiolitis. Apart from association at the allele level, these 4 SNPs also demonstrated association at the genotype level (P=.0056, P=.0285, P=.0372, and P=.0117 for the SNPs in VDR, JUN, IFNA5, and NOS2, respectively). The role of innate immunity as a process was reinforced by association of the whole group of innate immune SNPs when the global test for groups of genes was applied (P=.046).

    Conclusion: SNPs in innate immune genes are important in determining susceptibility to RSV bronchiolitis.

    The Journal of infectious diseases 2007;196;6;826-34

  • Skeletal muscle nNOS mu protein content is increased by exercise training in humans.

    McConell GK, Bradley SJ, Stephens TJ, Canny BJ, Kingwell BA and Lee-Young RS

    Department of Physiology, The University of Melbourne, Parkville, Victoria 3010, Australia. mcconell@unimelb.edu.au

    The major isoform of nitric oxide synthase (NOS) in skeletal muscle is the splice variant of neuronal NOS, termed nNOS mu. Exercise training increases nNOS mu protein levels in rat skeletal muscle, but data in humans are conflicting. We performed two studies to determine 1) whether resting nNOS mu protein expression is greater in skeletal muscle of 10 endurance-trained athletes compared with 11 sedentary individuals (study 1) and 2) whether intense short-term (10 days) exercise training increases resting nNOS mu protein (within whole muscle and also within types I, IIa, and IIx fibers) in eight sedentary individuals (study 2). In study 1, nNOS mu protein was approximately 60% higher (P < 0.05) in endurance-trained athletes compared with the sedentary participants. In study 2, nNOS mu protein expression was similar in types I, IIa, and IIx fibers before training. Ten days of intense exercise training significantly (P < 0.05) increased nNOS mu protein levels in types I, IIa, and IIx fibers, a finding that was validated by using whole muscle samples. Endothelial NOS and inducible NOS protein were barely detectable in the skeletal muscle samples. In conclusion, nNOS mu protein expression is greater in endurance-trained individuals when compared with sedentary individuals. Ten days of intense exercise is also sufficient to increase nNOS mu expression in untrained individuals, due to uniform increases of nNOS mu within types I, IIa, and IIx fibers.

    American journal of physiology. Regulatory, integrative and comparative physiology 2007;293;2;R821-8

  • Differential responses of VIPergic and nitrergic neurons in paediatric patients with Crohn's disease.

    Boyer L, Sidpra D, Jevon G, Buchan AM and Jacobson K

    Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

    Inflammatory bowel disease is a recurrent intestinal inflammatory disorder that in adults has been associated with changes in enteric nervous system neuropeptide expression. The aim of the present study was to determine whether similar changes were observed in paediatric Crohn's disease. The distribution of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) was determined in colonic tissues from children with ileo-colonic (n=4) and colonic (n=3) Crohn's disease. The submucosal plexus of inflamed regions showed significant increase in density of VIP immunoreactive neurons (margin, 48% vs. inflamed tissue, 82% of HuC/D positive neurons). The density of submucosal plexus nNOS immunoreactive neurons was too low to be reliably quantified. Using the pan-neuronal marker HuC/D, no significant difference in numbers of HuC/D positive submucosal neurons was evident except where neurons were normalized to length of tissue (margins, 3.6+/-0.7 vs. inflamed tissue, 4.0+/-0.6 neurons/ganglia, p=0.33; margins, 2.7+/-0.4 vs. inflamed tissue, 5.7+/-1.2, neurons/mm, p=0.03). In the myenteric plexus, there was a significant increase in the percent of NOS neurons (38% vs. 82% of HuC/D positive neurons) while there was no significant difference in percent of VIP neurons (4% vs. 8%). No difference in number of HuC/D positive myenteric neurons among margin and inflamed tissues was observed (margin, 12.2+/-3.0 vs. inflamed tissue, 12.5+/-5.1 neurons/ganglia, p=0.50; margins 9.1+/-2.1 vs. inflamed tissue, 13.7+/-2.3 neurons/mm, p=0.11). These data demonstrate that inflammation is associated with a differential expression of VIP and nNOS neuronal subpopulations within the two major enteric plexi, likely due to phenotypic switch. Such changes might contribute to the pathogenesis of IBD and ongoing symptoms even in quiescent disease.

    Autonomic neuroscience : basic & clinical 2007;134;1-2;106-14

  • Cardiomyocytes as effectors of nitric oxide signalling.

    Seddon M, Shah AM and Casadei B

    Department of Cardiology, Cardiovascular Division, King's College London, London, United Kingdom.

    Nitric oxide (NO) generated constitutively within the heart has long been known to influence myocardial function; however, the precise nature of these effects has been controversial--at least in part--because of the experimental use of non-isoform-selective inhibitors of NO synthases (NOS) and unwarranted extrapolation from results obtained with NO donors. Recent studies using NOS-selective inhibitors and genetically modified models are beginning to redress the balance. It is well established that agonist-stimulated release of NO from eNOS in the coronary endothelium exerts paracrine effects on cardiomyocytes, predominantly affecting the timing of relaxation as well as myocardial oxygen consumption. A significant recent advance has been the finding that both eNOS and nNOS are constitutively expressed in distinct subcellular locations within cardiomyocytes. The relative autocrine role of these isoforms in the cardiomyocyte remains to be fully clarified but evidence suggests that the autocrine effects of nNOS may include the modulation of basal inotropy and relaxation, beta-adrenergic responsiveness, and the force-frequency relationship. Myocardial eNOS, on the other hand, may be involved in mediating the inotropic response to sustained stretch. These effects may change significantly in the diseased heart where the expression, activity and/or coupling of NOS isoforms to downstream effectors may be altered. In this article, we review the current understanding of this important but complex field, focussing particularly on contractile function and on recent advances in knowledge regarding the autocrine functions of nNOS-derived NO.

    Cardiovascular research 2007;75;2;315-26

  • A genetic association analysis of cognitive ability and cognitive ageing using 325 markers for 109 genes associated with oxidative stress or cognition.

    Harris SE, Fox H, Wright AF, Hayward C, Starr JM, Whalley LJ and Deary IJ

    Department of Psychology, University of Edinburgh, Edinburgh, UK. Sarah.Harris@hgu.mrc.ac.uk <Sarah.Harris@hgu.mrc.ac.uk&gt;

    Background: Non-pathological cognitive ageing is a distressing condition affecting an increasing number of people in our 'ageing society'. Oxidative stress is hypothesised to have a major role in cellular ageing, including brain ageing.

    Results: Associations between cognitive ageing and 325 single nucleotide polymorphisms (SNPs), located in 109 genes implicated in oxidative stress and/or cognition, were examined in a unique cohort of relatively healthy older people, on whom we have cognitive ability scores at ages 11 and 79 years (LBC1921). SNPs showing a significant positive association were then genotyped in a second cohort for whom we have cognitive ability scores at the ages of 11 and 64 years (ABC1936). An intronic SNP in the APP gene (rs2830102) was significantly associated with cognitive ageing in both LBC1921 and a combined LBC1921/ABC1936 analysis (p < 0.01), but not in ABC1936 alone.

    Conclusion: This study suggests a possible role for APP in normal cognitive ageing, in addition to its role in Alzheimer's disease.

    Funded by: Medical Research Council: MC_U127561128

    BMC genetics 2007;8;43

  • Nongenomic, endothelium-independent effects of estrogen on human coronary smooth muscle are mediated by type I (neuronal) NOS and PI3-kinase-Akt signaling.

    Han G, Ma H, Chintala R, Miyake K, Fulton DJ, Barman SA and White RE

    Department of Pharmacology and Toxicology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2300, USA.

    Sex steroids exert profound and controversial effects on cardiovascular function. For example, estrogens have been reported to either ameliorate or exacerbate coronary heart disease. Although estrogen dilates coronary arteries from a variety of species, the molecular basis for this acute, nongenomic effect is unclear. Moreover, we know very little of how estrogen affects human coronary artery smooth muscle cells (HCASMC). The purpose of this study was to elucidate nongenomic estrogen signal transduction in HCASMC. We have used tissue (arterial tension studies), cellular (single-channel patch clamp, fluorescence), and molecular (protein expression) techniques to now identify novel targets of estrogen action in HCASMC: type I (neuronal) nitric oxide synthase (nNOS) and phosphatidylinositol 3-kinase (PI3-kinase)Akt. 17beta-Estradiol (E(2)) increased NO-stimulated fluorescence in HCASMC, and cell-attached patch-clamp experiments revealed that stimulation of nNOS leads to increased activity of calcium-activated potassium (BK(Ca)) channels in these cells. Furthermore, overexpression of nNOS protein in HCASMC greatly enhanced BK(Ca) channel activity. Immunoblot studies demonstrated that E(2) enhances Akt phosphorylation in HCASMC and that wortmannin, an inhibitor of PI3-kinase, attenuated E(2)-stimulated channel activity, NO production, Akt phosphorylation, and estrogen-stimulated coronary relaxation. These studies implicate the PI3-kinase/Akt signaling axis as an estrogen transduction component in vascular smooth muscle cells. We conclude, therefore, that estrogen opens BK(Ca) channels in HCASMC by stimulating nNOS via a transduction sequence involving PI3-kinase and Akt. These findings now provide a molecular mechanism that can explain the clinical observation that estrogen enhances coronary blood flow in patients with diseased or damaged coronary arteries.

    Funded by: NHLBI NIH HHS: HL 073890, HL 074279, HL 080402, HL 68026

    American journal of physiology. Heart and circulatory physiology 2007;293;1;H314-21

  • Polymorphisms of the neuronal and inducible nitric oxide synthase genes and the risk of cutaneous melanoma: a case-control study.

    Li C, Hu Z, Liu Z, Wang LE, Gershenwald JE, Lee JE, Prieto VG, Duvic M, Grimm EA and Wei Q

    Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Background: Nitric oxide (NO) is a multifunctional molecule that is produced by both neuronal NO synthase (nNOS) and inducible NO synthase (iNOS), and the expression of nNOS and iNOS is up-regulated in various cancer cells, including cutaneous melanoma (CM). The authors hypothesized that selected functional single-nucleotide polymorphisms (SNPs) in the nNOS and iNOS genes are associated with the risk of CM.

    Methods: In a hospital-based case-control study of 602 non-Hispanic white patients with CM and 603 matched, cancer-free controls, the authors genotyped the nNOS -84 guanine-to-adenine (G-->A), nNOS 276 cytosine-to-thymine (C-->T), iNOS Ex16+14C-->T, and iNOS 974G-->T SNPs and assessed their associations with the risk of CM in multivariate logistic regression models.

    Results: A significantly increased risk of CM was associated with the nNOS -84G-->A (adjusted odds ratio [OR], 1.49; 95% confidence interval [95% CI], 1.05-2.13) and -84AG+AA (OR, 1.48; 95% CI, 1.06-2.06) genotypes compared with the nNOS -84GG genotype, but not with other nNOS or iNOS SNPs. In a combined analysis, an increased risk of CM was associated with the nNOS -84AA+AG/276CT+TT genotype (OR, 1.70; 95% CI, 1.05-2.76) and the nNOS -84AA+AG/276CC genotype (OR, 1.70; 95% CI, 1.08-2.68) compared with the nNOS -84GG/276CT+TT genotypes. No altered risk of CM was associated with iNOS genotypes. In addition, there was statistical evidence of interaction of nNOS SNPs with having moles (P = .002) and sunburns (P = .017).

    Conclusions: Genetic variants of nNOS, but not iNOS, may be biomarkers for susceptibility to CM, and the risk of CM associated with sunburns and moles may be modulated by nNOS variant genotypes.

    Funded by: NCI NIH HHS: P30 CA-16672, P50 CA-093459, R01 CA-100264; NIEHS NIH HHS: R01 ES-11740

    Cancer 2007;109;8;1570-8

  • Activities of mitochondrial complexes correlate with nNOS amount in muscle from ALS patients.

    Sorarù G, Vergani L, Fedrizzi L, D'Ascenzo C, Polo A, Bernazzi B and Angelini C

    Department of Neurological and Psychiatric Sciences - University of Padova, Italy. janni010270@yahoo.com

    The pathogenesis of amyotrophic lateral sclerosis (ALS) is poorly understood. Increased levels of free radicals derived from nitric oxide (NO), the product of nitric oxide synthase (NOS), may damage mitochondrial function leading to motor neurone death. Previous studies demonstrated a specific impairment of mitochondrial function in skeletal muscle of ALS patients. In order to verify a pathogenetic relationship between neuronal NOS (nNOS) and mitochondrial function, we studied nNOS expression by Western blot and mitochondrial enzyme activity by spectrophotometric assays in muscle biopsies of 16 sporadic ALS patients and 16 controls subjects. We observed a reduced activity of respiratory chain complexes with mitochondrial encoded subunits and a lower nNOS amount in ALS muscles. There was a direct correlation between levels of nNOS and values of mitochondrial enzymes function. In ALS muscles we found normal levels of manganese superoxide dismutase (SOD2) that is assumed as related to mitochondrial DNA abnormalities. Our data suggest a beneficial role for NO to mitochondrial function and lead to the hypothesis of a common oxidative damage in motor neurones and skeletal muscle in sporadic ALS patients.

    Funded by: Telethon: GTF05003

    Neuropathology and applied neurobiology 2007;33;2;204-11

  • Application of HapMap data to the evaluation of 8 candidate genes for pediatric slow transit constipation.

    Garcia-Barcelo M, King SK, Miao X, So MT, Holden WT, Moore JH, Sutcliffe JR, Hutson JM and Tam PK

    Division of Paediatric Surgery, Department of Surgery, The University of Hong Kong, Hong Kong SAR, China.

    Background: Slow transit constipation (STC) affects up to 3% of all children and is an increasingly recognized cause of chronic constipation in children. We conducted a pilot study to investigate whether genes encoding neurotransmitters (TAC1, TAC3, VIP, NOS1) and receptors (TACR1, TACR2, TACR3, KIT) could be responsible for STC.

    Methods: One hundred seventeen tag single nucleotide polymorphisms (SNPs), distributed among the candidate genes, were selected from HapMap data and genotyped using Sequenom (San Diego, CA) technology in 35 affected families. Evaluation of association was performed by transmission disequilibrium test and multilocus analysis.

    Results: Five SNPs (rs3771863, rs4580655, rs11722288, rs4563545, and rs3782221) in the TACR1, TACR3, KIT, and NOS1 genes were found to be potentially associated with STC, although the significance of these results does not withstand correction for multiple testing.

    Conclusions: Our data indicate that 5 SNPs in the NOS1, TACR1, TACR3, and KIT genes could be involved in STC, especially rs3771863 in intron 1 of TACR1, which showed the highest association.

    Journal of pediatric surgery 2007;42;4;666-71

  • Physician-diagnosed asthma and acute chest syndrome: associations with NOS polymorphisms.

    Duckworth L, Hsu L, Feng H, Wang J, Sylvester JE, Kissoon N, Sandler E and Lima JJ

    Pharmacogenetics Center, Nemours Children's Clinic, Jacksonville, Florida 32207, USA.

    The main objectives of this paper were to test the hypothesis that polymorphisms in NOS1 and NOS3 genes associate with ACS in SCD patients and to characterize the association between physician-diagnosed asthma and acute chest syndrome (ACS). Case-control study of sickle cell disease patients >or=5 years old with ACS (cases; n=86) and those without ACS (controls; n=48) was carried out. Associations between ACS and the AAT repeat in intron 13 (formerly intron 20) of the NOS1, and with NOS3 T-786C polymorphism were explored. Physician-diagnosed asthma was determined by chart review, patient- or parent (guardian)-reported asthma, and drug use. Eighty five percent of participants with asthma had at least one episode of ACS compared to 14.6% of controls: adjusted odds ratio (OR) (95%CI) 5.46 (2.20,13.5), P= or<0.0001. Asthma correlated with the number of episodes of ACS (P<0.001). NOS1 AAT repeat polymorphism associated with the risk of ACS (P=0.001) in patients without physician-diagnosed asthma. No associations were found between the genotype of the NOS3 T-786C SNP and ACS. Physician-diagnosed asthma is a major risk factor for ACS. NOS1 AAT repeat polymorphism may contribute to physician-diagnosed asthma.

    Pediatric pulmonology 2007;42;4;332-8

  • No association between Parkinson's disease and three polymorphisms in the eNOS, nNOS, and iNOS genes.

    Huerta C, Sánchez-Ferrero E, Coto E, Blázquez M, Ribacoba R, Guisasola LM, Salvador C and Alvarez V

    Genética Molecular-Instituto de Investigación Nefrológica, Hospital Central de Asturias, Servicio de Salud del Principado de Asturias, Spain.

    Nitric oxide synthases (NOS) and mitochondrial DNA-polymorphisms have been associated with the risk of developing Parkinson's disease (PD). In this report, we genotyped 450 PD-patients and 200 controls for three polymorphisms in the endothelial, inducible and neuronal NOS-genes, and for the T4336C and A10398G mitochondrial DNA-polymorphisms. None of the eNOS (intron 4 VNTR), iNOS (exon 22 A/G), or nNOS (exon 29T/C) were significantly associated with PD. Mitochondrial 4336C increased the PD-risk among women (OR=6.13), while the 10398G had a protective effect (OR=0.52). We did not find significantly interactions between the NOS and mitochondrial polymorphisms in the risk for PD in our population.

    Neuroscience letters 2007;413;3;202-5

  • A NOS1 gene polymorphism associated with asthma and specific immunoglobulin E response to mite allergens in a Colombian population.

    Martínez B, Barrios K, Vergara C, Mercado D, Jiménez S, Gusmão L and Caraballo L

    Institute for Immunological Research, University of Cartagena, Cartagena, Colombia.

    Background: Nitric oxide (NO) is involved in asthma pathogenesis and is synthesized by three isoforms of NO synthase, one of them encoded by NOS1 gene. The CA-repeat and the C5266T SNP in NOS1 exon 29 have been associated with asthma and IgE levels. We thought to test the association of asthma and asthma-related phenotypes with the exon 29 CA-repeat and the C5266T SNP in a Colombian population sample.

    Methods: The CA-repeat and the C5266T SNP were genotyped in 167 asthmatics and 166 controls using PCR-based fragment length polymorphism and TaqMan assay. We also determined total and mite-specific IgE against Blomia tropicalis and Dermatophagoides pteronyssinus.

    Results: Three new CA-repeat alleles, 14, 23 and 24 repeats were detected. Allele comprising 16 repeats was associated with asthma (OR: 1.90 (CI 1.22-2.97, p(c) = 0.028) and low total (p(c) = 0.02) and specific IgE to B. tropicalis (p(c) < 0.0001) and D. pteronyssinus (p(c) < 0.0001). We found no association of the C5266T SNP and asthma or IgE levels.

    Conclusion: NOS1 exon 29 CA-repeat may be a risk factor for asthma susceptibility and mite specific IgE response in a Colombian population.

    International archives of allergy and immunology 2007;144;2;105-13

  • Fas (CD95) alters neuronal nitric oxide synthase expression to contribute in diabetic gastroparesis.

    Surendran S

    Medical hypotheses 2007;68;6;1427

  • Binding of Vac14 to neuronal nitric oxide synthase: Characterisation of a new internal PDZ-recognition motif.

    Lemaire JF and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 University Street, Montreal, Que., Canada H3A 2B4.

    PDZ domains mediate protein interactions primarily through either classical recognition of carboxyl-terminal motifs or PDZ/PDZ domain associations. Several studies have also described internal modes of PDZ recognition, most of which depend on beta-finger structures. Here, we describe a novel interaction between the PDZ domain of nNOS and Vac14, the activator of the PtdIns(3)P 5-kinase PIKfyve. Binding assays using various Vac14 deletion constructs revealed a beta-finger independent interaction that is based on a novel internal motif. Mutational analyses reveal essential residues within the motif allowing us to define a new type of PDZ domain interaction.

    FEBS letters 2006;580;30;6948-54

  • Role of Asp1393 in catalysis, flavin reduction, NADP(H) binding, FAD thermodynamics, and regulation of the nNOS flavoprotein.

    Konas DW, Takaya N, Sharma M and Stuehr DJ

    Department of Pathobiology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp(1393)) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (<or=1000x) cytochrome c reductase, ferricyanide reductase, and NADPH oxidase activities than the wild type. D1393N nNOSr also had lower reductase activities (<or=10x) but had greater NADPH oxidase activity than that of the wild type, as did its FNR fragment. Both mutants had an altered interaction between FAD and the nicotinamide ring of NADP(+), slower flavin reduction by NADPH, altered FAD midpoint potentials, a normal CaM response, and, in one case (D1393N), faster flavin oxidation by O(2) and a lack of FMN shielding in response to NADPH binding. The results suggest that the two mutants have compromised catalysis for two different reasons. In D1393V nNOSr, hydride transfer from NADPH to FAD is so slow that it compromises all downstream electron-transfer events. In D1393N nNOSr, the increased oxidation of reduced flavins by O(2) and thermodynamic destabilization of the FAD semiquinone uncouples or limits electron transfer to an extent that it inhibits downstream catalysis. These effects are due in part to the mutations eliminating (D1393V) or altering (D1393N) the native side-chain hydrogen-bonding properties of Asp(1393) as well as removing its negative charge.

    Funded by: NHLBI NIH HHS: HL76491; NIGMS NIH HHS: GM51491

    Biochemistry 2006;45;41;12596-609

  • Nitric oxide synthase in retina and optic nerve head of rat with increased intraocular pressure and effect of timolol.

    Vidal L, Díaz F, Villena A, Moreno M, Campos JG and de Vargas IP

    Department of Histology and Histopathology, School of Medicine, University of Malaga, Boulevard Louis Pasteur 32, 29071 Malaga, Spain. lvidal@uma.es

    We investigated the expression of nitric oxide synthase (NOS) isoforms -1, -2 and -3 in the retina and optic nerve head (ONH) in an experimental rat model of elevated intraocular pressure (IOP) before and after treatment with timolol, to assess whether its neuroprotective action is associated with the activity of these enzymes. Episcleral vein cauterization in unilateral eyes of Wistar rats was performed to produce elevated IOP. Histological sections of retina and ONH from animals with normal IOP, with elevated IOP, and elevated IOP treated with timolol, were studied by immunohistochemistry with antibodies to NOS-1, NOS-2, and NOS-3. In the control rats, NOS-1 was localized to photoreceptor inner segments, amacrine cells and bipolar cells in the retina, and in astrocytes, pericytes and vascular nitrergic terminals in the ONH. NOS-3 immunostaining localized to the endothelial cells. The rats with elevated IOP showed increased expression of NOS-1 in the plexiform layers of the retina and reactive astrocytes in the ONH. These cells also showed NOS-2 positivity. The rats treated with timolol showed reduced expression of NOS-1 in the retina and ONH. NOS-2 was only detected in a few groups of astrocytes in the ONH. NOS-3 was unchanged in both elevated IOP and timolol-treated groups. These results show that excessive levels of NO synthesized by the NOS-1 and -2 isoforms, considered neurotoxic, might contribute to the progressive lesions of retinal ganglion cell axons. Their reduction after treatment suggests a possible neuroprotective effect of timolol in neurons exposed to excessive amounts of NO.

    Brain research bulletin 2006;70;4-6;406-13

  • Association between achalasia and nitric oxide synthase gene polymorphisms.

    Mearin F, García-González MA, Strunk M, Zárate N, Malagelada JR and Lanas A

    Institute of Functional and Motor Digestive Disorders, Centro Médico Teknon, Barcelona, Spain.

    Background: Our group previously reported the absence of nitric oxide synthase (NOS) in the gastroesophageal junction of patients with achalasia. NOS exists in three distinct isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible isoform (iNOS). Some studies have shown that NO production is regulated by NOS polymorphisms.

    Aim: To assess whether some functional polymorphisms in the nNOS, iNOS, or eNOS genes are involved in susceptibility to suffer from achalasia.

    Methods: Genomic DNA from 80 unrelated Spanish Caucasian patients with sporadic achalasia and 144 healthy subjects matched for age (+/-5 yr) and gender was typed by PCR and RFLP methods for the 27-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 of the eNOS gene, a CA microsatellite repeat and a Nla III (C-->T) restriction fragment length polymorphism (RFLP) in exon 29 of the nNOS gene, and two nucleotide substitutions located in exon 16 (C-->T) and exon 22 (G-->A) of the iNOS gene.

    Results: No significant differences in carriage, genotype, and allele frequencies of the nNOS, iNOS, or eNOS gene polymorphisms were found between patients with achalasia and controls. Individuals homozygous for genotype iNOS22*A/A tended to be more frequent in achalasia (20%vs 11%, odds ratio [OR] 1.79, 95% confidence interval [CI] 0.89-3.59, p= 0.09) as were those homozygous for the rare eNOS*4a allele (6.2%vs 1.4%, OR 4.5, 95% CI 0.89-22.67, p= 0.1) although the difference did not reach statistical significance. No differences in genotype and allele distribution were found with respect to epidemiological and clinical characteristics of patients with achalasia.

    Conclusion: Our data suggest that NOS gene polymorphisms are not involved in the susceptibility to and nature of the clinical course of sporadic achalasia. However, studies in a greater number of patients are required to analyze the tendency toward a higher prevalence of genotypes iNOS22*A/A and eNOS*4a4a.

    The American journal of gastroenterology 2006;101;9;1979-84

  • Pulmonary nitric oxide synthases and nitrotyrosine: findings during lung development and in chronic lung disease of prematurity.

    Sheffield M, Mabry S, Thibeault DW and Truog WE

    Children's Mercy Hospitals and Clinics, Section of Neonatology, Department of Pediatrics, University of Missouri-Kansas City School of Medicine, Kansas City, Missouri 64108, USA.

    Background: Nitric oxide mediates and modulates pulmonary transition from fetal to postnatal life. NO is synthesized by 3 nitric oxide synthase isoforms. One key pathway of nitric oxide metabolism results in nitrotyrosine, a stable, measurable marker of nitric oxide production.

    Objective: The purpose of this study was to assess, by semiquantitative immunohistochemistry, nitric oxide synthase isoforms and nitrotyrosine at different airway and vascular tree levels in the lungs of neonates at different gestational ages and to compare results in control groups to those in infants with chronic lung disease.

    Formalin-fixed, paraffin-embedded, postmortem lung blocks were prepared for immunohistochemistry using antibodies to each nitric oxide synthase isoform and to nitrotyrosine. Blinded observers evaluated the airway and vascular trees for staining intensity (0-3 scale) at 5 levels and 3 levels, respectively. The control population consisted of infants from 22 to 42 weeks' gestation who died in < 48 hours. Results were compared with gestation-matched infants with varying severity of chronic lung disease.

    Results: In control and chronic lung disease groups, 22 to 42 weeks' gestation, staining for all 3 of the nitric oxide synthase isoforms was found in the airway epithelium from the bronchus to the alveolus or distal-most airspace. The abundance or distribution of nitric oxide synthase-3 staining in the airways did not show significant correlation with gestational age or severity of chronic lung disease. In the vascular tree, intense nitric oxide synthase-3 and moderate nitric oxide synthase-2 staining was found; nitric oxide synthase-1 was not consistently stained. Nitrotyrosine did stain in the pulmonary tree. Compared with controls where nitrotyrosine staining was minimal, regardless of gestation, in infants with chronic lung disease there was more than fourfold increase between severe chronic lung disease (n = 12) and either mild chronic lung disease or control infants (n = 16).

    Conclusions: All 3 of the nitric oxide synthase isoforms and nitrotyrosine are detectable by immunohistochemistry early in lung development. Nitric oxide synthase ontogeny shows no significant changes in abundance or distribution with advancing gestational age nor with chronic lung disease. Nitrotyrosine is significantly increased in severe chronic lung disease.

    Funded by: PHS HHS: R-01 70560

    Pediatrics 2006;118;3;1056-64

  • Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity.

    Raines KW, Cao GL, Lee EK, Rosen GM and Shapiro P

    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.

    nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.

    Funded by: NCI NIH HHS: CA-10529; NIBIB NIH HHS: EB-2034, P41 EB002034; NIGMS NIH HHS: R25 GM055036, R25-GM55036

    The Biochemical journal 2006;397;2;329-36

  • Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation.

    Cordelier P, Estève JP, Najib S, Moroder L, Vaysse N, Pradayrol L, Susini C and Buscail L

    INSERM U531, IFR31, Centre Hospitalier Universitaire Rangueil, 31432 Toulouse Cedex 4, France. pierre.cordelier@toulouse.inserm.fr

    Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.

    The Journal of biological chemistry 2006;281;28;19156-71

  • Transcription of human neuronal nitric oxide synthase mRNAs derived from different first exons is partly controlled by exon 1-specific promoter sequences.

    Bros M, Boissel JP, Gödtel-Armbrust U and Förstermann U

    Department of Dermatology, Johannes Gutenberg University, D-55101 Mainz, Germany.

    The human neuronal nitric oxide synthase (NOS1) gene is subject to extensive splicing. A total of 12 NOS1 mRNA species have been identified. They differ in their 5' ends and are derived from 12 different first exons (termed exons 1a to 1l). Various cell lines whose NOS1 first exon expression patterns were representative of human brain, skin, and skeletal muscle were identified. These included A673 neuroepithelioma cells, SK-N-MC neuroblastoma cells, HaCaT keratinocyte-like cells, and C2C12 myocyte-like cells. In these cell lines, correlations were found between the exon 1 variants preferentially expressed and the promoter activities of their cognate 5' flanking sequences. These data demonstrate that expression of the different exon 1-related splice variants of NOS1 mRNA is controlled directly (at least in part) by the associated 5' flanking sequences.

    Genomics 2006;87;4;463-73

  • Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) regulates endothelial nitric oxide synthase (eNOS) activity and its localization within the human vein endothelial cells (HUVEC) in culture.

    Di Pietro R, Mariggiò MA, Guarnieri S, Sancilio S, Giardinelli A, Di Silvestre S, Consoli A, Zauli G and Pandolfi A

    Department of Biomorphology, G. d'Annunzio University, Chieti-Pescara, Italy.

    We have recently demonstrated that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases endothelial nitric oxide synthase (eNOS) phosphorylation, NOS activity, and nitric oxide (NO) synthesis in cultured human umbilical vein endothelial cells (HUVEC), without inducing apoptotic cell death. Although an important factor that regulates eNOS activity is its localization within the cells, little is known about the role of TRAIL in the regulation of eNOS trafficking among cellular compartments and the cytoskeleton involvement in this machinery. Then, we did both quantitative and semi-quantitative evaluations with biochemical assays and immune fluorescence microscopy in the presence of specific inhibitors of NOS activity as well as of cytoskeletal microtubule structures. In our cellular model, TRAIL treatment not only increased NO levels but also caused a time-dependent NO migration of fluorescent spots from the plasma membrane to the inner part of the cells. In unstimulated cells, most of the eNOS was localized at the cell membranes. However, within 10 min following addition of TRAIL, nearly all the cells showed an increased cytoplasm localization of eNOS which appeared co-localized with the Golgi apparatus at a higher extent than in unstimulated cells. These effects were associated to an increased formation of trans-cytoplasm stress fibers with no significant changes of the microtubule network. Conversely, microtubule disruption and Golgi scattering induced with Nocodazole treatment inhibited TRAIL-increased NOS activity, indicating that, on cultured HUVEC, TRAIL ability to affect NO production by regulating eNOS sub-cellular distribution is mediated by cytoskeleton and Golgi complex modifications.

    Journal of cellular biochemistry 2006;97;4;782-94

  • A neuronal nitric oxide synthase (NOS-I) haplotype associated with schizophrenia modifies prefrontal cortex function.

    Reif A, Herterich S, Strobel A, Ehlis AC, Saur D, Jacob CP, Wienker T, Töpner T, Fritzen S, Walter U, Schmitt A, Fallgatter AJ and Lesch KP

    Department of Psychiatry and Psychotherapy (Section for Clinical and Molecular Psychobiology and Laboratory for Psychophysiology and Functional Imaging), Julius-Maximilians-University Würzburg, Würzburg, Germany. a.reif@gmx.net

    Nitric oxide (NO) is a gaseous neurotransmitter thought to play important roles in several behavioral domains. On a neurobiological level, NO acts as the second messenger of the N-methyl-D-aspartate receptor and interacts with both the dopaminergic as well as the serotonergic system. Thus, NO is a promising candidate molecule in the pathogenesis of endogenous psychoses and a potential target in their treatment. Furthermore, the chromosomal locus of the gene for the NO-producing enzyme NOS-I, 12q24.2, represents a major linkage hot spot for schizophrenic and bipolar disorder. To investigate whether the gene encoding NOS-I (NOS1) conveys to the genetic risk for those diseases, five NOS1 polymorphisms as well as a NOS1 mini-haplotype, consisting of two functional polymorphisms located in the transcriptional control region of NOS1, were examined in 195 chronic schizophrenic, 72 bipolar-I patients and 286 controls. Single-marker association analysis showed that the exon 1c promoter polymorphism was linked to schizophrenia (SCZ), whereas synonymous coding region polymorphisms were not associated with disease. Long promoter alleles of the repeat polymorphism were associated with less severe psychopathology. Analysis of the mini-haplotype also revealed a significant association with SCZ. Mutational screening did not detect novel exonic polymorphisms in patients, suggesting that regulatory rather than coding variants convey the genetic risk on psychosis. Finally, promoter polymorphisms impacted on prefrontal functioning as assessed by neuropsychological testing and electrophysiological parameters elicited by a Go-Nogo paradigm in 48 patients (continuous performance test). Collectively these findings suggest that regulatory polymorphisms of NOS1 contribute to the genetic risk for SCZ, and modulate prefrontal brain functioning.

    Molecular psychiatry 2006;11;3;286-300

  • Bipolar I disorder and schizophrenia: a 440-single-nucleotide polymorphism screen of 64 candidate genes among Ashkenazi Jewish case-parent trios.

    Fallin MD, Lasseter VK, Avramopoulos D, Nicodemus KK, Wolyniec PS, McGrath JA, Steel G, Nestadt G, Liang KY, Huganir RL, Valle D and Pulver AE

    Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21231, USA.

    Bipolar, schizophrenia, and schizoaffective disorders are common, highly heritable psychiatric disorders, for which familial coaggregation, as well as epidemiological and genetic evidence, suggests overlapping etiologies. No definitive susceptibility genes have yet been identified for any of these disorders. Genetic heterogeneity, combined with phenotypic imprecision and poor marker coverage, has contributed to the difficulty in defining risk variants. We focused on families of Ashkenazi Jewish descent, to reduce genetic heterogeneity, and, as a precursor to genomewide association studies, we undertook a single-nucleotide polymorphism (SNP) genotyping screen of 64 candidate genes (440 SNPs) chosen on the basis of previous linkage or of association and/or biological relevance. We genotyped an average of 6.9 SNPs per gene, with an average density of 1 SNP per 11.9 kb in 323 bipolar I disorder and 274 schizophrenia or schizoaffective Ashkenazi case-parent trios. Using single-SNP and haplotype-based transmission/disequilibrium tests, we ranked genes on the basis of strength of association (P<.01). Six genes (DAO, GRM3, GRM4, GRIN2B, IL2RB, and TUBA8) met this criterion for bipolar I disorder; only DAO has been previously associated with bipolar disorder. Six genes (RGS4, SCA1, GRM4, DPYSL2, NOS1, and GRID1) met this criterion for schizophrenia or schizoaffective disorder; five replicate previous associations, and one, GRID1, shows a novel association with schizophrenia. In addition, six genes (DPYSL2, DTNBP1, G30/G72, GRID1, GRM4, and NOS1) showed overlapping suggestive evidence of association in both disorders. These results may help to prioritize candidate genes for future study from among the many suspected/proposed for schizophrenia and bipolar disorders. They provide further support for shared genetic susceptibility between these two disorders that involve glutamate-signaling pathways.

    Funded by: NIMH NIH HHS: R01 MH058153, R01MH057314, R01MH58153

    American journal of human genetics 2005;77;6;918-36

  • Hypoxia induces a functionally significant and translationally efficient neuronal NO synthase mRNA variant.

    Ward ME, Toporsian M, Scott JA, Teoh H, Govindaraju V, Quan A, Wener AD, Wang G, Bevan SC, Newton DC and Marsden PA

    Division of Respirology, University of Toronto, Toronto, Ontario, Canada. wardm@smh.toronto.on.ca

    We tested the hypothesis that induction of neuronal NO synthase (nNOS) impairs vascular smooth muscle contractility after hypoxia. nNOS protein was increased in aorta, mesenteric arterioles, pulmonary arteries, brain, and diaphragm from rats exposed to 8% O2 for 48 hours and in human aortic SMCs after hypoxic incubation (1% O2). Ca-dependent NO synthase activity was increased in endothelium-denuded aortic segments from hypoxia-exposed rats. N-nitro-L-arginine methyl ester enhanced the contractile responses of endothelium-denuded aortic rings and mesenteric arterioles from hypoxia-exposed but not normoxic rats (P < 0.05). The hypoxia-inducible mRNA transcript expressed by human cells was found to contain a novel 5'-untranslated region, consistent with activation of transcription in the genomic region contiguous with exon 2. Translational efficiency of this transcript is markedly increased compared with previously described human nNOS mRNAs. Transgenic mice possessing a lacZ reporter construct under control of these genomic sequences demonstrated expression of the construct after exposure to hypoxia (8% O2, 48 hours) in the aorta, mesenteric arterioles, renal papilla, and brain. These results reveal a novel human nNOS promoter that confers the ability to rapidly upregulate nNOS expression in response to hypoxia with a functionally significant effect on vascular smooth muscle contraction.

    The Journal of clinical investigation 2005;115;11;3128-39

  • Nitric oxide synthase polymorphisms and asthma phenotypes in Chinese children.

    Leung TF, Liu EK, Tang NL, Ko FW, Li CY, Lam CW and Wong GW

    Department of Paediatrics, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR, China. tfleung@cuhk.edu.hk

    Background: Nitric oxide (NO) is a key factor for balancing T-helper type 1/T-helper type 2 immunity. Single nucleotide polymorphisms (SNPs) in nitric oxide synthase (NOS) genes have been associated with atopy and exhaled NO concentrations in Caucasians. We investigated the association between asthma traits and genetic polymorphisms in neuronal NO synthase (NOS1) and endothelial NO synthase (NOS3) in Chinese children.

    Methods: Asthmatic children between 5 and 18 years of age and non-allergic controls were recruited. Plasma total IgE was measured by microparticle immunoassay, whereas allergen-specific IgEs were measured by fluorescent enzyme immunoassay. Fractional exhaled NO concentration (FeNO) was measured by a chemiluminescence analyser. NOS1 C5266T and NOS3 G894T were genotyped by restriction fragment length polymorphism, and (AAT)n polymorphism in intron 20 of NOS1 was determined by GeneScan analysis.

    Results: The mean (SD) ages of 295 asthmatics and 174 controls were 11.1 (3.8) years and 11.6 (4.0) years, respectively (P=0.162). NOS1 C5266T and NOS3 G894T were not associated with asthma, atopy or FeNO. However, significantly more subjects with T/T in NOS1 C5266T had increased plasma total IgE as compared with those with C/T or C/C (P=0.017). This SNP was also associated with sensitization to Dermatophagoides pteronyssinus (P=0.049). Among asthmatic patients, log-transformed plasma total IgE levels were significantly higher among those homozygous for 5266T of NOS1 [mean (SD): 2.84 (0.44) for T/T, 2.68 (0.42) for C/T, 2.59 (0.69) for C/C; P=0.021]. This study found a significant inter-ethnic difference in the allele frequencies of AAT repeats, and this polymorphism was associated with high plasma total IgE levels (P=0.044) but not FeNO (P=0.158). NOS3 G894T was not associated with any asthma or atopy phenotype.

    Conclusions: NOS1 C5266T and AAT repeats affect plasma IgE concentrations in Chinese children. On the other hand, neither NOS1 nor NOS3 SNP was associated with FeNO or the risk of having asthma.

    Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2005;35;10;1288-94

  • A population genomics overview of the neuronal nitric oxide synthase (nNOS) gene and its relationship to migraine susceptibility.

    Johnson MP, Lea RA, Colson NJ, Macmillan JC and Griffiths LR

    Genomics Research Centre, School of Health Science, Griffith University, PMB 50 GCMC, Gold Coast 9726, Australia.

    The ubiquitous chemical messenger molecule nitric oxide (NO) has been implicated in a diverse range of biological activities including neurotransmission, smooth muscle motility and mediation of nociception. Endogenous synthesis of NO by the neuronal isoform of the nitric oxide synthase gene family has an essential role within the central and peripheral nervous systems in addition to the autonomic innervation of cerebral blood vessels. To investigate the potential role of NO and more specifically the neuronal nitric oxide synthase (nNOS) gene in migraine susceptibility, we investigated two microsatellite repeat variants residing within the 5' and 3' regions of the nNOS gene. Population genomic evaluation of the two nNOS repeat variants indicated significant linkage disequilibrium between the two loci. Z-DNA conformational sequence structures within the 5' region of the nNOS gene have the potential to enhance or repress gene promoter activity. We suggest that genetic analysis of this 5' repeat variant is the more functional variant expressing gene wide information that could affect endogenous NO synthesis and potentially result in diseased states. However, no association with migraine (with or without aura) was seen in our extensive case-control cohort (n = 579 affected with matched controls), when both the 5' and 3' genetic variants were investigated.

    Cellular and molecular biology (Noisy-le-Grand, France) 2005;51;3;285-92

  • Association of neuronal nitric oxide synthase C276T polymorphism with Alzheimer's disease.

    Galimberti D, Venturelli E, Gatti A, Lovati C, Fenoglio C, Mariani C, Forloni G, Bresolin N and Scarpini E

    Journal of neurology 2005;252;8;985-6

  • [Polymorphic markers of the NO synthase genes and genetic predisposition to diabetic polyneuropathy in patients with type 1 diabetes mellitus].

    Zotova EV, Voron'ko OE, Bursa TR, Galeev IV, Strokov IA and Nosikov VV

    The allele and genotype frequencies of polymorphic markers of NOS1, NOS2 and NOS3 genes, encoding three types of NO synthases, were compared in type 1 diabetes patients with and without diabetic polyneuropathty. 180 type 1 diabetes patients (T1DM) of Russian or Eastern Slavonic origin, living in Moscow city, were divided into two groups using non-overlapping (polar) phenotypes. 86 patients had overt DPN and T1DM duration in this group was less than 5 years (DPN+ group) and 94 patients had no clinical DPN and T1DM duration was more than 10 years (DPN- group). We have not found the significant differences of allele and genotype frequencies of polymorphic markers (CA)n of NOS1 gene, (CCTTT)n of NOS2 gene, ecNOS4a/4b and Glu298Asp of NOS3 gene that indicates that all these markers are not associated with diabetic polyneuropathty. Only in the case of (CCTTT)n marker of NOS2 gene we have found a tendency for the association of 14 allele with DPN development. The carriers of this allele have the lower risk of DPN in T1DM.

    Molekuliarnaia biologiia 2005;39;2;224-9

  • Family-based case-control study of cigarette smoking and Parkinson disease.

    Scott WK, Zhang F, Stajich JM, Scott BL, Stacy MA and Vance JM

    Department of Medicine and Duke Center for Human Genetics, Duke University Medical Center, Box 3445, Durham, NC 27710, USA. bill.scott@duke.edu

    Objective: To determine whether people with Parkinson disease (PD) are less likely to report a history of cigarette smoking than their unaffected siblings.

    Background: Previous studies reported that individuals with PD are half as likely to have smoked as those unaffected by PD. Other studies reported that smoking modified the risk of PD due to polymorphisms in the MAO-B and nNOS genes. Thus, genetic studies of PD should consider confounding or interaction with smoking history as well. The authors have collected detailed smoking histories on a family-based case-control sample ascertained for genetic studies of PD.

    Methods: In a matched case-control study of 140 sibships, individuals with PD (n = 143) were compared to sibling controls (n = 168). Cigarette smoking history was collected by a structured telephone interview. Conditional logistic regression was used to examine the relationship between smoking and PD while controlling for confounding by age and sex.

    Results: Ever smoking, current smoking, and increasing duration (in years), dose (in packs/day), and intensity (in pack-years) of smoking were significantly inversely associated with PD (p < 0.05). The association was not modified by sex, age at onset, or recency of exposure.

    Conclusions: Consistent with previous studies, individuals with Parkinson disease are significantly less likely to have smoked regularly than their unaffected siblings. This association was detected even though discordant sibling pairs are more likely to be overmatched for environmental exposures than unmatched case and control groups.

    Funded by: NINDS NIH HHS: NS39764

    Neurology 2005;64;3;442-7

  • Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase.

    Peng HM, Morishima Y, Jenkins GJ, Dunbar AY, Lau M, Patterson C, Pratt WB and Osawa Y

    Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

    It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.

    Funded by: NHLBI NIH HHS: HL65619; NIDDK NIH HHS: DK31573; NIEHS NIH HHS: ES08365; NIGMS NIH HHS: GM61728

    The Journal of biological chemistry 2004;279;51;52970-7

  • Nitric oxide synthase (NOS)-interacting protein interacts with neuronal NOS and regulates its distribution and activity.

    Dreyer J, Schleicher M, Tappe A, Schilling K, Kuner T, Kusumawidijaja G, Müller-Esterl W, Oess S and Kuner R

    Department of Molecular Pharmacology, Pharmacology Institute, University of Heidelberg, 69120 Heidelberg, Germany.

    Mechanisms governing the activity of neuronal nitric oxide synthase (nNOS), the major source of nitric oxide (NO) in the nervous system, are not completely understood. We report here a protein-protein interaction between nNOS and NOSIP (nitric oxide synthase-interacting protein) in rat brain in vivo. NOSIP and nNOS are concentrated in neuronal synapses and demonstrate significant colocalization in various regions of the central and peripheral nervous systems. NOSIP produces a significant reduction in nNOS activity in a neuroepithelioma cell line stably expressing nNOS. Furthermore, overexpression of NOSIP in cultured primary neurons reduces the availability of nNOS in terminal dendrites. These results thus suggest that the interaction between NOSIP and nNOS is functionally involved in endogenous mechanisms regulating NO synthesis. Furthermore, we found that the subcellular distribution and expression levels of NOSIP are dynamically regulated by neuronal activity in vitro as well as in vivo, suggesting that NOSIP may contribute to a mechanism via which neuronal activity regulates the synaptic availability and activity of nNOS.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;46;10454-65

  • Estrogen induces nitric oxide production via activation of constitutive nitric oxide synthases in human neuroblastoma cells.

    Xia Y and Krukoff TL

    Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

    Although it is becoming increasingly evident that nitric oxide (NO) mediates some of estrogen's actions in the brain, the effects of estrogen on NO production through NO synthases (NOS) in neuronal cells have not yet been identified. Here we assessed changes in NO production induced by 17beta-estradiol (E2) in cells of neuronal origin using human SK-N-SH neuroblastoma cells, which we show express all three isoforms of NOS. Involvement of NOS isoforms in E2-induced NO production was examined using isoform-specific NOS inhibitors. E2 (10(-10)-10(-6) m) induced rapid increases in NO release and changes in endothelial NOS (eNOS) expression, which were blocked by ICI 182,780, an antagonist of estrogen receptors. Increased levels of NO release and NOS activity induced by E2 were blocked by N5-(1-Imino-3-butenyl)-L-ornithine, a neuronal NOS inhibitor, and N(5)-(1-Iminoethyl)-L-ornithine, an eNOS inhibitor, but not by 1400W, an inducible NOS inhibitor. These results demonstrate that E2-stimulated NO production occurs via estrogen receptor-mediated activation of the constitutive NOSs, neuronal NOS and eNOS. The E2-induced NO increase was abolished when extracellular Ca2+ was removed from the medium or after the addition of nifedipine, an L-type channel blocker, and was partially inhibited using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester itself also caused an increase in NO release that was blocked by 1400W, suggesting that inducible NOS mediates this response. Together these data reveal that constitutive NOS activities are responsible for E2-induced NO production in neuroblastoma cells and that differential activation of NOS isoforms in these cells occurs in response to different treatments.

    Endocrinology 2004;145;10;4550-7

  • Differential expression of nitric oxide synthases (NOS 1-3) in human skeletal muscle following exercise countermeasure during 12 weeks of bed rest.

    Rudnick J, Püttmann B, Tesch PA, Alkner B, Schoser BG, Salanova M, Kirsch K, Gunga HC, Schiffl G, Lück G and Blottner D

    Department of Anatomy, Campus Benjamin Franklin, Charité University Medicine Berlin, Germany.

    Adaptive changes of major body systems in astronauts during spaceflight can be simulated by strict anti-orthostatic head-down tilt (HDT) bed rest (BR), a ground-based microgravity (microG) model that provides a meaningful opportunity to study atrophy mechanisms and possible countermeasures under controlled experimental conditions. As nitric oxide (NO) signaling is linked to muscle activity, we investigated altered expression of the three major isoforms of nitric oxide synthase (NOS 1-3) at cellular compartments during prolonged HDT BR without (control group) and with resistance exercise interventions (exercise group) using a flywheel ergometer (FWE). Atrophy detected in mixed (fast-slow) m. vastus lateralis (VL) and slow-type m. soleus (SOL) myofiber Types I and II (minus 35-40% of myofiber cross-sectional area) was prevented by FWE training. Concomitant to muscle atrophy, reduced NOS 1 protein and immunostaining was found in VL not in SOL biopsies. In trained VL, NOS 1 protein and immunostaining at myofibers II were significantly increased at the end of BR. Exercise altered NOS 2/caveolin 3 co-immunostaining patterns of subsarcolemmal focal accumulations in VL or SOL myofibers, which suggests reorganization of sarcolemmal microdomains. In trained VL, increased capillary-to-fiber (C/F) ratio and NOS 3 protein content were documented. Activity-linked NO signaling may be widespread in skeletal muscle cellular compartments that may be directly or indirectly impacted by adequate exercise countermeasure protocols to offset the negative effects induced by disuse, immobilization, or extended exposure to microgravity.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2004;18;11;1228-30

  • Nitric oxide synthase 1 is partly compensating for nitric oxide synthase 3 deficiency in nitric oxide synthase 3 knock-out mice and is elevated in murine and human cirrhosis.

    Biecker E, Neef M, Sägesser H, Shaw S, Koshy A and Reichen J

    Department of Clinical Pharmacology, University of Berne, Murtenstrasse, Switzerland.

    Background: The role of endothelial nitric oxide synthase 3 (NOS-3) in the hyperdynamic circulation associated with cirrhosis is established but not that of the neuronal (NOS-1) isoform. We therefore investigated aortic NOS-1 levels in NOS-3 knock-out (KO) and wildtype (WT) mice and in hepatic arteries of patients.

    Methods: Mice rendered cirrhotic by bile duct ligation (BDL) were compared with sham-operated controls. Hepatic arteries of cirrhotic patients were collected during liver transplantation; donor vessels served as controls. mRNA levels were quantified by real-time PCR, protein levels by Western blotting and NO production by Nomega-nitro-L-arginine methyl ester inhibitable arginine-citrulline assay.

    Results: Aortae of NOS-3 KO mice exhibited higher NOS-1mRNA (5.6-fold, P < 0.004) and protein levels (8.8-fold) compared with WT. NO production in aortae of NOS-3 KO mice was 52% compared with WT (P = 0.002). BDL increased NOS-1 mRNA (2.4-fold, P = 0.01) and protein (7.1-fold) levels in aortae of WT, but no further in the NOS-3 KO mice. Hepatic artery NOS-1 mRNA levels in cirrhotic patients were markedly increased compared with controls (24.5-fold, P = 0.0007).

    Conclusions: Increased NOS-1 mRNA and protein levels and partially maintained in vitro NO-production in aortae of NOS-3 KO mice suggest that NOS-1 may partially compensate for NOS-3 deficiency. BDL-induced increase in aortic NOS-1 mRNA and protein levels hint that not only NOS-3, but also NOS-1 may be involved in the regulation of systemic hyperdynamic circulation and portal hypertension. Upregulation of NOS-1 mRNA levels in hepatic arteries of portal hypertensive patients suggests possible clinical significance for these experimental findings.

    Liver international : official journal of the International Association for the Study of the Liver 2004;24;4;345-53

  • Calcium/calmodulin-dependent protein kinase I inhibits neuronal nitric-oxide synthase activity through serine 741 phosphorylation.

    Song T, Hatano N, Horii M, Tokumitsu H, Yamaguchi F, Tokuda M and Watanabe Y

    Department of Cell Physiology, Kagawa University, Faculty of Medicine, 1750-1 Ikenobe, Miki-cho, Kida-gun, Kagawa 761-0793, Japan.

    We demonstrate here that neuronal nitric-oxide synthase (nNOS) is phosphorylated and inhibited by a constitutively active form of Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM-K I1-293). Substitution of Ser741 to Ala in nNOS blocked the phosphorylation and the inhibitory effect. Mimicking phosphorylation at Ser741 by Ser to Asp mutation resulted in decreased binding of and activation by CaM, since the mutation was within the CaM-binding domain. CaM-K I1-293 gave phosphorylation of nNOS at Ser741 in transfected cells, resulting in 60-70% inhibition of nNOS activity. Wild-type CaM-K I also did phosphorylate nNOS at Ser741 in transfected cells, but either CaM-K II or CaM-K IV did not. These results raise the possibility of a novel cross-talk between nNOS and CaM-K I through the phosphorylation of Ser741 on nNOS.

    FEBS letters 2004;570;1-3;133-7

  • Expression, localization, and regulation of NOS in human mast cell lines: effects on leukotriene production.

    Gilchrist M, McCauley SD and Befus AD

    Institute for Systems Biology, Seattle, WA 98103, USA. mgilchrist@systemsbiology.org

    Nitric oxide (NO) is a potent radical produced by nitric oxide synthase (NOS) and has pleiotrophic activities in health and disease. As mast cells (MCs) play a central role in both homeostasis and pathology, we investigated NOS expression and NO production in human MC populations. Endothelial NOS (eNOS) was ubiquitously expressed in both human MC lines and skin-derived MCs, while neuronal NOS (nNOS) was variably expressed in the MC populations studied. The inducible (iNOS) isoform was not detected in human MCs. Both growth factor-independent (HMC-1) and -dependent (LAD 2) MC lines showed predominant nuclear eNOS protein localization, with weaker cytoplasmic expression. nNOS showed exclusive cytoplasmic localization in HMC-1. Activation with Ca(2+) ionophore (A23187) or IgE-anti-IgE induced eNOS phosphorylation and translocation to the nucleus and nuclear and cytoplasmic NO formation. eNOS colocalizes with the leukotriene (LT)-initiating enzyme 5-lipoxygenase (5-LO) in the MC nucleus. The NO donor, S-nitrosoglutathione (SNOG), inhibited, whereas the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME), potentiated LT release in a dose-dependent manner. Thus, human MC lines produce NO in both cytoplasmic and nuclear compartments, and endogenously produced NO can regulate LT production by MCs.

    Blood 2004;104;2;462-9

  • NIDD, a novel DHHC-containing protein, targets neuronal nitric-oxide synthase (nNOS) to the synaptic membrane through a PDZ-dependent interaction and regulates nNOS activity.

    Saitoh F, Tian QB, Okano A, Sakagami H, Kondo H and Suzuki T

    Department of Neuroplasticity Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan.

    Targeting of neuronal nitric-oxide synthase (nNOS) to appropriate sites in a cell is mediated by interactions with its PDZ domain and plays an important role in specifying the sites of reaction of nitric oxide (NO) in the central nervous system. Here we report the identification and characterization of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) (GenBank accession number AB098078), which increases nNOS enzyme activity by targeting the nNOS to the synaptic plasma membrane in a PDZ domain-dependent manner. The deduced NIDD protein consisted of 392 amino acid residues and possessed five transmembrane segments, a zinc finger DHHC domain, and a PDZ-binding motif (-EDIV) at its C-terminal tail. In vitro pull-down assays suggested that the C-terminal tail region of NIDD specifically interacted with the PDZ domain of nNOS. The PDZ dependence was confirmed by an experiment using a deletion mutant, and the interaction was further confirmed by co-sedimentation assays using COS-7 cells transfected with NIDD and nNOS. Both NIDD and nNOS were enriched in synaptosome and synaptic plasma membrane fractions and were present in the lipid raft and postsynaptic density fractions in the rat brain. Co-localization of these proteins was also observed by double staining of the proteins in cultured cortical neurons. Thus, NIDD and nNOS were co-localized in the brain, although the colocalizing regions were restricted, as indicated by the distribution of their mRNA expression. Most important, co-transfection of NIDD and nNOS increased NO-producing nNOS activity. These results suggested that NIDD plays an important role in the regulation of the NO signaling pathway at postsynaptic sites through targeting of nNOS to the postsynaptic membrane.

    The Journal of biological chemistry 2004;279;28;29461-8

  • The neuronal nitric oxide synthase is upregulated in mouse skin repair and in response to epidermal growth factor in human HaCaT keratinocytes.

    Boissel JP, Ohly D, Bros M, Gödtel-Armbrust U, Förstermann U and Frank S

    Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany. boissel@mail.uni-mainz.de

    Expression of nNOS mRNA was found in normal human and mouse skin tissue. Upon wounding, we observed a rapid downregulation of nNOS mRNA and protein in wounds of mice; however, when repair continued, nNOS mRNA was strongly upregulated and nNOS protein expression peaked at late stages of healing. Immunohistochemistry revealed wound keratinocytes as the cellular source of nNOS. In line with the in vivo situation, we found a basal expression of nNOS in the human keratinocyte cell line HaCaT. A marked stimulation of nNOS expression in the cells was achieved with epidermal growth factor receptor (EGFR) ligands such as epidermal growth factor (EGF), heparin-binding EGF, transforming growth factor-alpha and two alternate splicing forms of the neuregulin gene. EGF-induced induction of nNOS was completely inhibited by the specific EGFR antagonist PD153035 and by the EGFR and Janus kinase 2/3 inhibitor AG490. Activation of EGFR might contribute to the observed upregulation of nNOS also in skin repair, as we found a spatial and temporal correlation of phosphorylated EGFR (Y1173) with nNOS expression at the wound site. Thus, in addition to the inducible- and endothelial-type NOS isoforms, also nNOS expression is regulated in the process of cutaneous wound repair.

    The Journal of investigative dermatology 2004;123;1;132-9

  • Contribution of nNOS- and eNOS-derived NO to microvascular smooth muscle NO exposure.

    Kavdia M and Popel AS

    Biomedical Engineering, University of Arkansas, Fayetteville, AR 72701, USA. mkavdia@uark.edu

    Nitric oxide (NO) plays an important role in autocrine and paracrine manner in numerous physiological processes, including regulation of blood pressure and blood flow, platelet aggregation, and leukocyte adhesion. In vascular wall, most of the bioavailable NO is believed to derive from endothelial cell NO synthase (eNOS). Recently, neuronal NOS (nNOS) has been identified as a source of NO in the vicinity of microvessels and has been shown to participate in vascular function. Thus NO can be produced and transported to the vascular smooth muscle cells from 1). endothelial cells and 2). perivascular nerve fibers, mast cells, and other nNOS-containing sources. In this study, a mathematical model of NO diffusion-reaction in a cylindrical arteriolar segment was formulated. The model quantifies the relative contribution of these NO sources and the smooth muscle availability of NO in a tissue containing an arteriolar blood vessel. The results indicate that a source of NO derived through nNOS in the perivascular region can be a significant contributor to smooth muscle NO. Predicted smooth muscle NO concentrations are as high as 430 nM, which is consistent with reported experimental measurements ( approximately 400 nM). In addition, we used the model to analyze the smooth muscle NO availability in 1). eNOS and nNOS knockout experiments, 2). the presence of myoglobin, and 3). the presence of cell-free Hb, e.g., Hb-based oxygen carriers. The results show that NO release by nNOS would significantly affect available smooth muscle NO. Further experimental and theoretical studies are required to account for distribution of NOS isoforms and determine NO availability in vasculatures of different tissues.

    Funded by: NHLBI NIH HHS: HL-18292

    Journal of applied physiology (Bethesda, Md. : 1985) 2004;97;1;293-301

  • nNOS is involved in estrogen mediated neuroprotection in neuroblastoma cells.

    Wen Y, Perez EJ, Green PS, Sarkar SN and Simpkins JW

    Department of Pharmacology and Neuroscience, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Blvd, Fort Worth, TX 76107l, USA.

    Estrogens exert neuroprotective activity in both in vivo and in vitro model systems. Herein, we report that both 17beta-estradiol and low concentrations of nitric oxide (NO) attenuate hydrogen peroxide (H2O2) induced toxicity in SK-N-SH cells, which express the neuronal nitric oxide synthase (nNOS). 17beta-estradiol rapidly induced an increase in NO levels. A nNOS inhibitor was able to block the neuroprotection of 17beta-estradiol. Cyclic guanylyl mono-phosphate (cGMP) also protected against H2O2 induced toxicity, while NO's protection was attenuated by ODQ, a soluble guanylyl cyclase (sGC) inhibitor. In SK-N-SH cells, the major estrogen receptor isoforms is estrogen receptor beta. Our current study suggests that increased activity of nNOS may be involved in the neuroprotection conferred by 17beta-estradiol.

    Neuroreport 2004;15;9;1515-8

  • Changes in the dimeric state of neuronal nitric oxide synthase affect the kinetics of secretagogue-induced insulin response.

    Lajoix AD, Pugnière M, Roquet F, Mani JC, Dietz S, Linck N, Faurie F, Ribes G, Petit P and Gross R

    CNRS UMR 5160, Institut de Biologie, 4 Boulevard Henri IV, 34960 Montpellier Cedex 2, France.

    We previously showed that pancreatic beta-cells express a neuronal isoform of nitric oxide synthase (nNOS) that controls insulin secretion by exerting two enzymatic activities: nitric oxide (NO) production and cytochrome c reductase activity. We now bring evidence that two inhibitors of nNOS, N-omega-nitro-l-arginine methyl ester (l-NAME) and 7-nitroindazole (7-NI), increase glucose-induced insulin secretion but affect beta-cell function differently. In the presence of l-NAME, insulin response is monophasic, whereas 7-NI preserves the normal biphasic secretory pattern. In addition, the alterations of beta-cell functional response induced by the inhibitors also differ by their sensitivity to a substitutive treatment with sodium nitroprusside, a chemical NO donor. These differences are probably related to the nature of the two inhibitors. Indeed, using low-temperature SDS-PAGE and real-time analysis of nNOS dimerization by surface plasmon resonance, we could show that 7-NI, which competes with arginine and tetrahydrobiopterin (BH(4)), an essential cofactor for nNOS dimer formation, inhibits dimerization of the enzyme, whereas the substrate-based inhibitor l-NAME stabilizes the homodimeric state of nNOS. The latter effect could be reproduced by the two endogenous inhibitors of NOS, N-omega-methyl-l-arginine and asymmetric dimethylarginine, and resulted interestingly in a reduced ability of the protein inhibitor of nNOS (PIN) to dissociate nNOS dimers. We conclude that intracellular factors able to induce abnormalities in the nNOS monomer/dimer equilibrium could lead to pancreatic beta-cell dysfunction.

    Diabetes 2004;53;6;1467-74

  • Genetic analysis of 22 candidate genes for hypertension in the Japanese population.

    Iwai N, Tago N, Yasui N, Kokubo Y, Inamoto N, Tomoike H and Shioji K

    National Cardiovascular Center, Suita, Osaka, Japan. iwai@ri.ncvc.go.jp

    Objective: We performed association studies between 118 single-nucleotide polymorphisms (SNPs) of 22 candidate genes (or gene family) and hypertension in a Japanese population.

    The study population consisted of 1880 subjects representing the general population in Japan, recruited from the Suita study. The candidate genes were selected based on their functions, including insulin resistance (APM1, CD36, HSD11B1), oxidative stress (CYBA, GPX1, GSTMs), steroid hormone (ESR1, ESR2, HSD11B2), renal functions (PTGS2, KLK1, NPHS1, NPHS2, SGK, SLC12A1, PTGES), and others related to cardiovascular physiology (GJA4, NOS1, NTRK3, P2RX4, SPP1, ALDH2).

    Results: Multiple logistic analyses, with age and body mass index as covariates, indicated that 13 SNPs (eight genes), six SNPs (four genes) and 11 SNPs (four genes) were associated with hypertension (P < 0.05) in the total, male, and female populations, respectively. PTGS2 seems to be a promising candidate gene for hypertension in men. GSTM3 and SLC12A1 seem to be promising candidate genes for hypertension in women. Especially, a polymorphism in SLC12A1 was significantly associated with hypertension in women even after correction by the Bonferroni method (corrected P = 0.0236). Multiple logistic analyses, with age and body mass index as covariates, indicated that the prevalence of hypertension in females was significantly higher in subjects with the CC genotype than in those with the TT + TC genotypes (P < 0.0001, odds ratio = 1.967, 95% confidence interval = 1.430-2.712).

    Conclusion: Although the present results should be replicated in other study populations for confirmation, the present results suggest that SLC12A1 may contribute to hypertension in Japanese women.

    Journal of hypertension 2004;22;6;1119-26

  • Association analysis of a neural nitric oxide synthase gene polymorphism and antipsychotics-induced tardive dyskinesia in Chinese schizophrenic patients.

    Wang YC, Liou YJ, Liao DL, Bai YM, Lin CC, Yu SC and Chen JY

    Department of Psychiatry, Yu-Li Veterans Hospital, Taipei, Taiwan.

    Recent findings from rodent studies with chronic administration of antipsychotic drugs have indicated the role of neural nitric oxide synthase (NOS1) on the susceptibility of tardive dyskinesia (TD). In the present study, the association between a 3'-untranslated region C267T (3'-UTR C267T) polymorphism of the NOS1 gene and TD as well as TD severity was investigated in 251 Chinese schizophrenic patients with long-term antipsychotic treatment (TD: 128, non-TD: 123). After adjusting the effects of confounding factors, there was no significant association between NOS1 3'-UTR C276T genotypes and TD occurrence (p=0.758). With in the TD group, we could not discover a significant correlation between NOS1 3'-UTR C276T genotypes and the scores of abnormal involuntary movement scale (AIMS) (p=0.219 and 0.774). We concluded that the NOS1 3'-UTR C276T polymorphism might not play a major role in the susceptibility of TD development, or on the severity of TD.

    Journal of neural transmission (Vienna, Austria : 1996) 2004;111;5;623-9

  • Neuronal nitric oxide synthase gene polymorphism and IgE-mediated allergy in the Central European population.

    Hollá LI, Schüller M, Bucková D and Vácha J

    Department of Pathological Physiology, Masaryk University Brno, Brno, Czech Republic.

    Background: Several findings suggest that nitric oxide (NO) plays a significant role in the regulation of the Th1/Th2 balance and contributes to the development of allergic diseases. Our study investigates a possible association of C/T transition located 276-bp downstream from the translation termination site in exon 29 of the human nitric oxide synthase type 1 (NOS1) gene with immunoglobulin E (IgE)-mediated allergic diseases in the Czech population.

    Methods: The study included 688 subjects - 368 patients with clinically manifested allergic diseases and 320 unrelated controls with negative familial history of asthma/atopy. The NOS1 genotypes were determined by polymerase chain reaction (PCR) and restriction analysis by Eco72I.

    Results: No significant differences were found for allele or genotype frequencies of the 5266 C/T polymorphism in exon 29 of the NOS1 gene between IgE-mediated allergic diseases (or asthma alone) and healthy subjects. However, this common polymorphism showed a significant association with signs of atopy, especially with total serum IgE levels [log(e) IgE levels (mean +/- SD): CC genotype = 4.34 +/- 1.40; CT genotype = 4.58 +/- 1.53; TT genotype = 5.01 +/- 1.61; P < 0.05).

    Conclusions: Our findings suggest that NOS1 gene may participate in the pathogenesis of high total serum IgE levels in allergic diseases in our population. These findings provide support for NOS1 as a candidate gene for IgE-mediated allergy.

    Allergy 2004;59;5;548-52

  • 1-Methyl-4-phenylpyridinium-induced apoptosis in cerebellar granule neurons is mediated by transferrin receptor iron-dependent depletion of tetrahydrobiopterin and neuronal nitric-oxide synthase-derived superoxide.

    Shang T, Kotamraju S, Kalivendi SV, Hillard CJ and Kalyanaraman B

    Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, 53226, USA.

    In this study, we investigated the molecular mechanisms of toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), an ultimate toxic metabolite of a mitochondrial neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, that causes Parkinson-like symptoms in experimental animals and humans. We used rat cerebellar granule neurons as a model cell system for investigating MPP(+) toxicity. Results show that MPP(+) treatment resulted in the generation of reactive oxygen species from inhibition of complex I of the mitochondrial respiratory chain, and inactivation of aconitase. This, in turn, stimulated transferrin receptor (TfR)-dependent iron signaling via activation of the iron-regulatory protein/iron-responsive element interaction. MPP(+) caused a time-dependent depletion of tetrahydrobiopterin (BH(4)) that was mediated by H(2)O(2) and transferrin iron. Depletion of BH(4) decreased the active, dimeric form of neuronal nitric-oxide synthase (nNOS). MPP(+)-mediated "uncoupling" of nNOS decreased *NO and increased superoxide formation. Pretreatment of cells with sepiapterin to promote BH(4) biosynthesis or cell-permeable iron chelator and TfR antibody to prevent iron-catalyzed BH(4) decomposition inhibited MPP(+) cytotoxicity. Preincubation of cerebellar granule neurons with nNOS inhibitor exacerbated MPP(+)-induced iron uptake, BH(4) depletion, proteasomal inactivation, and apoptosis. We conclude that MPP(+)-dependent aconitase inactivation, Tf-iron uptake, and oxidant generation result in the depletion of intracellular BH(4), leading to the uncoupling of nNOS activity. This further exacerbates reactive oxygen species-mediated oxidative damage and apoptosis. Implications of these results in unraveling the molecular mechanisms of neurodegenerative diseases (Parkinson's and Alzheimer's disease) are discussed.

    Funded by: NHLBI NIH HHS: 1P01HL68769-01; NINDS NIH HHS: NS39958, R01 NS039958

    The Journal of biological chemistry 2004;279;18;19099-112

  • Expression of nNOS and soluble guanylate cyclase in schizophrenic brain.

    Baba H, Suzuki T, Arai H and Emson PC

    Department of Psychiatry, Juntendo University, School of Medicine, Tokyo, Japan. GZZ01551@nifty.ne.jp

    Recent evidence suggests that nitric oxide (NO) systems are affected in the pathophysiology of schizophrenia. We quantified levels of neuronal NO synthase (nNOS) and soluble guanylate cyclase (sGC) subunit mRNAs in the prefrontal cortex of post-mortem brains from individuals with schizophrenia and controls using real-time quantitative PCR, to determine whether levels of nNOS and sGC subunits are altered in 'schizophrenic' brains. Neuronal NOS expression in the prefrontal cortex was significantly higher in individuals with schizophrenia, whereas no significant changes were found in sGC subunit mRNAs in people with schizophrenia or in controls. Abnormalities of nNOS expression in the brain might contribute to the development of schizophrenia.

    Neuroreport 2004;15;4;677-80

  • Single-nucleotide promoter polymorphism alters transcription of neuronal nitric oxide synthase exon 1c in infantile hypertrophic pyloric stenosis.

    Saur D, Vanderwinden JM, Seidler B, Schmid RM, De Laet MH and Allescher HD

    Department of Internal Medicine II, Technical University of Munich, Ismaningerstrasse 22, 81675 Munich, Germany. dieter.saur@lrz.tum.de

    Infantile hypertrophic pyloric stenosis (IHPS), characterized by enlarged pyloric musculature and gastric-outlet obstruction, is associated with altered expression of neuronal nitric oxide synthase (nNOS). Here we have studied molecular mechanisms by which nNOS gene expression is altered in pyloric tissues of 16 infants with IHPS and 9 controls. A significant decreased expression of total nNOS mRNA was found by quantitative RT-PCR in IHPS after normalization against GAPDH, which predominantly affected exon 1c with a reduction of 88% compared with controls (P < 0.001). After normalization against the neuronal-specific gene PGP9.5, expression of exon 1c was still decreased (P < 0.001), whereas expression of exon 1f was increased significantly (P = 0.001), indicating a compensatory up-regulation of this nNOS mRNA variant. DNA samples of 16 IHPS patients and 81 controls were analyzed for nNOS exon 1c promoter mutations and single-nucleotide polymorphism (SNP). Sequencing of the 5'-flanking region of exon 1c revealed mutations in 3 of 16 IHPS tissues, whereas 81 controls showed the wild-type sequence exclusively. Carriers of the A allele of a previously uncharacterized nNOS exon 1c promoter SNP (-84G --> A) had increased risk for development of IHPS (odds ratio, 8.0; 95% confidence interval, 2.5-25.6). Reporter gene assays revealed an unchanged promoter activity for mutations but a approximately 30% decrease for the -84A SNP (P < 0.001). In summary, our findings indicate that genetic alterations in the nNOS exon 1c regulatory region influence expression of the nNOS gene and may contribute to the pathogenesis of IHPS.

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;6;1662-7

  • Nitric oxide inhibition of ERK1/2 activity in cells expressing neuronal nitric-oxide synthase.

    Raines KW, Cao GL, Porsuphatana S, Tsai P, Rosen GM and Shapiro P

    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.

    Neuronal nitric-oxide synthase (nNOS) is a constitutively expressed enzyme responsible for the production of nitric oxide (NO*) from l-arginine and O2. Nitric oxide is an intra- and intercellular messenger that mediates a diversity of signaling pathways in target cells. In the absence of l-arginine, nNOS has been shown to generate superoxide (O2*). Superoxide, either directly or through its self-dismutation to H2O2, is likewise believed to be a cell-signaling agent. Because nNOS can generate NO* and O2*, we examined the activation of cellular signal transduction pathways in nNOS-transfected cells grown in the presence or absence of l-arginine. Spin trapping/EPR spectroscopy confirmed that stimulated nNOS-transfected cells grown in an l-arginine environment secreted NO* into the surrounding milieu. Production of NO* blocked Ca2+ ionophore-induced activation of the ERK1/2 through a mechanism involving inhibition of the Ras G-protein and Raf-1 kinase. In contrast, ERK activation was largely unaffected in nNOS-transfected cells grown in l-arginine-free media. Inhibition of nNOS-generated NO* with the competitive NOS inhibitor, NG-nitro-l-arginine methyl ester, in cells grown in l-arginine restored ERK1/2 activation to levels similar to that found when nNOS was activated in l-arginine-free media. These findings indicate that nNOS can differentially regulate the ERK signal transduction pathway in a manner dependent on the presence of l-arginine and the production of NO*.

    Funded by: NCRR NIH HHS: RR-12257; NIBIB NIH HHS: EB-2034; NIGMS NIH HHS: R25 GM55036

    The Journal of biological chemistry 2004;279;6;3933-40

  • Posttranscriptional regulation of neuronal nitric oxide synthase expression by IFN-gamma.

    Chesler DA, McCutcheon JA and Reiss CS

    Department of Biology, New York University, New York, NY 10003, USA.

    In this report, the mechanism through which interferon-gamma (IFN-gamma) regulates the expression of nitric oxide synthase (NOS-1) in neurons was examined. We have shown previously that IFN-gamma treatment of cells results in a two log inhibition of vesicular stomatitis virus (VSV) production. This inhibition of VSV replication is dependent both in vitro and in vivo on nitric oxide (NO) production by NOS-1. Furthermore, this effect is associated with the increased expression and activity of NOS-1 following IFN-gamma treatment. In vitro, exposure to IFN-gamma prior to infection with VSV is a prerequisite to establish an effective antiviral state, indicating the necessity for a priming event. Neuroblastoma cells (NB41A3) were treated with IFN-gamma or medium and examined for changes in NOS-1 protein and mRNA expression. NOS-1 protein expression was found to be increased after IFN-gamma treatment, and this was associated with increases in both neosynthesis and NOS-1 protein stability. NOS-1 transcription and mRNA levels were unaffected by IFN-gamma treatment. These data demonstrate that IFN-gamma regulates NOS-1 expression through posttranscriptional and posttranslational mechanisms.

    Funded by: NIAID NIH HHS: AI039029; NIDCD NIH HHS: DC003536; NINDS NIH HHS: NS039746

    Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 2004;24;2;141-9

  • Genetic association analysis of neuronal nitric oxide synthase gene polymorphism with tardive dyskinesia.

    Shinkai T, Ohmori O, Matsumoto C, Hori H, Kennedy JL and Nakamura J

    Department of Psychiatry, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, 807-8555, Japan. takahiro_shinkai@camh.net

    Possible involvement of oxidative stress in the pathophysiology of tardive dyskinesia (TD) has been proposed. Long-term administration of neuroleptics alters dopaminergic turnover, yielding the increase of the formation of reactive oxygen species (ROS), which may lead to TD through neuronal toxicity as a consequence of oxidative stress. In the present study, the relationship between TD and a polymorphism of the neuronal nitric oxide synthase (NOS1) gene whose reaction product, nitric oxide (NO), is involved in oxidative stress was studied in 171 Japanese patients with schizophrenia, including 41 patients meeting TD criteria. The C/T polymorphism in exon 29 of the NOS1 gene was genotyped using polymerase chain reaction (PCR) amplification followed by restriction enzyme digestion. No significant difference in genotype frequencies was detected between subjects with and without TD (chi2 = 1.54, df = 2, p = 0.46). In addition, there was no difference in allele frequencies (chi2 = 0.42, df = 1, p = 0.51). These results suggest that the NOS1 gene polymorphism may not confer increased susceptibility to TD, although more investigations on other populations are warranted.

    Neuromolecular medicine 2004;5;2;163-70

  • Intron 4 polymorphism of the endothelial nitric oxide synthase gene is associated with the development of lupus nephritis.

    Lee YH, Kim HJ, Rho YH, Choi SJ, Ji JD and Song GG

    Division of Rheumatology, Department of Internal Medicine, College of Medicine, Korea University, Seoul, Korea.

    The objective was to investigate whether the functional polymorphism of intron 4 in the endothelial nitric oxide synthase (eNOS) gene is associated with susceptibility to systemic lupus erythematosus (SLE) and its clinical features. The 27-bp repeat polymorphism in intron 4 of the eNOS gene was determined by polymerase chain reaction in 88 SLE patients and 95 healthy control subjects. Clinical manifestations were analysed in each patient and correlated with the genotypes. The genotype distribution of the intron 4 of the eNOS did not differ between SLE patients and control subjects (aa, ab, bb genotypes 0, 15, 73 versus 2, 19, 74 controls respectively, chi-squared = 2.21, 2 df, P = 0.331). In the lupus patients according to the intron 4 genotypes of the eNOS, there was no clinically significant difference in age at onset, anti-dsDNA titre, C3, C4 level, SLEDAI, SLICC/ACR Damage Index, or autoantibodies such as RF, anti-Ro, La, RNP, Sm, or phospholipid antibodies. However, renal involvement was higher in patients with ab genotypes than in those with bb genotypes (53% versus 26%), but it did not reach statistical significance (P = 0.062). Logistic regression showed that having the ab genotype was a significant risk factor for the development of lupus nephritis (odds ratio = 3.28, 95%CI: 1.04-10.2, P = 0.04). In conclusion, our data show that the eNOS ab genotypes may be associated with the development of lupus nephritis, suggesting individuals who carry the 'a' allele are more susceptible to lupus nephritis than those with the 'b' allele.

    Lupus 2004;13;3;188-91

  • Association analysis for the CA repeat polymorphism of the neuronal nitric oxide synthase (NOS1) gene and schizophrenia.

    Liou YJ, Tsai SJ, Hong CJ and Liao DL

    Schizophrenia research 2003;65;1;57-9

  • Expression of nitric oxide synthases in keratinocytes after UVB irradiation.

    Chang HR, Tsao DA, Wang SR and Yu HS

    Department of Biomedical Engineering, I-Shou University, Section 1, 1 Shiuecheng Road, Dashu Shiang, Kaohsiung County, Taiwan. huoy@isu.edu.tw

    The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.

    Archives of dermatological research 2003;295;7;293-6

  • Nitric oxide synthases and protein oxidation in the quadriceps femoris of patients with chronic obstructive pulmonary disease.

    Barreiro E, Gea J, Corominas JM and Hussain SN

    Room L3.05, 687 Pine Ave. West, Montreal, PQ, H3A 1A1 Canada.

    Skeletal muscle dysfunction contributes to poor exercise performance in patients with chronic obstructive pulmonary disease (COPD). Increased oxygen radicals and nitric oxide (NO) have been proposed as mechanisms. In this study, we assessed the levels of protein oxidation (carbonyl formation), lipid peroxidation (4-hydroxy-2-nonenal formation), catalase and Mn-superoxide dismutase (Mn-SOD) expressions, nitric oxide synthases (NOSs), and protein tyrosine nitration in quadriceps muscles of 12 patients with patients with COPD and 6 control subjects. Lipid peroxidation was elevated in muscles of patients with patients with COPD as compared with control subjects, but protein oxidation was not. Muscle Mn-SOD but not catalase protein expression was significantly higher (200%) in patients with patients with COPDas compared with control subjects. Expression of neuronal NOS and endothelial NOS isoforms did not differ between control subjects and patients with COPD, whereas no inducible NOS protein expression was detected in limb muscles of the two groups of subjects. In patients with COPD, neuronal NOS expression correlated negatively with the degree of the airway obstruction (%FEV1 predicted). 3-Nitrotyrosine levels were significantly elevated in muscles of patients with COPDas compared with control subjects, and correlated positively with nNOS protein levels. These results indicate the development of both oxidative and nitrosative stresses in the quadriceps of patients with COPD, suggesting their involvement in muscle dysfunction.

    American journal of respiratory cell and molecular biology 2003;29;6;771-8

  • Interplay of Cu,Zn superoxide dismutase and nitric oxide synthase in neurodegenerative processes.

    Rotilio G, Aquilano K and Ciriolo MR

    Department of Biology, University of Rome Tor Vergata, Rome, Italy. rotilio@bio.uniroma2.it

    Reactive oxygen and nitrogen species (ROS and RNS) have been extensively recognized as important signaling molecules implicated in physiological processes such as gene expression, cell differentiation and immune activation. Nevertheless, continuous production of these species may produce oxidative and/or nitrosative stress resulting in cell damage and ultimately leading to cell death. Due to the high oxygen consumption and relative poor antioxidant defense, the central nervous system is highly susceptible to ROS- and RNS-mediated toxicity. Actually, the oxidative and nitrosative stress have been implicated in the pathogenesis of neurodegeneration of a large variety of neurological disorders. This review will cover some aspects of the involvement of ROS- and RNS-mediated apoptotic processes occurring in cellular models of familial amyotrophic lateral sclerosis (FALS), in particular the cases associated with mutations in SOD1, the gene encoding Cu,Zn superoxide dismutase (Cu,Zn SOD). A possible role for proteasome in the inhibition of neurodegenerative process by balancing ROS and RNS species is envisaged on the basis of evidence provided by results obtained from studies on this experimental model.

    IUBMB life 2003;55;10-11;629-34

  • Mechanistic studies on the intramolecular one-electron transfer between the two flavins in the human neuronal nitric-oxide synthase and inducible nitric-oxide synthase flavin domains.

    Guan ZW, Kamatani D, Kimura S and Iyanagi T

    Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, 678-1279 Japan.

    Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.

    The Journal of biological chemistry 2003;278;33;30859-68

  • Immunocytochemical distribution of nitric oxide synthase in the human seminal vesicle: a light and electron microscopical study.

    Uckert S, Stanarius A, Stief CG, Wolf G, Jonas U and Machtens S

    Hannover Medical School, Department of Urology, 30625 Hannover, Germany.

    Although nitric oxide (NO) has been proven to be one of the most important non-adrenergic, non-cholinergic mediators in the control of human reproductive tract organs, to date information on the significance of NO-mediated signal transduction in the control of human seminal vesicle (SV) function is still sparse.()Recent investigations have underlined the significance of NO in the maintenance of sperm capacitation and viscosity of the seminal plasma as well as in the control of mammalian seminal vesicle smooth muscle tone. In order to further investigate the functional impact of NO on the regulation of normal SV function, we examined the distribution of NADPH-diaphorase (NADPH-d), endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) in the cellular anatomy of human SV by means of light and electron microscopical immunocytochemistry (LM, EM) in combination with the tyramide signal amplification technique. Human SV were obtained from 15 patients who had undergone surgery for pelvic malignancies (carcinoma of the prostate or urinary bladder). SV specimens were fixed, sectioned and examined by LM and EM for the presence of NAPDH-d, eNOS and nNOS using specific antibodies and advanced staining procedures. LM revealed a dense NADPH-d reaction in glandular epithelial structures, whereas no substantial labeling was detected in the fibromuscular stroma. EM showed that the NADPH-d reaction product was abundantly detectable attached to membranes of the endoplasmic reticulum, mitochondria and the nuclei of glandular epithelial cells. nNOS staining was found in nerve fibers branching within the SV tissue. eNOS staining was present in small vessels but was only observed to a minor degree in glandular and subglandular structures and the smooth muscle stroma. Our results support the hypothesis that human SV is a site of NO production. The distribution of NADPH-d may give rise to the speculation that NO is mainly involved in the regulation of SV secretory activity. The sparse correlation between NADPH-d-, eNOS- and nNOS-staining might hint at the existence of a previously unidentified NOS isoform in human SV.

    Urological research 2003;31;4;262-6

  • Inhibition of neural nitric oxide synthase attenuates the chloride secretory response to stroking in human jejunum.

    King BN, Haque SM, Stoner MC, Ellis ZM and Kellum JM

    Department of Surgery, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA 23298, USA.

    Background: Stroking of human jejunal mucosa induces serotonin release and a rise in short-circuit current (DeltaI(sc)). Nitric oxide is known to function as a nonadrenergic, noncholinergic neurotransmitter in response to neural serotonin receptor activation in the rat. We hypothesize that neural nitrergic mechanisms mediate the chloride secretory response to mucosal stroking in human jejunum.

    Methods: Segments of normal proximal jejunum were obtained from patients having gastric bypass surgery for obesity. Muscle-stripped segments of jejunum were mounted in Ussing chambers under short-circuit conditions. The neural nitric oxide synthase inhibitor, l-thiocitrulline, was added to experimental tissues. Mucosal stroking of control and experimental segments was then performed.

    Results: Pretreatment with l-thiocitrulline attenuated the short circuit rise seen after stroking in the experimental group when compared with the control (5.4 +/- 1.5 microA/cm(2) vs 8.0 +/- 1.6 microA/cm(2); P <.05, Student t test, paired data, n = 11), but did not affect baseline I(sc) before stroking. Serotonin released by stroking was not different in experimental versus control tissue.

    Conclusions: The significantly reduced DeltaI(sc) in the group pretreated with the neural nitric oxide synthase inhibitor suggests that nitric oxide liberated from enteric neurons participates in the chloride secretory response to stroking in human jejunum in vitro.

    Funded by: NIDDK NIH HHS: DK-43899

    Surgery 2003;134;2;255-9

  • NOS1 polymorphism is associated with atopy but not exhaled nitric oxide levels in healthy children.

    Ali M, Khoo SK, Turner S, Stick S, Le Souëf P and Franklin P

    Department of Paediatrics, University of Western Australia, Perth, Australia. niluwana@cyllene.uwa.edu.au

    Exhaled nitric oxide (FENO) is raised in atopy. The mechanism for this is unclear. The aim of this study was to investigate whether the number of AAT repeats in intron 20 of the NOS1 gene, recently associated with variations in FENO in adults with asthma and cystic fibrosis, was associated with the raised FENO in healthy atopic children. Eighty-seven healthy children (44 girls, 42 atopic, age range 6-18 years) underwent measurements of FENO, spirometry, airway responsiveness and skin prick testing. Genotyping was carried out to determine the number of AAT repeats. There was no association between the number of AAT repeats and FENO in either the whole sample of healthy children (n = 87) or in the subsample of healthy atopics (n = 42). However, a greater number of atopic children had two high repeat alleles compared with non-atopic children (33.3% vs. 13.6%, respectively, p = 0.03). This suggests that variations in the NOS1 gene may contribute to atopy without this relationship being reflected by FENO.

    Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology 2003;14;4;261-5

  • Expression of NOS1 and soluble guanylyl cyclase by human kidney epithelial cells: morphological evidence for an autocrine/paracrine action of nitric oxide.

    Jarry A, Renaudin K, Denis MG, Robard M, Buffin-Meyer B, Karam G, Buzelin F, Paris H, Laboisse CL and Vallette G

    INSERM U539, Faculty of Medicine, Nantes, France. ajarry@sante.univ-nantes.fr

    Background: Nitric oxide plays an important role in the kidney through effects on both renal hemodynamics and tubular functions. Tubular epithelial cells are thus a target for nitric oxide. However, as to whether tubular epithelial cells endogeneously produce nitric oxide under physiologic conditions in human kidney is currently unknown. The aim of the present study was to characterize and localize in situ the nitric oxide synthase (NOS) isoforms (NOS1, NOS2, and NOS3) expressed in human normal kidney, and soluble guanylyl cyclase, the well-known target for nitric oxide.

    Methods: Five complementary experimental approaches were used: (1) detection of NOS reductase activity by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry, (2) immunolocalization of the NOS isoforms (NOS1, NOS2, NOS3), (3) immunoblot analysis, (4) quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of NOS mRNA, and (5) measurement of NOS activity as the conversion rate of l-[14C]-arginine to l-[14C]-citrulline. In addition, in situ detection of soluble guanylyl cyclase was assessed by immunohistochemistry.

    Results: All these techniques led to consistent results showing that epithelial cells of most tubules along the human nephron exhibit functional NOS1, with a corticomedullary gradient observed both at the protein and mRNA levels. Moreover, epithelial cells expressing NOS1 also express soluble guanylyl cyclase, indicating that these cells possess the machinery for autocrine/paracrine effect of nitric oxide.

    Conclusion: The present study demonstrates that NOS1 is strongly expressed in most tubules of the human nephron and therefore invites to consider epithelial cells as one of the major source of nitric oxide in the human kidney under physiologic conditions.

    Kidney international 2003;64;1;170-80

  • Simultaneous analysis of five genetic risk factors in Polish patients with Alzheimer's disease.

    Styczynska M, Religa D, Pfeffer A, Luczywek E, Wasiak B, Styczynski G, Peplonska B, Gabryelewicz T, Golebiowski M, Kobrys M and Barcikowska M

    Department of Neurodegenerative Disorders, Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Str., 02-106 Warsaw, Poland. desperat@cmdik.pan.pl

    As Alzheimer's disease (AD) is a complex disease, we decided to estimate how previously reported genetic polymorphisms interact to increase the risk for the disease. Five candidate genes were chosen: apolipoprotein E (APOE), alpha 2-macroglobulin, cathepsin D, myeloperoxidase and nitric oxide synthase. Genotyping was performed in 100 cases of late-onset AD and 100 healthy controls. We found a highly significant difference in APOE epsilon 4 distribution between groups (P<0.005). However, no evidence of association for other studied loci was found. Cumulative analysis of five genetic polymorphisms was performed, but it also failed to reveal any synergistic effect of candidate genes greater than that caused by APOE itself. Our results suggest that the APOE epsilon 4 allele is the only known genetic risk factor for late-onset, sporadic AD.

    Neuroscience letters 2003;344;2;99-102

  • Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase.

    Watanabe Y, Song T, Sugimoto K, Horii M, Araki N, Tokumitsu H, Tezuka T, Yamamoto T and Tokuda M

    Department of Cell Physiology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kida-gun, Japan. yasuwata@kms.ac.jp

    Post-synaptic density-95 (PSD-95) is a neuronal scaffolding protein that associates with N -methyl-D-aspartate (NMDA) receptors and links them to intracellular signalling molecules. In neurons, neuronal nitric oxide synthase (nNOS) binds selectively to the second PDZ domain (PDZ2) of PSD-95, thereby exhibiting physiological activation triggered via NMDA receptors. We have demonstrated previously that Ca(2+)/calmodulin-dependent protein kinase IIalpha (CaM-K IIalpha) directly phosphorylates nNOS at residue Ser(847), and can attenuate the catalytic activity of the enzyme in neuronal cells [Komeima, Hayashi, Naito and Watanabe (2000) J. Biol. Chem. 275, 28139-28143]. In the present study, we examined how CaM-K II participates in the phosphorylation by analysing the functional interaction between nNOS and PSD-95 in cells. The results showed that PSD-95 directly promotes the nNOS phosphorylation at Ser(847) induced by endogenous CaM-K II. In transfected cells, this effect of PSD-95 required its dual palmitoylation and the PDZ2 domain, but did not rely on its guanylate kinase domain. CaM-K Ialpha and CaM-K IV failed to phosphorylate nNOS at Ser(847) in transfected cells. Thus PSD-95 mediates cellular trafficking of nNOS, and may be required for the efficient phosphorylation of nNOS at Ser(847) by CaM-K II in neuronal cells.

    The Biochemical journal 2003;372;Pt 2;465-71

  • Cellular compartmentalization of phosphorylated eIF2alpha and neuronal NOS in human temporal lobe epilepsy with hippocampal sclerosis.

    Petrov T, Rafols JA, Alousi SS, Kupsky WJ, Johnson R, Shah J, Shah A and Watson C

    Department of Anatomy and Cell Biology, School of Medicine, Wayne State University, 540 East Canfield Ave., Detroit, MI 48201, USA. tpetrov@med.wayne.edu

    Hippocampal sclerosis (HS) is the most common neuropathologic finding in patients with medically refractory temporal lobe epilepsy (TLE). The mechanisms resulting in neuronal injury and cell loss in HS are incompletely understood, but inhibition of protein synthesis may play a pivotal role in these processes. This study examined the relationships between two molecules known to be involved in reduced protein synthesis in animals subjected to traumatic brain injury. Translational initiation of protein synthesis is inhibited when 2alpha (eIF2alpha) is phosphorylated. Recently, nitric oxide (NO) has been shown to reduce protein synthesis by inducing phosphorylation of eIF2alpha. We performed immunocytochemistry for eIF2alpha(P) and histochemistry (NADPH-D reaction) for nitric oxide synthase (NOS) to determine the distribution of these molecules in hippocampi removed from patients undergoing anterior temporal lobectomy (ATL) for medically intractable TLE due to HS. The greatest number of eIF2alpha(P) positive cells was in the CA1 sector of the hippocampus, followed by the hilus of the dentate gyrus. NADPH-D positive neurons were observed most often in the hilus. Labeling in both instances involved neuronal cell body cytoplasm and varicose processes. Combination of both staining procedures revealed close relationships between differentially labeled neurons within the hilus. The results suggest that NO participates in the phosphorylation of eIF2alpha since we demonstrated that nNOS processes are closely related to eIF2alpha(P) positive cells. This may occur through activation of kinases such as PERK, which was recently revealed. In human, TLE protein synthesis inhibition may occur at the translational level since the eIF2alpha (P) labeling is cytoplasmic. Protein synthesis inhibition may contribute to neuronal cell injury and death in HS.

    Funded by: NINDS NIH HHS: NS 39860

    Journal of the neurological sciences 2003;209;1-2;31-9

  • Effects of sex and of gene variants in constitutive nitric oxide synthases on exhaled nitric oxide.

    Grasemann H, Storm van's Gravesande K, Buscher R, Drazen JM and Ratjen F

    Department of Pediatrics, University of Essen, Germany. hartmutg@hotmail.com

    Genetic factors may contribute to the variability of exhaled nitric oxide in healthy individuals. We studied exhaled nitric oxide and genetic variants in both neuronal and endothelial nitric oxide synthases in 105 healthy nonsmoking and smoking subjects. Genomic DNA was screened for a repeat polymorphism in intron 20 of the neuronal nitric oxide synthase gene and for the 894G/T mutation of the endothelial nitric oxide synthase gene. Exhaled nitric oxide was significantly higher in males than females among both nonsmokers (p < 0.0001) and smokers (p = 0.003). No association was found between exhaled nitric oxide and the endothelial nitric oxide synthase gene variant. However, healthy nonsmoking females with greater numbers of repeats (i.e., both alleles with 12 or more repeats) in neuronal nitric oxide synthase had significantly lower nitric oxide levels than did females with fewer numbers of repeats (i.e., at least one allele with fewer than 12 repeats) (13.6 +/- 1.6 versus 19.4 +/- 1.6 ppb, p = 0.02). No association was found between exhaled nitric oxide and neuronal nitric oxide synthase genotype in males. These data suggest that variants in the neuronal nitric oxide synthase gene contribute to the variability of airway nitric oxide concentrations in healthy females.

    American journal of respiratory and critical care medicine 2003;167;8;1113-6

  • Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase.

    Guan ZW and Iyanagi T

    Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678-1279, Japan.

    The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.

    Archives of biochemistry and biophysics 2003;412;1;65-76

  • Conservation of a Pumilio-Nanos complex from Drosophila germ plasm to human germ cells.

    Jaruzelska J, Kotecki M, Kusz K, Spik A, Firpo M and Reijo Pera RA

    Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California at San Francisco, San Francisco, CA 94143-0546, USA.

    Germ cells are the cells which ultimately give rise to mature sperm and eggs. In model organisms such as flies and worms, several genes that are required for formation and maintenance of germ cells have been identified and their interactions are rapidly being delineated. By contrast, little is known of the genes required for development of human germ cells and it is not clear whether findings from model organisms will translate into knowledge of human germ cell development, especially given observations that reproductive pathways may evolve more rapidly than somatic pathways. The Pumilio and Nanos genes have been especially well-characterized in model organisms and encode proteins that interact and are required for development of germ stem cells in one or both sexes. Here we report the first characterization of a mammalian Nanos homolog, human NANOS1 ( NOS1). We show that human NOS1 protein interacts with the human PUMILIO-2 (PUM2) protein via highly conserved domains to form a stable complex. We also show that in men, the NOS1 and PUM2 proteins are particularly abundant in germline stem cells. These observations mirror those in distant species and document for the first time a conserved protein-protein interaction in germ cells from flies to humans. These results suggest the possibility that the interaction of PUM2 and NOS1 may play a conserved role in germ cell development and maintenance in humans as in model organisms.

    Development genes and evolution 2003;213;3;120-6

  • Endothelial nitric oxide synthase polymorphism in preeclampsia.

    Häkli T, Romppanen EL, Hiltunen M, Helisalmi S, Punnonen K and Heinonen S

    Department of Obstetrics and Gynecology, Kuopio University and University Hospital, Kuopio, Finland.

    Objective: We wished to determine whether genetic variability in the gene encoding endothelial nitric oxide synthase (eNOS) modifies individual susceptibility to the development of preeclampsia.

    Methods: The study involved 132 preeclamptic and 113 healthy control pregnant women who were genotyped for the Glu298Asp polymorphism in the eNOS gene. Chi(2) analysis was used to assess genotype and allele frequency differences between preeclamptic women and controls.

    Results: A statistically similar allelic distribution of eNOS Glu298Asp polymorphism was observed in the two groups, with the frequency of the variant G allele being 74.6% in the preeclampsia group and 67.7% in the control group (P = .091; odds ratio 1.40, 95% confidence interval 0.95, 2.01). Accordingly, the genotype distribution of the NOS polymorphism in the preeclamptic and control groups was found to be similar (P = .233).

    Conclusion: These genotype data in subjects from eastern Finland were not suggestive of an important contribution of the Glu298Asp polymorphism in the NOS gene on preeclampsia across populations. However, the observed association between the G allele and disease risk, of borderline significance, may imply that other polymorphism(s) in the gene may modify disease risk.

    Journal of the Society for Gynecologic Investigation 2003;10;3;154-7

  • Transcription of different exons 1 of the human neuronal nitric oxide synthase gene is dynamically regulated in a cell- and stimulus-specific manner.

    Boissel JP, Zelenka M, Gödtel-Armbrust U, Feuerstein TJ and Förstermann U

    Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany.

    An extensive screening of the human neuronal nitric oxide synthase (nNOS) mRNAs in various human tissues and cell lines unraveled an extreme complexity in the transcription of this gene. Using 5' rapid amplification of cDNA ends (5'-RACE), ten different exons 1 (named 1a-1l) were identified. They were spliced in a cell-specific manner to a common exon 2, which bears the translational start site. Three first exons (1d, 1g and 1f) were used predominantly for the transcription of the nNOS gene (146 out of 197 5'-RACE clones contained these exons). Exon 1k was found alone, but in many instances was interposed between exons 1b, 1d, 1g, 1i or 1j and the common exon 2. In addition to the cell-specific heterogeneity of human nNOS transcripts, nNOS is highly regulated at the transcriptional level. In resting A673 neuroepithelioma cells, the prevalent nNOS transcript was the exon 1g mRNA (with minor expression of exons 1d+1k and exon 1f mRNAs). When the cells were treated with dibutyryl-cAMP, nNOS mRNA was markedly upregulated. This upregulation was solely due to an increase in exon 1f mRNA, while the expression of the other mRNA species remained unchanged. Human HaCat keratinocyte-like cells expressed the exon 1i+1k and 1i nNOS transcripts under basal conditions. When stimulated with epidermal growth factor, only the exon 1i+1k transcript was upregulated. Although these nNOS transcripts do not differ in their translated region, the various mRNAs may trigger post-transcriptional effects such as changes in mRNA stability and translation efficiency.

    Biological chemistry 2003;384;3;351-62

  • Translational regulation of human neuronal nitric-oxide synthase by an alternatively spliced 5'-untranslated region leader exon.

    Newton DC, Bevan SC, Choi S, Robb GB, Millar A, Wang Y and Marsden PA

    Renal Division and the Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario M5S 1A8, Canada.

    Expression of the neuronal nitric-oxide synthase (nNOS) mRNA is subject to complex cell-specific transcriptional regulation, which is mediated by alternative promoters. Unexpectedly, we identified a 89-nucleotide alternatively spliced exon located in the 5'-untranslated region between exon 1 variants and a common exon 2 that contains the translational initiation codon. Alternative splicing events that do not affect the open reading frame are distinctly uncommon in mammals; therefore, we assessed its functional relevance. Transient transfection of reporter RNAs performed in a variety of cell types revealed that this alternatively spliced exon acts as a potent translational repressor. Stably transfected cell lines confirmed that the alternatively spliced exon inhibited translation of the native nNOS open reading frame. Reverse transcription-PCR and RNase protection assays indicated that nNOS mRNAs containing this exon are common and expressed in both a promoter-specific and tissue-restricted fashion. Mutational analysis identified the functional cis-element within this novel exon, and a secondary structure prediction revealed that it forms a putative stem-loop. RNA electrophoretic mobility shift assay techniques revealed that a specific cytoplasmic RNA-binding complex interacts with this motif. Hence, a unique splicing event within a 5'-untranslated region is demonstrated to introduce a translational control element. This represents a newer model for the translational control of a mammalian mRNA.

    The Journal of biological chemistry 2003;278;1;636-44

  • Does endothelial nitric oxide synthase gene variation play a role in the occurrence of hypertension in pregnancy?

    Grandone E, Colaizzo D, Martinelli P, Pavone G, Errico M, Vecchione G and Margaglione M

    Atherosclerosis and Thrombosis Unit, Division of Obstetrics and Gynaecology, I.R.C.C.S., "Casa Sollievo della Sofferenza", S. Giovanni Rotondo (FG), Italy. ate.tro@operapadrepio.it

    Objective: Nitric oxide is suggested to play a role in the development of preeclampsia.

    Methods: We studied 61 patients with gestational hypertension (GH), 77 with GH and significant proteinuria (urine protein excretion > or = 300 mg/24 h), 82 with essential hypertension (EH) and 188 normotensive women with at least one normal pregnancy.

    A polymorphism within the constitutive endothelial nitric oxide synthase (ecNOS) gene in various types of hypertension in pregnancy was explored.

    Results: Allelic and genotypic frequencies did not differ between controls and case groups. A significant difference was observed between the frequency of the rare allele in GH patients and that in EH group (chi2: 4.47, P <.04). This difference approximated the significance when GH subjects with or without proteinuria were grouped (chi2 square: 3.33; P =.068). Cigarette smoking or gravidity did not interact with the ecNOS polymorphism in identifying different types of hypertension in this setting.

    Conclusion: Our findings argue against an association between ecNOS polymorphism and preeclampsia and support the hypothesis for a different pathogenesis of GH in respect to EH.

    Hypertension in pregnancy 2003;22;2;149-55

  • Analysis of nitric oxide synthase genes in cluster headache.

    Sjöstrand C, Modin H, Masterman T, Ekbom K, Waldenlind E and Hillert J

    Department of Neurology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden. christina.sjostrand@neurotek.ki.se

    The aetiology of cluster headache is still not yet completely understood, but the potential relevance of genetic factors has been recognized during recent years. Nitric oxide (NO) plays a critical role in the regulation of vasodilation, neurotransmission, inflammation and many other events throughout the body. NO also appears to be an important mediator of vascular headache pathophysiology. In this study we have performed an association analysis of five polymorphic microsatellite markers in the three different NO synthase (NOS) genes; nNOS (NOS1), iNOS (NOS2A) and eNOS (NOS3). Ninety-one cluster headache patients diagnosed according to International Headache Society criteria and 111 matched controls were studied. Phenotype and allele frequencies were similarly distributed in patients and controls except for an iNOS (NOS2A) pentanucleotide repeat allele which was significantly more common in controls. We observed a higher phenotype frequency of this allele in our control group compared with rates in control groups of other studies, whereas the frequency in our patients was similar to that in controls from previous reports. Thus, we conclude that it is unlikely that genetic variations within the NOS genes contribute greatly to cluster headache susceptibility.

    Cephalalgia : an international journal of headache 2002;22;9;758-64

  • Expression of nitric oxide synthase isoforms in human pregnant myometrium at term.

    Bao S, Rai J and Schreiber J

    Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. bao@borcim.wustl.edu

    Objective: To determine whether nitric oxide synthase isoforms are expressed in human myometrium, to define their localization, and to examine the possibility that one or more may play a role in initiating labor.

    Methods: Human myometrial biopsies were collected from term laboring and nonlaboring patients. The mRNA expression was assessed using in situ hybridization. The relative expression levels were also assessed by Affymetrix GeneChip hybridization. The protein expression was determined using Western blot analysis, and localization was shown using immunostaining.

    Results: Both inducible nitric oxide synthase and brain nitric oxide synthase were expressed in myometrial cells and blood vessel endothelial cells. Endothelial nitric oxide synthase was expressed exclusively in blood vessel endothelial cells. There were no differences in distribution or quantity of nitric oxide synthase expression between term labor and nonlabor myometrium.

    Conclusions: All three isotypes of nitric oxide synthases were expressed in human pregnant term uterus. Their expression did not change in the myometrium of the laboring uterus.

    Funded by: NICHD NIH HHS: R03 HD35964

    Journal of the Society for Gynecologic Investigation 2002;9;6;351-6

  • Further immunohistochemical evidence for impaired NO signaling in the hypothalamus of depressed patients.

    Bernstein HG, Heinemann A, Krell D, Mawrin C, Bielau H, Danos P, Diekmann S, Keilhoff G, Bogerts B and Baumann B

    Department of Psychiatry, University of Magdeburg, D-39120 Magdeburg, Germany. Hans-Gert.Bernstein@medizin.uni-magdeburg.de

    The cellular expression of nitric oxide synthase (NOS) was studied in neurons of the Nuc. suprachiasmaticus (SCN) of depressed patients and matched controls. The number of NOS-immunoreactive SCN neurons was significantly reduced in depression. We conclude that affective disorders are accompanied by impaired hypothalamic NO signaling.

    Annals of the New York Academy of Sciences 2002;973;91-3

  • Polymorphisms of the IL-1beta and IL-1beta-inducible genes in ulcerative colitis.

    Nohara H, Saito Y, Higaki S, Okayama N, Hamanaka Y, Okita K and Hinoda Y

    Department of Gastroenterology and Hepatology, Yamaguchi University School of Medicine, 1-1-1 Minami-kogushi, Ube 755-8505, Japan.

    Background: Ulcerative colitis (UC) is a chronic disorder of undetermined etiology, but a genetic predisposition to UC is well recognized. Among cytokines induced in UC, interleukin 1 (IL-1) appears to have a central role because of its immunological upregulatory and proinflammatory activities. The aim of this study was to assess whether UC is associated with polymorphisms of the IL-1beta gene and three additional genes inducible with IL-1beta in Japanese subjects.

    Methods: A total of 96 patients with UC and 106 ethnically matched controls were genotyped at polymorphic sites in IL-1beta, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), and inducible nitric oxide synthase (iNOS) genes, using polymerase chain reaction (PCR)-based methods.

    Results: There was no significant difference in genotype distributions of IL-1beta, MMP-1, MMP-3, and iNOS genes between controls and UC patients in a Japanese population. Also, no significant association of those polymorphisms with various clinical parameters of the patients was found. However, concerning association of age at onset with clinical factors in UC, the frequency of pancolitis was significantly higher in UC patients with age at onset being less than 30 years than in those more than 30 years of age (P = 0.049).

    Conclusions: No association of the IL-1beta and three IL-1beta-inducible gene polymorphisms with UC was observed in a Japanese population.

    Journal of gastroenterology 2002;37 Suppl 14;107-10

  • Assembly and activation of heme-deficient neuronal NO synthase with various porphyrins.

    Bender AT, Kamada Y, Kleaveland PA and Osawa Y

    Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, MI 48109-0632, USA.

    The heme prosthetic group of NO synthase is critical for catalytic activity as well as assembly of the enzyme to the native homodimeric form. In the current study, we examined if structurally different metal porphyrins could substitute for the native heme prosthetic group in neuronal NO synthase (nNOS) with regard to assembly and catalysis. We established, with the use of a recently developed in vitro method that functionally reconstitutes heme-deficient apo-nNOS, that Fe-mesoporphyrin IX or Fe-deuteroporphyrin IX can substitute for heme and lead to assembly of a functional nNOS, albeit with lower activity. Fe-protoporphyrin IX dimethyl ester or the metal free protoporphyrin IX, however, lead to minimal assembly of nNOS. Protoporphyrin IX compounds where the native Fe was substituted with Zn, Mn, Co, or Sn lead to assembly of nNOS, but no detectable NO was synthesized in the presence of NADPH and L-arginine. Thus, the presence of the metal and propionic acid groups, but not the vinyl moieties, of heme are important structural features in assembly of nNOS. These studies establish that the mechanism of assembly and catalysis of nNOS can be probed with structurally diverse metal porphyrins.

    Funded by: NIEHS NIH HHS: ES08365

    Journal of inorganic biochemistry 2002;91;4;625-34

  • Interaction of neuronal nitric oxide synthase with alpha1-adrenergic receptor subtypes in transfected HEK-293 cells.

    Pupo AS and Minneman KP

    Department of Pharmacology, Emory University, Atlanta, GA 30322, USA. aspupo@ibb.unesp.br

    Background: The C-terminal four amino acids (GEEV) of human alpha1A-adrenergic receptors (ARs) have been reported to interact with the PDZ domain of neuronal nitric oxide synthase (nNOS) in a yeast two-hybrid system. The other two alpha1-AR subtypes have no sequence homology in this region, raising the possibility of subtype-specific protein-protein interactions.

    Results: We used co-immunoprecipitation and functional approaches with epitope-tagged alpha1-ARs to examine this interaction and the importance of the C-terminal tail. Following co-transfection of HEK-293 cells with hexahistidine/Flag (HF)-tagged alpha1A-ARs and nNOS, membranes were solubilized and immunoprecipitated with anti-FLAG affinity resin or anti-nNOS antibodies. Immunoprecipitation of HFalpha1A-ARs resulted in co-immunoprecipitation of nNOS and vice versa, confirming that these proteins interact. However, nNOS also co-immunoprecipitated with HFalpha1B- and HFalpha1D-ARs, suggesting that the interaction is not specific to the alpha1A subtype. In addition, nNOS co-immunoprecipitated with each of the three HFalpha1-AR subtypes which had been C-terminally truncated, suggesting that this interaction does not require the C-tails; and with Flag-tagged beta1- and beta2-ARs. Treatment of PC12 cells expressing HFalpha1A-ARs with an inhibitor of nitric oxide formation did not alter norepinephrine-mediated activation of mitogen activated protein kinases, suggesting nNOS is not involved in this response.

    Conclusions: These results show that nNOS does interact with full-length alpha1A-ARs, but that this interaction is not subtype-specific and does not require the C-terminal tail, raising questions about its functional significance.

    BMC pharmacology 2002;2;17

  • Complex regulation of human neuronal nitric-oxide synthase exon 1c gene transcription. Essential role of Sp and ZNF family members of transcription factors.

    Saur D, Seidler B, Paehge H, Schusdziarra V and Allescher HD

    Department of Internal Medicine II, Technische Universität München, Germany. Saur@lrz.tu-muenchen.de

    Neuronal nitric-oxide synthase (nNOS) is expressed in a variety of human tissues and shows a complex transcriptional regulation with the presence of nine alternative first exons (1a-1i) resulting in nNOS transcripts with differing 5'-untranslated regions. We previously demonstrated that nNOS exon 1c, one of the predominant transcripts in the human gastrointestinal tract, is driven by a separate promoter (Saur, D., Paehge, H., Schusdziarra, V., and Allescher, H. D. (2000) Gastroenterology 118, 849-858). The present study focused on the quantitative expression of nNOS first exon variants in different human tissues and the characterization of the basal nNOS exon 1c promoter. In human brain, skeletal muscle, colon, and TGW-nu-I neuroblastoma cells, first exon expression patterns were analyzed by quantitative real-time reverse transcription-PCR. In these tissues/cells exon 1c was one of the most abundant first exons of nNOS. By transient transfections of TGW-nu-I and HeLa cells with reporter plasmids containing a series of 5' and 3' deletions in the exon 1c regulatory region, the minimal TATA-less promoter was localized within 44 base pairs. Gel mobility shift assays of this cis-regulatory region revealed a high complexity of the basal promoter with a cooperative binding of several transcription factors, like Sp and ZNF family members. When the Sp binding site of the minimal promoter construct was mutated, promoter activity was completely abolished in both cell lines, whereas mutation of the common binding site of ZNF76 and ZNF143 resulted in a decrease of 53% in TGW-nu-I and 37% in HeLa cells. In Drosophila Schneider cells expression of Sp1, the long Sp3 isoform, ZNF76 and ZNF143 potently transactivated the nNOS exon 1c promoter. These results identify the critical regulatory region for the nNOS exon 1c basal promoter and stress the functional importance of multiple protein complexes involving Sp and ZNF families of transcription factors in regulating nNOS exon 1c transcription.

    The Journal of biological chemistry 2002;277;28;25798-814

  • Neuronal nitric oxide synthase ligand and protein vibrations at the substrate binding site. A study by FTIR.

    Ingledew WJ, Smith SM, Salerno JC and Rich PR

    School of Biology, University of St. Andrews, St. Andrews KY16 9JF, UK. wji@st-andrews.ac.uk

    Improvements in sensitivity and data processing of Fourier transform infrared (FTIR) spectroscopy enable it to be used to detect changes in protein structure at the atomic level. This paper reports a study of neuronal nitric oxide synthase (nNOS) by FTIR difference spectroscopy in the 1000-2500 cm(-1) range where vibrational bands of ligands, prosthetic groups, and protein and amino acid side chains are found. We have exploited the photolyzable CO compound of the ferrous heme of nNOS to produce light-induced CO photolysis difference spectra and to compare spectra after hydrogen/deuterium exchange. In (reduced) minus (reduced plus CO) difference spectra, negative bands at 1931 and 1907 cm(-1) are observed due to photolysis of multiple forms of ferrous heme-ligated CO, similar to those observed by resonance Raman spectroscopy [Wang et al. (1997) Biochemistry 36, 4595-4606]. Photolysis of the ferrous heme CO compound is accompanied by hitherto unreported changes in the 1000-2000 cm(-1) region that arise from changes of protein backbone, substrate, amino acid side chain, and cofactor vibrations. Preliminary assignments of vibrations are made on the basis of frequencies and the effects of hydrogen/deuterium exchange, and in the light of known atomic structures.

    Biochemistry 2002;41;26;8377-84

  • Cardiac nitric oxide synthase 1 regulates basal and beta-adrenergic contractility in murine ventricular myocytes.

    Ashley EA, Sears CE, Bryant SM, Watkins HC and Casadei B

    University Department of Cardiovascular Medicine, John Radcliffe Hospital, Oxford, UK. euan.ashley@cardiov.ox.ac.uk

    Background: Evidence indicates that myocardial NO production can modulate contractility, but the source of NO remains uncertain. Here, we investigated the role of a type 1 NO synthase isoform (NOS1), which has been recently localized to the cardiac sarcoplasmic reticulum, in the regulation of basal and beta-adrenergic myocardial contraction.

    Contraction was assessed in left ventricular myocytes isolated from mice with NOS1 gene disruption (NOS1(-/-) mice) and their littermate controls (NOS1(+/+) mice) at 3 stimulation frequencies (1, 3, and 6 Hz) in basal conditions and during beta-adrenergic stimulation with isoproterenol (2 nmol/L). In addition, we examined the effects of acute specific inhibition of NOS1 with vinyl-L-N-5-(1-imino-3-butenyl)-L-ornithine (L-VNIO, 500 micromol/L). NOS1((-/-)) myocytes exhibited greater contraction at all frequencies (percent cell shortening at 6 Hz, 10.7+/-0.92% in NOS1(-/-) myocytes versus 7.21+/-0.8% in NOS1(+/+) myocytes; P<0.05) with a flat frequency-contraction relationship. Time to 50% relaxation was increased in NOS1(-/-) myocytes at all frequencies (at 6 Hz, 26.53+/-1.4 ms in NOS1(-/-) myocytes versus 21.27+/-1.3 ms in NOS1(+/+) myocytes; P<0.05). L-VNIO prolonged time to 50% relaxation at all frequencies (at 6 Hz, 21.28+/-1.7 ms in NOS1(+/+) myocytes versus 26.45+/-1.4 ms in NOS1(+/+)+L-VNIO myocytes; P<0.05) but did not significantly increase basal contraction. However, both NOS1(-/-) myocytes and NOS1(+/+) myocytes treated with L-VNIO showed a greatly enhanced contraction in response to beta-adrenergic stimulation (percent increase in contraction at 6 Hz, 25.2+/-10.8 in NOS1(+/+) myocytes, 68.2+/-11.2 in NOS1(-/-) myocytes, and 65.1+/-13.2 in NOS1(+/+)+L-VNIO myocytes; P<0.05).

    Conclusions: NOS1 disruption enhances basal contraction and the inotropic response to beta-adrenergic stimulation in murine ventricular myocytes. These findings indicate that cardiac NOS1-derived NO plays a significant role in the autocrine regulation of myocardial contractility.

    Circulation 2002;105;25;3011-6

  • Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON.

    Jaffrey SR, Benfenati F, Snowman AM, Czernik AJ and Snyder SH

    Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    The specificity of the reactions of nitric oxide (NO) with its neuronal targets is determined in part by the precise localizations of neuronal NO synthase (nNOS) within the cell. The targeting of nNOS is mediated by adapter proteins that interact with its PDZ domain. Here, we show that the nNOS adapter protein, CAPON, interacts with synapsins I, II, and III through an N-terminal phosphotyrosine-binding domain interaction, which leads to a ternary complex comprising nNOS, CAPON, and synapsin I. The significance of this ternary complex is demonstrated by changes in subcellular localization of nNOS in mice harboring genomic deletions of both synapsin I and synapsin II. These results suggest a mechanism for specific actions of NO at presynaptic sites.

    Funded by: NIDA NIH HHS: DA00074, K05 DA000074; NIMH NIH HHS: MH-18501, R01 MH018501, R37 MH018501; Telethon: 1131

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;5;3199-204

  • Arginase in glomerulonephritis.

    Waddington SN

    Gene Therapy Group, Section of Cell and Molecular Biology, Faculty of Medicine, Imperial College School of Science Technology and Medicine, Sir Alexander Fleming Building, Imperial College Road, London, England SW7 2AZ, UK. s.waddington@ic.ac.uk

    l-Arginine is converted to nitric oxide and citrulline by the enzyme nitric oxide synthase (NOS). Its in vivo inhibition has led to the revelation of a multitude of diverse, often conflicting functions in the inflammatory melee. l-Arginine is also converted to ornithine and urea by the enzyme arginase as a part of the hepatic urea cycle. However, a more holistic interpretation of the two pathways and the associated metabolism (summarized in Fig. 1) has led to its reassessment as a pathologically significant enzyme. This is reflected by the continued increase over the past five years of the number of publications discussing both nitric oxide and arginase. The strong association between inflammation and high arginase and NOS activity is epitomized by immune complex-induced glomerulonephritis and other glomerulonephritides. Arginase is encoded by two recently discovered genes (Arginase I and Arginase II). There is now substantial evidence for an interaction between both arginase isoforms and all three NOS isoforms in pathological situations. This review considers the relationship between Arginases I and II and the inflammation-associated isoform of NOS called NOS II. In particular, it consolidates the current understanding of arginase and associated metabolic pathways, and highlights some of the issues that are often overlooked in such studies.

    Kidney international 2002;61;3;876-81

  • Interactions between inducible nitric oxide synthase and heme oxygenase-1 in glomerulonephritis.

    Datta PK, Gross EJ and Lianos EA

    Division of Nephrology, Department of Medicine, UMDNJ-Robert Wood Johnson Medical School, P.O. Box 19, MEB 412,New Brunswick, NJ, USA.

    Background: In anti-glomerular basement membrane (GBM) nephritis, inducible nitric oxide synthase (iNOS) and heme oxygenase (HO-1) are co-induced. Moreover, in glomerular mesangial cells iNOS-derived nitric oxide (NO) production stimulates HO-1 while HO-1 activation reduces iNOS expression/activity. These observations prompted us to explore regulatory interactions between iNOS and HO-1 in anti-GBM nephritis.

    Methods: Rats with anti-GBM nephritis were pretreated with the iNOS inhibitor l-N6-(1-iminoethyl) lysine (L-NIL) or with the HO-1 inducer hemin. Glomerular HO-1 levels were assessed by Western blot analysis. iNOS activity was assessed by calculating conversion of l-arginine to l-citrulline.

    Results: iNOS inhibition reduced glomerular HO-1 levels without altering the inflammatory response to anti-GBM antibody induced injury. Induction of HO-1 reduced glomerular iNOS activity.

    Conclusions: In anti-GBM nephritis iNOS up-regulates HO-1 presumably via high output NO production. Suprainduction of HO-1 attenuates iNOS activity. This negative feedback interaction points to HO-1 as a target for pharmacologic manipulation to reduce activity of prooxidant heme containing enzymes such as iNOS.

    Kidney international 2002;61;3;847-50

  • Nasal nitric oxide levels in cystic fibrosis patients are associated with a neuronal NO synthase (NOS1) gene polymorphism.

    Grasemann H, Storm van's Gravesande K, Gärtig S, Kirsch M, Büscher R, Drazen JM and Ratjen F

    Children's Hospital, University of Essen, Essen, Germany.

    Nitric oxide (NO) plays an important role in a number of physiological processes in the airways, including host defense. Although the exact cellular and molecular source of the NO formation in airways is unknown, there is recent evidence that neuronal NO synthase (NOS1) contributes significantly to NO in the lower airways of cystic fibrosis (CF) patients. NOS1 protein has been shown to be expressed in nasal epithelium, suggesting an involvement of NOS1-derived NO in upper airway biology. We here hypothesized that nasal NO concentrations in CF patients are related to genotype variants in the NOS1 gene. Measurements of nasal NO concentration and pulmonary function were performed in 40 clinically stable CF patients. Genomic DNA from all patients was screened for an intronic AAT-repeat polymorphism in the NOS1 gene using polymerase chain reaction and simple sequence length polymorphism (SSLP) analysis. The allele size at that locus was significantly (P = 0.001) associated with upper airway NO. Mean (+/- SD) nasal NO concentrations were 40.5 +/- 5.2 ppb in CF patients (n = 12) with high repeat numbers (i.e., both alleles > or =12 repeats) and 72.6 +/- 7.4 ppb in patients (n = 28) with low repeat numbers (i.e., at least one allele <12 repeats). Furthermore, in the group of CF patients harboring NOS1 genotypes associated with low nasal NO, colonization of airways with P. aeruginosa was significantly more frequent than in patients with NOS1 genotypes associated high nasal NO concentrations (P = 0.0022). We conclude that (1) the variability in CF nasal NO levels are related to naturally occurring variants in the NOS1 gene, and (2) that nasal NOS1-derived NO affects the susceptibility of CF airways to infection with P. aeruginosa.

    Nitric oxide : biology and chemistry 2002;6;2;236-41

  • An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells.

    Wang Y, Newton DC, Miller TL, Teichert AM, Phillips MJ, Davidoff MS and Marsden PA

    Renal Division and Department of Medicine, St. Michael's Hospital and University of Toronto, Toronto, Ontario, Canada.

    Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of beta-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. beta-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P(450)scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, beta-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of beta-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, beta-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of the male gonad suggests an important role in the regulation of testosterone release and represents an intriguing model with which to dissect the molecular basis of Leydig cell-specific gene expression.

    The American journal of pathology 2002;160;1;369-80

  • Allelic association of the neuronal nitric oxide synthase (NOS1) gene with schizophrenia.

    Shinkai T, Ohmori O, Hori H and Nakamura J

    Department of Psychiatry, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-ku, Kitakyushu, 807-8555, Japan.

    Nitric oxide (NO) has been identified as a widespread and multifunctional biological messenger molecule in the central nervous system (CNS), with possible roles in neurotransmission, neurosecretion, synaptic plasticity, and tissue injury in many neurological disorders, including schizophrenia. Neuronal NO is widely produced in the brain from L-arginine catalyzed by neuronal NO synthase (NOS1). We therefore hypothesized that the NOS1 gene may play a role in the pathophysiology of schizophrenia. In the present study, we examined the genetic association between a novel single nucleotide polymorphism (SNP: a C-->T transition located 276 base pairs downstream from the translation termination site) of the human NOS1 gene, which is located in chromosome 12q24, and schizophrenia (215 Japanese patients with schizophrenia and 182 healthy controls). The allele frequencies of the polymorphism in exon 29 of the NOS1 gene differed significantly between patients with schizophrenia and controls (chi(2) = 20.10, df = 1, P = 0.000007; relative risk = 1.92; 95% confidence interval = 1.44-2.55). Our results suggest that the NOS1 gene polymorphism may confer increased susceptibility to schizophrenia.

    Molecular psychiatry 2002;7;6;560-3

  • Binding of neuronal nitric-oxide synthase (nNOS) to carboxyl-terminal-binding protein (CtBP) changes the localization of CtBP from the nucleus to the cytosol: a novel function for targeting by the PDZ domain of nNOS.

    Riefler GM and Firestein BL

    Department of Cell Biology and Neuroscience, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854-8082, USA.

    Recent work suggests a role for PDZ domains in the targeting of binding partners to specific sites in the cell. To identify whether the PDZ domain of neuronal nitric-oxide synthase (nNOS) can play such a role, we performed affinity chromatography of brain extract with the nNOS PDZ domain. We identified the carboxyl-terminal-binding protein (CtBP), a phosphoprotein first identified as a binding partner to adenovirus E1A, as a nNOS binding partner. CtBP interacts with the PDZ domain of nNOS, and this interaction can be competed with peptide that binds to the PDZ peptide-binding site. In addition, binding of CtBP to nNOS is dependent on its carboxyl-terminal sequence -DXL, residues conserved between species that fit the canonical sequence for nNOS PDZ binding. Immunoprecipitation studies show that CtBP and nNOS associate in the brain. When CtBP is expressed in Madin-Darby canine kidney cells, its distribution is primarily nuclear; however, when CtBP is co-expressed with nNOS, its localization becomes more cytosolic. This change in CtBP localization does not occur when its carboxyl-terminal nNOS PDZ binding motif is mutated or when CtBP is co-expressed with postsynaptic density 95, another PDZ domain-containing protein. Taken together, our data suggest a new function for nNOS as a regulator of CtBP nuclear localization.

    The Journal of biological chemistry 2001;276;51;48262-8

  • Association of neuronal nitric oxide synthase (nNOS) with alpha1-syntrophin at the sarcolemma.

    Miyagoe-Suzuki Y and Takeda SI

    Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187-8502, Japan.

    alpha1-syntrophin is a PDZ-containing dystrophin-associated protein, expressed predominantly in striated muscle and brain. alpha1-syntrophin null mice generated by gene targeting technique showed no overt muscular dystrophic phenotype. Though other dystrophin-associated proteins were localized at the sarcolemma, neuronal nitric oxide synthase (nNOS) was selectively lost from the membrane fraction but remained in the cytoplasm. Thus, the alpha1-syntrophin null mice are useful in the elucidation of the functional importance of nNOS targeting at the sarcolemma. In addition, the mice would facilitate identification of other signaling molecules, which are targeted to dystrophin complex via interaction with alpha1-syntrophin.

    Microscopy research and technique 2001;55;3;164-70

  • The plasmamembrane calmodulin-dependent calcium pump: a major regulator of nitric oxide synthase I.

    Schuh K, Uldrijan S, Telkamp M, Rothlein N and Neyses L

    Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany.

    The plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA) (Shull, G.E., and J. Greeb. 1988. J. Biol. Chem. 263:8646-8657; Verma, A.K., A.G. Filoteo, D.R. Stanford, E.D. Wieben, J.T. Penniston, E.E. Strehler, R. Fischer, R. Heim, G. Vogel, S. Mathews, et al. 1988. J. Biol. Chem. 263:14152-14159; Carafoli, E. 1997. Basic Res. Cardiol. 92:59-61) has been proposed to be a regulator of calcium homeostasis and signal transduction networks of the cell. However, little is known about its precise mechanisms of action. Knock-out of (mainly neuronal) isoform 2 of the enzyme resulted in hearing loss and balance deficits due to severe inner ear defects, affecting formation and maintenance of otoconia (Kozel, P.J., R.A. Friedman, L.C. Erway, E.N. Yamoah, L.H. Liu, T. Riddle, J.J. Duffy, T. Doetschman, M.L. Miller, E.L. Cardell, and G.E. Shull. 1998. J. Biol. Chem. 273:18693-18696). Here we demonstrate that PMCA 4b is a negative regulator of nitric oxide synthase I (NOS-I, nNOS) in HEK293 embryonic kidney and neuro-2a neuroblastoma cell models. Binding of PMCA 4b to NOS-I was mediated by interaction of the COOH-terminal amino acids of PMCA 4b and the PDZ domain of NOS-I (PDZ: PSD 95/Dlg/ZO-1 protein domain). Increasing expression of wild-type PMCA 4b (but not PMCA mutants unable to bind PDZ domains or devoid of Ca2+-transporting activity) dramatically downregulated NO synthesis from wild-type NOS-I. A NOS-I mutant lacking the PDZ domain was not regulated by PMCA, demonstrating the specific nature of the PMCA-NOS-I interaction. Elucidation of PMCA as an interaction partner and major regulator of NOS-I provides evidence for a new dimension of integration between calcium and NO signaling pathways.

    The Journal of cell biology 2001;155;2;201-5

  • Fine mapping and single nucleotide polymorphism association results of candidate genes for asthma and related phenotypes.

    Immervoll T, Loesgen S, Dütsch G, Gohlke H, Herbon N, Klugbauer S, Dempfle A, Bickeböller H, Becker-Follmann J, Rüschendorf F, Saar K, Reis A, Wichmann HE and Wjst M

    GSF-National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany. immervoll@gsf.de

    Several genome-wide screens for asthma and related phenotypes have been published to date but data on fine-mapping are scarce. For higher resolution we performed a fine-mapping study with 2 cM average spacing in often discussed asthma candidate regions (2p, 5q, 6p, 7p, 9q, 11p, and 12q) to narrow down the regions of interest. All participants of a Caucasian family study (97 families with at least two affected sib pairs) were genotyped for 49 supplementary polymorphic dinucleotide markers. Our results indicate increased evidence for linkage on chromosome 6p, 9q, and 12q. These candidate regions were further analyzed with SNP polymorphisms in the endothelin 1 (EDN1), lymphotoxin alpha (LTA), and neuronal nitric oxide synthase (NOS1) genes. In addition, IL4 -590C>T and IL10 -592C>A, localized on chromosomes 5q and 1q, respectively, have been analyzed for SNP association. Of the six SNPs tested, four revealed weak association with the examined phenotypes. These are the IL10 -592C>A SNP in the interleukin 10 gene (p=0.036 for eosinophil cell counts), the 4124T>C SNP in EDN1 (p=0.044 for asthma), the 3391C>T SNP in NOS1 with eosinophil cell counts (p=0.0086), and the 5266C>T polymorphism, also in the NOS1 gene, for high IgE levels (p=0.022). In summary, fine mapping data enable us to confine asthma candidate regions, while variants of EDN1 and NOS1, or nearby genes, may play an important role in this context.

    Human mutation 2001;18;4;327-36

  • Neuronal nitric oxide synthase: a substrate for SHP-1 involved in sst2 somatostatin receptor growth inhibitory signaling.

    Lopez F, Ferjoux G, Cordelier P, Saint-Laurent N, Estève JP, Vaysse N, Buscail L and Susini C

    INSERM U 531, IFR 31, CHU Rangueil, 31403 Toulouse Cedex 4, France.

    Somatostatin receptor sst2 is an inhibitory G protein-coupled receptor, which inhibits normal and tumor cell growth by a mechanism involving the tyrosine phosphatase SHP-1. We reported previously that SHP-1 associates transiently with and is activated by sst2 and is a critical component for sst2 growth inhibitory signaling. Here, we demonstrate that in Chinese hamster ovary cells expressing sst2, SHP-1 is associated at the basal level with the neuronal nitric oxide synthase (nNOS). Following sst2 activation by the somatostatin analog RC-160, SHP-1 rapidly recruits nNOS tyrosine dephosphorylates and activates it. The resulting NO activates guanylate cyclase and inhibits cell proliferation. Coexpression of a catalytically inactive SHP-1 mutant with sst2 blocks RC-160-induced nNOS dephosphorylation and activation, as well as guanylate cyclase activation. In mouse pancreatic acini, RC-160 treatment reduces nNOS tyrosine phosphorylation accompanied by an increase of its activity. By opposition, in acini from viable motheaten (mev/mev) mice, which express a markedly inactive SHP-1, RC-160 has no effect on nNOS activity. Finally, expression of a dominant-negative form of nNOS prevents both RC-160-induced p27 up-regulation and cell proliferation inhibition. We therefore identified nNOS as a novel SHP-1 substrate critical for sst2-induced cell-growth arrest.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2001;15;12;2300-2

  • No linkage or association of the nitric oxide synthase genes to multiple sclerosis.

    Modin H, Dai Y, Masterman T, Svejgaard A, Sørensen PS, Oturai A, Ryder LP, Spurkland A, Vartdal F, Laaksonen M, Sandberg-Wollheim M, Myhr KM, Nyland H and Hillert J

    Department of Neurology, R54, Karolinska Institutet, Huddinge University Hospital, S-121 86, Huddinge, Sweden. Helena.Modin@Neurotec.ki.se

    Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) of unknown etiology. Nitric oxide (NO) is a free radical that participates in a variety of biological processes. It is an important mediator in the immune response. Several studies indicate involvement of NO in the pathogenesis of MS. We studied five markers within the three NO synthase genes with regards to susceptibility and disease course in 156 affected sib-pairs and in 96 "benign" and 96 "severe" definite MS patients and 148 controls. We found no significant association or evidence for linkage in our data sets.

    Journal of neuroimmunology 2001;119;1;95-100

  • Sepsis is associated with reciprocal expressional modifications of constitutive nitric oxide synthase (NOS) in human skeletal muscle: down-regulation of NOS1 and up-regulation of NOS3.

    Lanone S, Mebazaa A, Heymes C, Valleur P, Mechighel P, Payen D, Aubier M and Boczkowski J

    Institut National de la Santé et de la Recherche Médicale (INSERM) U408 and IFR 02, Faculté X. Bichat, Hôpital Lariboisière, Paris, France.

    Objective: To study the expression (mRNA and protein) and activity of the constitutive isoforms of nitric oxide synthase (NOS1 and NOS3) in a skeletal muscle of septic patients.

    Design: Prospective study.

    Setting: An adult trauma/surgical intensive care unit in an urban teaching hospital.

    Patients: Sixteen septic patients and 21 controls.

    Interventions: None.

    Samples of the rectus abdominis muscle were obtained during surgical procedure. NOS mRNA, protein, and activity were detected by reverse-transcriptase polymerase chain reaction, Western blot, and the conversion of [3H]L-arginine to [3H]L-citrulline, respectively. The main results of this study are as follows: a) Levels of NOS1 mRNA and protein were significantly higher than those of NOS3 in the rectus abdominis muscle of control patients; b) NOS1 expression was down-regulated in septic patients, whereas NOS3 was up-regulated; c) these modulations were associated with a reduction in constitutive NOS activity; and d) modifications of NOS1 and NOS3 protein expression were correlated significantly with the severity of sepsis, assessed by the Simplified Acute Physiology Score II.

    Conclusions: Sepsis induces reciprocal expressional modifications of NOS1 and NOS3 in human skeletal muscle, which decreases muscular constitutive NOS activity. These modifications may have implications for muscle impairment in septic patients.

    Critical care medicine 2001;29;9;1720-5

  • The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney.

    Kakoki M, Zou AP and Mattson DL

    Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.

    The role of nitric oxide (NO) produced by NO synthase 1 (NOS1) in the renal vasculature remains undetermined. In the present study, we investigated the influence of systemic inhibition of NOS1 by intravenous administration of N(omega)-propyl-L-arginine (L-NPA; 1 mg. kg(-1). h(-1)) and N(5)-(1-imino-3-butenyl)-L-ornithine (v-NIO; 1 mg. kg(-1). h(-1)), highly selective NOS1 inhibitors, on renal cortical and medullary blood flow and interstitial NO concentration in Sprague-Dawley rats. Arterial blood pressure was significantly decreased by administration of both NOS1-selective inhibitors (-11 +/- 1 mmHg with L-NPA and -7 +/- 1 mmHg with v-NIO; n = 9/group). Laser-Doppler flowmetry experiments demonstrated that blood flow in the renal cortex and medulla was not significantly altered following administration of either NOS1-selective inhibitor. In contrast, the renal interstitial level of NO assessed by an in vivo microdialysis oxyhemoglobin-trapping technique was significantly decreased in both the renal cortex (by 36-42%) and medulla (by 32-40%) following administration of L-NPA (n = 8) or v-NIO (n = 8). Subsequent infusion of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 50 mg. kg(-1). h(-1)) to rats pretreated with either of the NOS1-selective inhibitors significantly increased mean arterial pressure by 38-45 mmHg and significantly decreased cortical (25-29%) and medullary (37-43%) blood flow. In addition, L-NAME further decreased NO in the renal cortex (73-77%) and medulla (62-71%). To determine if a 40% decrease in NO could alter renal blood flow, a lower dose of L-NAME (5 mg. kg(-1). h(-1); n = 8) was administered to a separate group of rats. The low dose of L-NAME reduced interstitial NO (cortex 39%, medulla 38%) and significantly decreased blood flow (cortex 23-24%, medulla 31-33%). These results suggest that NOS1 does not regulate basal blood flow in the renal cortex or medulla, despite the observation that a considerable portion of NO in the renal interstitial space appears to be produced by NOS1.

    Funded by: NHLBI NIH HHS: HL-29587; NIDDK NIH HHS: DK-50739

    American journal of physiology. Regulatory, integrative and comparative physiology 2001;281;1;R91-7

  • Heat-shock protein 90 augments neuronal nitric oxide synthase activity by enhancing Ca2+/calmodulin binding.

    Song Y, Zweier JL and Xia Y

    Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.

    Heat-shock protein 90 (hsp90) has been shown to facilitate neuronal NO synthase (nNOS, type 1) activity in vivo. But the direct effect of hsp90 on purified nNOS has not been determined yet. Moreover, the mechanism underlying the action of hsp90 is not known. nNOS activity is primarily initiated and regulated by the binding of Ca(2+)/calmodulin (CaM). Therefore, we explored whether hsp90 modulates nNOS activity by affecting CaM binding. Recombinant rat nNOS was purified from the stably transfected cells by affinity chromatography. hsp90 increased nNOS activity in a dose-dependent manner with an EC(50) of 24.1+/-6.4 nM. In the presence of hsp90, the CaM-nNOS dose-response curve was shifted markedly to the left and the maximal activity was also elevated. Further in vitro protein-binding experiments confirmed that hsp90 increased the binding of CaM to nNOS. Taken together, these data indicate that hsp90 directly augments nNOS catalytic function and that this effect is, at least partially, mediated by CaM-binding enhancement.

    Funded by: NIA NIH HHS: AG00835

    The Biochemical journal 2001;355;Pt 2;357-60

  • NOSIP, a novel modulator of endothelial nitric oxide synthase activity.

    Dedio J, König P, Wohlfart P, Schroeder C, Kummer W and Müller-Esterl W

    Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University, D-55099 Mainz, Germany.

    Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2001;15;1;79-89

  • AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation.

    Chen ZP, McConell GK, Michell BJ, Snow RJ, Canny BJ and Kemp BE

    St. Vincent's Institute of Medical Research, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia.

    AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-alpha1 and -alpha2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser(79) (AMPK phosphorylation site) approximately 8.5-fold. Under these conditions, there is also an approximately 5.5-fold increase in phosphorylation of neuronal NO synthase-mu (nNOSmu;) at Ser(1451). These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSmu to protect the muscle from ischemia/metabolic stress.

    American journal of physiology. Endocrinology and metabolism 2000;279;5;E1202-6

  • Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON.

    Fang M, Jaffrey SR, Sawa A, Ye K, Luo X and Snyder SH

    Department of Neuroscience, School of Medicine, The Johns Hopkins University, Baltimore, Maryland 21205, USA.

    Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins. Here we identify a selective interaction of the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. We find that Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). We also find that nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced.

    Funded by: NIDA NIH HHS: DA00074; NIMH NIH HHS: MH-18501

    Neuron 2000;28;1;183-93

  • Inhibition of neuronal nitric-oxide synthase by calcium/ calmodulin-dependent protein kinase IIalpha through Ser847 phosphorylation in NG108-15 neuronal cells.

    Komeima K, Hayashi Y, Naito Y and Watanabe Y

    Departments of Pharmacology and Ophthalmology, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan.

    We have previously demonstrated that phosphorylation of neuronal nitric-oxide synthase (nNOS) at Ser(847) by Ca(2+)/calmodulin-dependent protein kinases (CaM kinases) attenuates the catalytic activity of the enzyme in vitro (Hayashi Y., Nishio M., Naito Y., Yokokura H., Nimura Y., Hidaka H., and Watanabe Y. (1999) J. Biol. Chem. 274, 20597-20602). In the present study we determined that CaM kinase IIalpha (CaM-K IIalpha) can directly phosphorylate nNOS on Ser(847), leading to a reduction of nNOS activity in cells. The phosphorylation abilities of purified CaM kinase Ialpha (CaM-K Ialpha), CaM-K IIalpha, and CaM-kinase IV (CaM-K IV) on Ser(847) were analyzed using the synthetic peptide nNOS-(836-859) (Glu-Glu-Arg-Lys-Ser-Tyr-Lys-Val-Arg-Phe-Asn-Ser-Val-Ser-Ser-Tyr-Ser- Asp-Ser-Arg-Lys-Ser-Ser-Gly) from nNOS as substrate. The relative V(max)/K(m) ratios of CaM kinases for nNOS-(836-859) were found to be as follows: CaM-K IIalpha, 100; CaM-K Ialpha, 54.5; CaM-K IV, 9.1. Co-transfection of constitutively active CaM-K IIalpha1-274 but not inactive CaM-K IIalpha1-274, generated by mutation of Lys(42) to Ala, with nNOS into NG108-15 cells, resulted in increased Ser(847) phosphorylation in the presence of okadaic acid, an inhibitor of protein phosphatase (PP)1 and PP2A, with a concomitant inhibition of NOS enzyme activity. In addition, this latter decrease could be reversed by treatment with exogenous PP2A. Cells expressing mutant nNOS (S847A) proved resistant to phosphorylation and a decrease of NOS activity. Thus, our results indicate that Ca(2+) triggers cross-talk signal transduction between CaM kinase and NO and CaM-K IIalpha phosphorylating nNOS on Ser(847), which in turn decreases the gaseous second messenger NO in neuronal cells.

    The Journal of biological chemistry 2000;275;36;28139-43

  • The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of beta2-syntrophin and nNOS in pancreatic beta-cells.

    Ort T, Maksimova E, Dirkx R, Kachinsky AM, Berghs S, Froehner SC and Solimena M

    Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

    Funded by: PHS HHS: N533145

    European journal of cell biology 2000;79;9;621-30

  • Formation of a native-like beta-hairpin finger structure of a peptide from the extended PDZ domain of neuronal nitric oxide synthase in aqueous solution.

    Wang P, Zhang Q, Tochio H, Fan JS and Zhang M

    Department of Biochemistry, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, P. R. China.

    Neuronal nitric oxide synthase (nNOS) is targeted to the cell membrane via interactions of its extended PDZ domain with PDZ domains of membrane-associated proteins including PSD-95 and alpha1-syntrophin. The formation of heterodimers between the nNOS PDZ domain and the PDZ domains of nNOS-binding proteins requires a stretch of continuous amino-acid residues C-terminal to the canonical nNOS PDZ domain. In this work, we show that a 27-residue peptide comprising the C-terminal extension of the extended nNOS PDZ domain is capable of binding to PSD-95. The structure of the 27-residue peptide in aqueous solution was determined using multidimensional NMR-spectroscopic techniques. The free peptide adopts a native-like beta-hairpin finger structure in aqueous solution. The results indicate that the C-terminal extension peptide of the nNOS PDZ domain may represent a relatively independent structural unit in the mediation of the interaction between nNOS and PDZ domain-containing proteins including PSD-95 and alpha1-syntrophin.

    European journal of biochemistry 2000;267;11;3116-22

  • Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation.

    Golser R, Gorren AC, Leber A, Andrew P, Habisch HJ, Werner ER, Schmidt K, Venema RC and Mayer B

    Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.

    Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.

    Funded by: NHLBI NIH HHS: HL57201, HL62152

    The Journal of biological chemistry 2000;275;8;5291-6

  • RNA diversity has profound effects on the translation of neuronal nitric oxide synthase.

    Wang Y, Newton DC, Robb GB, Kau CL, Miller TL, Cheung AH, Hall AV, VanDamme S, Wilcox JN and Marsden PA

    Renal Division and Department of Medicine, St. Michael's Hospital, University of Toronto, Toronto, ON M4X 1B1, Canada.

    A comprehensive analysis of the structure of neuronal nitric oxide synthase (nNOS; EC 1.14.13.39) mRNA species revealed NOS1 to be the most structurally diverse human gene described to date in terms of promoter usage. Nine unique exon 1 variants are variously used for transcript initiation in diverse tissues, and each is expressed from a unique 5'-flanking region. The dependence on unique genomic regions to control transcription initiation in a cell-specific fashion burdens the transcripts with complex 5'-mRNA leader sequences. Elaborate splicing patterns that involve alternatively spliced leader exons and exon skipping have been superimposed on this diversity. Highly structured nNOS mRNA 5'-untranslated regions, which have profound effects on translation both in vitro and in cells, contain cis RNA elements that modulate translational efficiency in response to changes in cellular phenotype.

    Funded by: NHLBI NIH HHS: HL48667

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;21;12150-5

  • PSD-95 assembles a ternary complex with the N-methyl-D-aspartic acid receptor and a bivalent neuronal NO synthase PDZ domain.

    Christopherson KS, Hillier BJ, Lim WA and Bredt DS

    Department of Physiology, and Program in Biomedical Sciences, University of California, San Francisco, California 94143-0444, USA.

    Nitric oxide (NO) biosynthesis in cerebellum is preferentially activated by calcium influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors, suggesting that there is a specific link between these receptors and neuronal NO synthase (nNOS). Here, we find that PSD-95 assembles a postsynaptic protein complex containing nNOS and NMDA receptors. Formation of this complex is mediated by the PDZ domains of PSD-95, which bind to the COOH termini of specific NMDA receptor subunits. In contrast, nNOS is recruited to this complex by a novel PDZ-PDZ interaction in which PSD-95 recognizes an internal motif adjacent to the consensus nNOS PDZ domain. This internal motif is a structured "pseudo-peptide" extension of the nNOS PDZ that interacts with the peptide-binding pocket of PSD-95 PDZ2. This asymmetric interaction leaves the peptide-binding pocket of the nNOS PDZ domain available to interact with additional COOH-terminal PDZ ligands. Accordingly, we find that the nNOS PDZ domain can bind PSD-95 PDZ2 and a COOH-terminal peptide simultaneously. This bivalent nature of the nNOS PDZ domain further expands the scope for assembly of protein networks by PDZ domains.

    Funded by: NINDS NIH HHS: R01-NS34822

    The Journal of biological chemistry 1999;274;39;27467-73

  • Human immunodeficiency virus infection, inducible nitric oxide synthase expression, and microglial activation: pathogenetic relationship to the acquired immunodeficiency syndrome dementia complex.

    Rostasy K, Monti L, Yiannoutsos C, Kneissl M, Bell J, Kemper TL, Hedreen JC and Navia BA

    Pediatric Neurology, The Floating Hospital, Boston, MA, USA.

    The regional expression of immune-mediated and neurotoxic events in the human immunodeficiency virus (HIV)-infected brain in relationship to the acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) and brain pathology remains uncertain. The extent of gp41, inducible nitric oxide synthase (iNOS), and HLA-DR expression was examined in the frontal lobe and basal ganglia of 25 patients at varying stages of ADC. The expression of gp41 and iNOS was present predominantly in perivascular cells and most often in the basal ganglia. Staining for gp41 correlated significantly with iNOS in the basal ganglia, whereas the severity of staining for gp41 and iNOS in the basal ganglia and white matter was significantly greater in subjects with moderate to severe dementia compared with those with milder impairment. The degree of macrophage staining in the white matter and basal ganglia also correlated significantly with ADC severity and was more abundant than gp41 or iNOS staining, particularly in the white matter. Logistic regression analysis revealed that staining for iNOS and gp41 increased linearly with ADC severity and was significantly more abundant in the basal ganglia compared with the white matter. Double-immunolabeling studies colocalized iNOS predominantly to macrophage/microglia and to gp41-positive cells. The expression of iNOS and gp41 in the basal ganglia combined with immune activation contributes to the development and progression of the clinical syndrome.

    Funded by: NINDS NIH HHS: NS36524

    Annals of neurology 1999;46;2;207-16

  • Regulation of neuronal nitric-oxide synthase by calmodulin kinases.

    Hayashi Y, Nishio M, Naito Y, Yokokura H, Nimura Y, Hidaka H and Watanabe Y

    Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, Japan.

    Phosphorylation of neuronal nitric-oxide synthase (nNOS) by Ca2+/calmodulin (CaM)-dependent protein kinases (CaM kinases) including CaM kinase Ialpha (CaM-K Ialpha), CaM kinase IIalpha (CaM-K IIalpha), and CaM kinase IV (CaM-K IV), was studied. It was found that purified recombinant nNOS was phosphorylated by CaM-K Ialpha, CaM-K IIalpha, and CaM-K IV at Ser847 in vitro. Replacement of Ser847 with Ala (S847A) prevented phosphorylation by CaM kinases. Phosphorylated recombinant wild-type nNOS at Ser847 (approximately 0.5 mol of phosphate incorporation into nNOS) exhibited a 30% decrease of Vmax with little change of both the Km for L-arginine and Kact for CaM relative to unphosphorylated enzyme. The activity of mutant S847D was decreased to a level 50-60% as much as the wild-type enzyme. The decreased NOS enzyme activity of phosphorylated nNOS at Ser847 and mutant S847D was partially due to suppression of CaM binding, but not to impairment of dimer formation which is thought to be essential for enzyme activation. Inactive nNOS lacking CaM-binding ability was generated by mutation of Lys732-Lys-Leu to Asp732-Asp-Glu (Watanabe, Y., Hu, Y., and Hidaka, H. (1997) FEBS Lett. 403, 75-78). It was phosphorylated by CaM kinases, as was the wild-type enzyme, indicating that CaM-nNOS binding was not required for the phosphorylation reaction. We developed antibody NP847, which specifically recognize nNOS in its phosphorylated state at Ser847. Using the antibody NP847, we obtained evidence that nNOS is phosphorylated at Ser847 in rat brain. Thus, our results suggest that CaM kinase-induced phosphorylation of nNOS at Ser847 alters the activity control of this enzyme.

    The Journal of biological chemistry 1999;274;29;20597-602

  • Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex.

    Hillier BJ, Christopherson KS, Prehoda KE, Bredt DS and Lim WA

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA.

    The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.

    Science (New York, N.Y.) 1999;284;5415;812-5

  • Interaction of neuronal nitric-oxide synthase and phosphofructokinase-M.

    Firestein BL and Bredt DS

    Department of Physiology, UCSF, San Francisco, California 94143-0444, USA. bonfire@itsa.ucsf.edu

    Neurons that express neuronal nitric-oxide synthase (nNOS) are resistant to NO-induced neurotoxicity; however, the mechanism by which these neurons are protected is not clear. To identify proteins possibly involved in this process, we performed affinity chromatography with the nNOS PDZ domain, a N-terminal motif that mediates protein interactions. Using this method to fractionate soluble tissue extracts, we identified the muscle isoform of phosphofructokinase (PFK-M) as a protein that binds to nNOS both in brain and skeletal muscle. PFK-M interacts with the PDZ domain of nNOS, and nNOS-PFK-M binding can be competed by peptides that bind to the PDZ domain of nNOS. We found that nNOS is significantly associated with PFK-M in skeletal muscle because nNOS can be immunodepleted from cytosolic skeletal muscle extracts using an antibody directed against PFK-M. In brain, nNOS and PFK-M are both enriched in synaptosomes, and specifically, in the synaptic vesicle fraction, where they can interact. At the cellular level, PFK-M is enriched in neurons that express nNOS protein. As fructose-1, 6-bisphosphate, the product of PFK activity, is neuroprotective, the interaction of nNOS and PFK may contribute to neuroprotection of nNOS positive cells.

    Funded by: NINDS NIH HHS: R01-NS36017

    The Journal of biological chemistry 1999;274;15;10545-50

  • Human immunodeficiency virus-1-infected macrophages induce inducible nitric oxide synthase and nitric oxide (NO) production in astrocytes: astrocytic NO as a possible mediator of neural damage in acquired immunodeficiency syndrome.

    Hori K, Burd PR, Furuke K, Kutza J, Weih KA and Clouse KA

    Division of Cytokine Biology and the Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD, USA.

    Nitric oxide (NO) plays an important role in normal neural cell function. Dysregulated or overexpression of NO contributes to neurologic damage associated with various pathologies, including human immunodeficiency virus (HIV)-associated neurological disease. Previous studies suggest that HIV-infected monocyte-derived macrophages (MDM) produce low levels of NO in vitro and that inducible nitric oxide synthase (iNOS) is expressed in the brain of patients with neurologic disease. However, the levels of NO could not account for the degree of neural toxicity observed. In this study, we found that induction of iNOS with concomitant production of NO occurred in primary human astrocytes, but not in MDM, when astrocytes were cocultured with HIV-1-infected MDM. This coincided with decreased HIV replication in infected MDM. Supernatants from cocultures of infected MDM and astrocytes also stimulated iNOS/NO expression in astrocytes, but cytokines known to induce iNOS expression (interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha) were not detected. In addition, the recombinant HIV-1 envelope protein gp41, but not rgp120, induced iNOS in cocultures of uninfected MDM and astrocytes. This suggests that astrocytes may be an important source of NO production due to dysregulated iNOS expression and may constitute one arm of the host response resulting in suppression of HIV-1 replication in the brain. It also leads us to speculate that neurologic damage observed in HIV disease may ensue from prolonged, high level production of NO.

    Blood 1999;93;6;1843-50

  • Rate and severity of HIV-associated dementia (HAD): correlations with Gp41 and iNOS.

    Adamson DC, McArthur JC, Dawson TM and Dawson VL

    Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    Background: Fifteen to thirty percent of AIDS patients develop some type of neurologic disorder during the course of their illness and the vast majority of these neurologic disorders will be HIV-associated dementia (HAD). These patients can exhibit varying degrees of severity and rates of progression of HAD. Neuropathologic variables that are associated with the rate of progression of HAD are not known.

    Tissue was collected at autopsy from the Johns Hopkins University HIV Neurology Program. Seventy-one AIDS patients of this prospectively characterized population were followed until death to obtain information on dementia severity and the rate of neurological progression. Immunoblot analysis of immunological nitric oxide synthase (iNOS), HAM56, gp41, p24, gp120, and beta-tubulin was performed and the levels of iNOS, HAM56, gp41, and p24 were normalized to beta-tubulin and analyzed for significance by means of the Kruskal-Wallis test for multiple groups.

    Results: We have identified unique groups within this spectrum and designated them slow, moderate, and rapid progressors. Slow and moderate progressors' neurological progression occurs over a course of months to years, whereas the rapid progressors' disease shows rapid increases in severity over weeks to months. In the present study we demonstrate that the severity and rate of progression of HAD correlates significantly with levels of the HIV-1 coat protein, gp41, iNOS, and HAM56, a marker of microglial/macrophage activation.

    Conclusion: The severity and rate of progression of HAD correlates with indices of immune activation as well as levels of iNOS and gp41. There appears to be a threshold effect in which high levels of gp41, iNOS, and immune activation are particularly associated with severe (Memorial Sloan-Kettering score 3 to 4) and rapidly progressive HAD.

    Funded by: NINDS NIH HHS: NS 26643

    Molecular medicine (Cambridge, Mass.) 1999;5;2;98-109

  • Nitric oxide synthase in cardiac sarcoplasmic reticulum.

    Xu KY, Huso DL, Dawson TM, Bredt DS and Becker LC

    Department of Medicine, Division of Cardiology, Johns Hopkins Medical Institutions, Baltimore, MD 21224, USA.

    NO. is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO. produced by SR-associated NOS inhibits SR Ca2+ uptake. Ca2+-dependent biochemical conversion of L-arginine to L-citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO. was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOS mu antibody, suggesting the possibility of a new nNOS-type isoform. 45Ca uptake by cardiac SR vesicles, catalyzed by Ca2+-ATPase, was inhibited by NO. produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca2+ concentration and modulate SR Ca2+ ion active transport in the heart.

    Funded by: NHLBI NIH HHS: HL 52175, HL33360, P50 HL52315

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;2;657-62

  • Neuronal nitric-oxide synthase is regulated by the Hsp90-based chaperone system in vivo.

    Bender AT, Silverstein AM, Demady DR, Kanelakis KC, Noguchi S, Pratt WB and Osawa Y

    Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR.hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS. hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS. hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.

    Funded by: NIDDK NIH HHS: DK31573; NIEHS NIH HHS: ES08365; NIGMS NIH HHS: GM07767

    The Journal of biological chemistry 1999;274;3;1472-8

  • Mechanisms and structural determinants of HIV-1 coat protein, gp41-induced neurotoxicity.

    Adamson DC, Kopnisky KL, Dawson TM and Dawson VL

    Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.

    Of the individuals with human immunodeficiency virus type 1 (HIV-1) infection, 20-30% will develop the neurological complication of HIV-associated dementia (HAD). The mechanisms underlying HAD are unknown; however, indirect immunologically mediated mechanisms are theorized to play a role. Recently, the HIV-1 coat protein gp41 has been implicated as a major mediator of HAD through induction of neurocytokines and subsequent neuronal cell death. Using primary mixed cortical cultures from neuronal nitric oxide synthase (NOS) null (nNOS-/-) mice and immunological NOS null (iNOS-/-) mice, we establish iNOS-derived NO as a major mediator of gp41 neurotoxicity. Neurotoxicity elicited by gp41 is markedly attenuated in iNOS-/- cultures compared with wild-type and nNOS-/- cultures. The NOS inhibitor L-nitroarginine methyl ester is neuroprotective in wild-type and nNOS-/- cultures, confirming the role of iNOS-derived NO in gp41 neurotoxicity. Confirming that iNOS-/- cultures lack iNOS, gp41 did not induce iNOS in iNOS-/- cultures, but it markedly induced iNOS in wild-type and nNOS-/- cultures. We elucidate the region of gp41 that is critical for iNOS induction and neuronal cell death by monitoring iNOS induction with overlapping peptides spanning gp41. We show that the N-terminal region of gp41, which we designate as the neurotoxic domain, induces iNOS protein activity and iNOS-dependent neurotoxicity at picomolar concentrations in a manner similar to recombinant gp41 protein. Our experiments suggest that gp41 is eliciting the induction of iNOS through potential cell surface receptors or binding sites because the induction of iNOS is dose dependent and saturable and occurs at physiologically relevant concentrations. These data confirm that the induction of iNOS by gp41 and the production of NO are primary mediators of neuronal damage and identify a neurotoxic domain of gp41 that may play an important role in HAD.

    Funded by: NINDS NIH HHS: NS 26643, T32 NS 07392

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;1;64-71

  • Protein inhibitor of neuronal nitric-oxide synthase, PIN, binds to a 17-amino acid residue fragment of the enzyme.

    Fan JS, Zhang Q, Li M, Tochio H, Yamazaki T, Shimizu M and Zhang M

    Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.

    Neuronal nitric-oxide synthase (nNOS) is the primary nitric oxide (NO) regulator in neurons. The activity of the enzyme is inhibited by a protein inhibitor called PIN. We were able to purify large quantities of PIN overexpressed in bacterial cells. Analytical ultracentrifugation and chemical cross-linking studies showed that PIN exists as a monomer at low concentrations. The protein forms a high order aggregate at elevated concentrations. We have shown, using NMR spectroscopy, that the previously identified PIN-binding domain (PINB) of nNOS (residues 161-245) adopts a random coil structure in solution. By titrating 15N-labeled PINB with unlabeled PIN, the PIN-binding region of nNOS was precisely mapped to a 17-residue peptide fragment from Met-228 to His-244 of nNOS. NMR titration experiments also showed that PIN binds to nNOS with a 1:2 stoichiometry. A synthetic peptide corresponding to the identified PIN-binding region of nNOS was used to study the interaction between PIN and nNOS in detail. The functional implications of the results obtained from this study are discussed.

    The Journal of biological chemistry 1998;273;50;33472-81

  • Binding of dynein light chain (PIN) to neuronal nitric oxide synthase in the absence of inhibition.

    Rodríguez-Crespo I, Straub W, Gavilanes F and Ortiz de Montellano PR

    Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense, Madrid, 28040, Spain.

    PIN, an 89-amino-acid polypeptide found in a rat hippocampal cDNA library using the yeast two-hybrid system and various neuronal nitric oxide synthase (nNOS) fragments as bait, was reported to be an inhibitor of nNOS (Science 274, 774-778, 1996). PIN reportedly inhibited nNOS selectively and did not interact with either the endothelial or inducible nitric oxide synthase isoforms. Inhibition was attributed to the ability of PIN to dissociate the catalytically active nNOS homodimer. PIN is a dynein light chain (J. Biol. Chem. 271, 19358-19366, 1996), which suggested that PIN may serve as an axonal transport protein for nNOS. We have synthesized a rat PIN cDNA by recursive polymerase chain reaction and have expressed the protein in Escherichia coli. Recombinant PIN is a folded dimeric, mostly alpha-helical protein with a single deeply buried tryptophan residue. We have also expressed and purified the nNOS fragment to which PIN reportedly binds (residues 163-245). This recombinant peptide has a disordered secondary structure. Gel-filtration experiments show that PIN binds to both the full-length nNOS and nNOS fragment. However, PIN neither inhibits nNOS activity nor dissociates the nNOS dimer into monomeric species. PIN thus possibly functions as a dynein light chain involved in nNOS axonal transport but is not an inhibitor of the enzyme. Our results agree with the proposal (Cell 82, 743-752, 1995) that the PIN recognition sequence in nNOS both lies outside the catalytic core and is not part of the monomer-monomer contact region.

    Funded by: NIGMS NIH HHS: GM25515

    Archives of biochemistry and biophysics 1998;359;2;297-304

  • Isolation and characterization of a novel, human neuronal nitric oxide synthase cDNA.

    Larsson B and Phillips SC

    Department of Clinical Pharmacology, Lund University Hospital, Lund, Sweden.

    A splice variant of nNOS has recently been identified in both rat and mouse which contains an in-frame insertion of 34 conserved amino acids between the N-terminal oxygenase and the C-terminal reductase domains. In the present study we report the isolation and characterization of a similar, but not identical (76% amino acid identity), human variant (nNOSmu) which arises from the splicing in of an additional exon between exons 16 and 17 of the human nNOS gene. Furthermore, we describe two additional splice variants which, if translated, would give rise to truncated forms of nNOS lacking the C-terminal reductase domain. These additional variants and nNOSmu; have a distinct and more restricted expression pattern compared to nNOS. Further studies are required to elucidate the possible physiological roles of these novel human nNOS splice variants in NO signaling.

    Biochemical and biophysical research communications 1998;251;3;898-902

  • Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophin-associated proteins.

    Gee SH, Madhavan R, Levinson SR, Caldwell JH, Sealock R and Froehner SC

    Department of Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7545, USA.

    Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, alpha1, beta1, and beta2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from alpha1-, beta1-, and beta2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;1;128-37

  • CAPON: a protein associated with neuronal nitric oxide synthase that regulates its interactions with PSD95.

    Jaffrey SR, Snowman AM, Eliasson MJ, Cohen NA and Snyder SH

    The Johns Hopkins University School of Medicine, Department of Neuroscience, Pharmacology and Molecular Sciences, Baltimore, Maryland 21205, USA.

    Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ-PDZ domain interaction between PSD95 and nNOS. Here, we report the identification of a novel nNOS-associated protein, CAPON, which is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes.

    Funded by: NIDA NIH HHS: DA00074; NIGMS NIH HHS: GM-07309; NIMH NIH HHS: MH-10341; ...

    Neuron 1998;20;1;115-24

  • Interaction of neuronal nitric-oxide synthase with caveolin-3 in skeletal muscle. Identification of a novel caveolin scaffolding/inhibitory domain.

    Venema VJ, Ju H, Zou R and Venema RC

    Department of Pediatrics, Medical College of Georgia, Augusta, Georgia 30912, USA.rvenema@mail.mcg.edu

    Neuronal nitric-oxide synthase (nNOS) has been shown previously to interact with alpha1-syntrophin in the dystrophin complex of skeletal muscle. In the present study, we have examined whether nNOS also interacts with caveolin-3 in skeletal muscle. nNOS and caveolin-3 are coimmunoprecipitated from rat skeletal muscle homogenates by antibodies directed against either of the two proteins. Synthetic peptides corresponding to the membrane-proximal caveolin-3 residues 65-84 and 109-130 and homologous caveolin-1 residues 82-101 and 135-156 potently inhibit the catalytic activity of purified, recombinant nNOS. Purified nNOS also binds to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. In vitro binding is completely abolished by preincubation of nNOS with either of the two caveolin-3 inhibitory peptides. Interactions between nNOS and caveolin-3, therefore, appear to be direct and to involve two distinct caveolin scaffolding/inhibitory domains. Other caveolin-interacting enzymes, including endothelial nitric-oxide synthase and the c-Src tyrosine kinase, are also potently inhibited by each of the four caveolin peptides. Inhibitory interactions mediated by two different caveolin domains may thus be a general feature of enzyme docking to caveolin proteins in plasmalemmal caveolae.

    Funded by: NHLBI NIH HHS: HL57201

    The Journal of biological chemistry 1997;272;45;28187-90

  • A novel, testis-specific mRNA transcript encoding an NH2-terminal truncated nitric-oxide synthase.

    Wang Y, Goligorsky MS, Lin M, Wilcox JN and Marsden PA

    Renal Division and Department of Medicine, St. Michael's Hospital, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

    mRNA diversity represents a major theme of neuronal nitric-oxide synthase (nNOS) gene expression in somatic cells/tissues. Given that gonads often express unique and biologically informative variants of complex genes, we determined whether unique variants of nNOS are expressed in the testis. Analysis of cDNA clones isolated from human testis identified a novel, testis-specific nNOS (TnNOS) mRNA transcript. A predicted 3294-base pair open reading frame encodes an NH2-terminal truncated protein of 1098 amino acids. Measurement of calcium-activated L-[14C]citrulline formation and nitric oxide release in CHO-K1 cells stably transfected with the TnNOS cDNA indicates that this protein is a calcium-dependent nitric-oxide synthase with catalytic activity comparable to that of full-length nNOS. TnNOS transcripts exhibit novel 5' mRNA sequences encoded by two unique exons spliced to exon 4 of the full-length nNOS. Characterization of the genomic structure indicates that exonic regions used by the novel TnNOS are expressed from intron 3 of the NOS1 gene. Although lacking canonical TATA and CAAT boxes, the 5'-flanking region of the TnNOS exon 1 contains multiple putative cis-regulatory elements including those implicated in testis-specific gene expression. The downstream promoter of the human nNOS gene, which directs testis-specific expression of a novel NH2-terminal truncated nitric-oxide synthase, represents the first reported example in the NOS gene family of transcriptional diversity producing a variant NOS protein.

    Funded by: NHLBI NIH HHS: HL 48667; NIDDK NIH HHS: DK 45462

    The Journal of biological chemistry 1997;272;17;11392-401

  • Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41.

    Adamson DC, Wildemann B, Sasaki M, Glass JD, McArthur JC, Christov VI, Dawson TM and Dawson VL

    Department of Neurology, Johns Hopkins University School of Medicine, 600 North Wolfe Street, Pathology 2-210, Baltimore, MD 21287, USA. valina.dawson@qmail.bs.jhu.edu

    Indirect mechanisms are implicated in the pathogenesis of the dementia associated with human immunodeficiency virus-type 1 (HIV-1) infection. Proinflammatory molecules such as tumor necrosis factor alpha and eicosanoids are elevated in the central nervous system of patients with HIV-1-related dementia. Nitric oxide (NO) is a potential mediator of neuronal injury, because cytokines may activate the immunologic (type II) isoform of NO synthase (iNOS). The levels of iNOS in severe HIV-1-associated dementia coincided with increased expression of the HIV-1 coat protein gp41. Furthermore, gp41 induced iNOS in primary cultures of mixed rat neuronal and glial cells and killed neurons through a NO-dependent mechanism. Thus, gp41-induced NO formation may contribute to the severe cognitive dysfunction associated with HIV-1 infection.

    Funded by: NIAID NIH HHS: AI35042; NINDS NIH HHS: NS07392, NS22643; ...

    Science (New York, N.Y.) 1996;274;5294;1917-21

  • Cloning and characterization of postsynaptic density 93, a nitric oxide synthase interacting protein.

    Brenman JE, Christopherson KS, Craven SE, McGee AW and Bredt DS

    Department of Physiology, University of California at San Francisco School of Medicine, 94143-0444, USA.

    Nitric oxide (NO) formation in brain is regulated by the calcium/calmodulin dependence of neuronal NO synthase (nNOS). Calcium influx through NMDA-type glutamate receptors is efficiently coupled to nNOS activity, whereas many other intracellular calcium pathways are poorly coupled. To elucidate possible mechanisms responsible for this coupling, we performed yeast two-hybrid screening to identify proteins that interact with nNOS. Two nNOS interacting proteins were identified: the postsynaptic density proteins PSD-93 and PSD-95. Here, we report the cloning and characterization of PSD-93. PSD-93 is expressed in discrete neuronal populations as well as in specific non-neuronal cells, and it exhibits complex molecular diversity attributable to tissue-specific alternative splicing. PSD-93, like PSD-95, binds to nNOS and to the NMDA receptor 2B. PSD-93, however, is unique among PSD-95/SAP-90 family members in its expression in Purkinje neuron cell bodies and dendrites. We also demonstrate that the PDZ domain at the N terminus of nNOS is required, but it is not sufficient for interaction with PSD-93/95. Given that PSD-93 and PSD-95 each contain multiple potential binding sites for nNOS and the NMDA receptor, complexes involving oligomers of PSD-93/95 may help account for the functional as well as the physical coupling of nNOS to NMDA receptors.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;23;7407-15

  • PIN: an associated protein inhibitor of neuronal nitric oxide synthase.

    Jaffrey SR and Snyder SH

    Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

    The neurotransmitter functions of nitric oxide are dependent on dynamic regulation of its biosynthetic enzyme, neuronal nitric oxide synthase (nNOS). By means of a yeast two-hybrid screen, a 10-kilodalton protein was identified that physically interacts with and inhibits the activity of nNOS. This inhibitor, designated PIN, appears to be one of the most conserved proteins in nature, showing 92 percent amino acid identity with the nematode and rat homologs. Binding of PIN destabilizes the nNOS dimer, a conformation necessary for activity. These results suggest that PIN may regulate numerous biological processes through its effects on nitric oxide synthase activity.

    Funded by: NIDA NIH HHS: DA00074; NIGMS NIH HHS: GM-07309

    Science (New York, N.Y.) 1996;274;5288;774-7

  • Neuronal isoform of nitric oxide synthase is expressed at low levels in human retina.

    Park CS, Gianotti C, Park R and Krishna G

    Section of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1760, USA.

    1. The expression of neuronal isoform of nitric oxide synthase (nNOS) was studied in human retinal tissues. The cDNA sequence was cloned in human retinal poly (A)+ RNA by the RT-PCR method and encompassed an open-reading frame of 4,302 bp encoding 1,434 amino acids. This sequence showed a possibility of genetic polymorphism in comparison to human brain form. 2. Restriction fragment length polymorphism (RFLP) patterns of a partial cDNA fragment suggest that there is genetic polymorphism in the neuronal form of NOS. Important differences were observed in a certain region between human retinal and brain froms. This region is a result of frame shift by the addition of three cytidines. In this study, regions from human brain (cerebellum) and skeletal muscle as well as retina were sequenced to confirm the difference in this region. The sequences from these tissues were completely identical. This indicated that genetic polymorphism of nNOS gene was due to single base substitution and not frame shift phenomenon by addition or deletion of bases. 3. The nNOS mRNA of approximately 12 kb was detected by northern blot analysis. The lower level of the expression was distinguished in comparison to those of human brain and skeletal muscle. The cDNA transiently transfected into CHO-K1 cells expressed a protein which contained a significant level of NOS activity. The size of the nNOS was found to be approximately 160 kDa by both in vitro and in vivo translation systems. This NOS was calcium dependent and the K(m) for arginine was 4.4 microM. 4. The Ca+2, L-arginine and NADPH dependency along with the inhibitory effect of N-nitro-L-arginine on NOS activity were evaluated. The finding of a constitutive from of NOS in human retina, which is calcium-NADPH dependent, gives further credence to the possible role of nitric oxide in retinal function and neuronal diseases.

    Cellular and molecular neurobiology 1996;16;4;499-515

  • Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains.

    Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC and Bredt DS

    Department of Physiology, School of Medicine, University of California at San Fancisco, California 94143, USA.

    Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

    Cell 1996;84;5;757-67

  • Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity.

    Chen PF, Tsai AL and Wu KK

    Vascular Biology Research Center, University of Texas Health Science Center at Houston 77030, USA.

    Tetrahydrobiopterin (BH4) is an essential cofactor for all three isoforms of nitric oxide synthase (NOS). However, its binding sites and functional roles remain elusive. Here, we demonstrated that cys-99 of human endothelial NOS (ecNOS) is critical for BH4 involvement in NOS catalytic activity and stability. Mutation of cys-99 to alanine in ecNOS resulted in loss of catalytic activity which could be restored to the level of wild type by adding a high concentration of exogenous BH4 to the crude extract. Purified C99A mutant was unstable and its maximal activity was only about 20% of the purified wild type activity. Comparison of BH4 concentration-dependent citrulline formation between C99A and the wild type revealed that the BH4 concentrations required for generating half-maximal citrulline were 10-fold higher for C99A. Purified C99A had no detectable BH4 and had a reduced heme content when compared to the purified wild type, but retained the ability of forming CO-ferrous heme complex and had the same Km value for L-arginine (approximately 4 microM) as the wild type. These findings indicate that Cys-99 is critically involved in BH4 binding. Mutation of this residue leads to reduced affinity for BH4 and the resultant enzyme instability and irreversible heme loss.

    Funded by: NHLBI NIH HHS: HL-50675; NIGMS NIH HHS: GM-44911; NINDS NIH HHS: NS-23327

    Biochemical and biophysical research communications 1995;215;3;1119-29

  • Human immunodeficiency virus 1 envelope proteins induce interleukin 1, tumor necrosis factor alpha, and nitric oxide in glial cultures derived from fetal, neonatal, and adult human brain.

    Koka P, He K, Zack JA, Kitchen S, Peacock W, Fried I, Tran T, Yashar SS and Merrill JE

    Department of Neurology, University of California, School of Medicine, Los Angeles 90024, USA.

    Although microglia are the only cells found to be productively infected in the central nervous system of acquired immunodeficiency disease syndrome (AIDS) patients, there is extensive white and gray matter disease nonetheless. This neuropathogenesis is believed to be due to indirect mechanisms other than infection with human immunodeficiency virus 1 (HIV-1). Cytokines and toxic small molecules have been implicated in the clinical and histopathological findings in CNS AIDS. Previously, we have demonstrated in rodent glial cultures the presence of biologically active epitopes of gp120 and gp41 that are capable of inducing interleukin 1 and tumor necrosis factor alpha. In this study, we map the HIV-1 envelope epitopes that induce nitric oxide, inducible nitric oxide synthase, interleukin 1, and tumor necrosis factor alpha in human glial cultures. Epitopes in the carboxy terminus of gp120 and the amino terminus of gp41 induce these proinflammatory entities. In addition, we compare HIV-1 infection and pathology in glial cells derived from human brain taken at different states of maturation (fetal, neonatal, and adult brain) in an effort to address some of the clinical and histological differences seen in vivo. This study demonstrates that, in the absence of virus infection and even in the absence of distinct viral tropism, human glia respond like rodent glia to non-CD4-binding epitopes of gp120/gp41 with cytokine and nitric oxide production. Differences among fetal, neonatal, and adult glial cells' infectivity and cytokine production indicate that, in addition to functional differences of glia at different stages of development, cofactors in vitro and in vivo may also be critical in facilitating the biological responses of these cells to HIV-1.

    Funded by: NIMH NIH HHS: 5T32MHI19200; NINDS NIH HHS: R0-1 NS30768

    The Journal of experimental medicine 1995;182;4;941-51

  • Nitric oxide synthase complexed with dystrophin and absent from skeletal muscle sarcolemma in Duchenne muscular dystrophy.

    Brenman JE, Chao DS, Xia H, Aldape K and Bredt DS

    Department of Physiology, University of California, San Francisco School of Medicine 94143-0444, USA.

    Nitric oxide (NO) is synthesized in skeletal muscle by neuronal-type NO synthase (nNOS), which is localized to sarcolemma of fast-twitch fibers. Synthesis of NO in active muscle opposes contractile force. We show that nNOS partitions with skeletal muscle membranes owing to association of nNOS with dystrophin, the protein mutated in Duchenne muscular dystrophy (DMD). The dystrophin complex interacts with an N-terminal domain of nNOS that contains a GLGF motif. mdx mice and humans with DMD evince a selective loss of nNOS protein and catalytic activity from muscle membranes, demonstrating a novel role for dystrophin in localizing a signaling enzyme to the myocyte sarcolemma. Aberrant regulation of nNOS may contribute to preferential degeneration of fast-twitch muscle fibers in DMD.

    Cell 1995;82;5;743-52

  • Death of cultured human neuroblastoma cells induced by HIV-1 gp120 is prevented by NMDA receptor antagonists and inhibitors of nitric oxide and cyclooxygenase.

    Corasaniti MT, Melino G, Navarra M, Garaci E, Finazzi-Agrò A and Nisticò G

    Faculty of Pharmacy, University of Reggio Calabria, Cantanzaro, Italy.

    The cytotoxic effects of the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 were studied in human CHP100 neuroblastoma cell cultures. Incubation of neuroblastoma cultures with gp120 (1 pM-10 nM) induces cell death which is not concentration-related. The significant cell death evoked by 10 pM gp120 was prevented by neutralization of the viral protein with a monoclonal anti-gp120 (IgG) antibody. In addition, gp120-induced cytotoxicity was inhibited by [DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid] (CGP37849; 100 microM), [(+/-)-3R*, 4as*, 6R*, 8aR*-6-(phosphonomethyl) decahydro-isoquinoline-3-carboxylic acid] (LY274614; 100 microM), MK801 (dizocilpine; 200 nM) and 7-chloro kynurenic acid (100 microM), selective antagonists of the NMDA receptor complex; by contrast, (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 100 microM), a non-NMDA antagonist, was ineffective. Prevention of the lethality elicited by the HIV-1 coat protein was also obtained by incubating neuroblastoma cells with gp120 in Ca(2+)-free medium. The lethal effects induced by gp120 involve activation of L-arginine-nitric oxide (NO) pathway since these were prevented by haemoglobin (10 microM), a NO-trapping agent, and by D-arginine (1 mM), the less active enantiomer of the endogenous precursor of NO synthesis. Cytoprotection was also afforded by N omega-nitro-L-arginine methyl ester (L-NAME; 200 microM), an inhibitor of NO synthase, and this was reversed by L-arginine (1 mM). Interestingly, indomethacin and flufenamic acid (10 microM), two inhibitors of cyclooxygenase, protected neuroblastoma cells from death induced by gp120. Furthermore, indomethacin prevented the neuroblastoma cell death evoked by exposure of cultures to sodium nitroprusside (SNP; 0.2-1.6 mM), a NO donor. Finally significant cytotoxic effects were observed after incubation of neuroblastoma cells with prostaglandin E2 (0.1-10 microM). In conclusion, the present data suggest that death of human CHP100 neuroblastoma cells in culture produced by gp120 involves NO and PGE2 production.

    Neurodegeneration : a journal for neurodegenerative disorders, neuroprotection, and neuroregeneration 1995;4;3;315-21

  • Three members of the nitric oxide synthase II gene family (NOS2A, NOS2B, and NOS2C) colocalize to human chromosome 17.

    Bloch KD, Wolfram JR, Brown DM, Roberts JD, Zapol DG, Lepore JJ, Filippov G, Thomas JE, Jacob HJ and Bloch DB

    Cardiovascular Research Center, Massachusetts General Hospital, Boston 02114, USA.

    Nitric oxide synthases (NOSs) are a family of enzymes responsible for the synthesis of nitric oxide from L-arginine and molecular oxygen. Three human NOS enzymes (I, II, and III) with differing cellular distribution and regulatory mechanisms have been identified. To determine whether additional NOSs are encoded in the human genome, a bovine NOS II-related cDNA was used to screen two human genomic libraries. Clones containing three independent genes were isolated. One clone encoded the previously identified NOS II gene (NOS2A). The two other genes specified amino acids homologous, but not identical, to human NOS II (NOS2B and NOS2C). Southern blot hybridization demonstrated that all three genes are present in the human genome. DNA from human-mouse somatic cell hybrids were used to determine the chromosomal location of the NOS II-related genes. All three NOS II-related genes colocalized to human chromosome 17 between bands p13.1 and q25. These observations suggest that there is more than one NOS II-related gene in the human genome. This finding may have important implications for the design of NOS isoform-specific inhibitors.

    Funded by: NHLBI NIH HHS: HL45895, K08 HL004237; NIAMS NIH HHS: AR01866

    Genomics 1995;27;3;526-30

  • Structural organization of the human neuronal nitric oxide synthase gene (NOS1).

    Hall AV, Antoniou H, Wang Y, Cheung AH, Arbus AM, Olson SL, Lu WC, Kau CL and Marsden PA

    Renal Division, St. Michael's Hospital, University of Toronto, Ontario, Canada.

    Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmission, the regulation of body fluid homeostasis, neuroendocrine physiology, control of smooth muscle motility, sexual function, and myocyte/myoblast biology, among others. Restriction enzyme mapping, subcloning, and DNA sequence analysis of bacteriophage- and yeast artificial chromosome-derived human genomic DNA indicated that the mRNA for neuronal NO synthase is dispersed over a minimum of 160 kilobases of human genomic DNA. Analysis of intron-exon splice junctions predicted that the open reading frame is encoded by 28 exons, with translation initiation and termination in exon 2 and exon 29, respectively. Determination of transcription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleotides downstream from a TATA box. Sequence inspection of 5'-flanking regions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CREB/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences. Diversity appears to represent a major theme apparent upon analysis of human neuronal NO synthase mRNA transcripts. A microsatellite of the dinucleotide variety was detected within the 3'-untranslated region of exon 29. Multiple alleles were evident in normal individuals indicating the existence of allelic mRNA sequence variation. Characterization of variant human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA diversity due to alternative splicing. The latter deletion of a 175-nucleotide exon introduces a frame-shift and premature stop codon indicating the potential existence of a novel NH2 terminus protein. In summary, analysis of the human neuronal NO synthase locus reveals a complex genomic organization and mRNA diversity that is both allelic and structural.

    The Journal of biological chemistry 1994;269;52;33082-90

  • HIV coating gp 120 glycoprotein-dependent prostaglandin E2 release by human cultured astrocytoma cells is regulated by nitric oxide formation.

    Mollace V, Colasanti M, Rodino P, Lauro GM and Nistico G

    Department of Biology, University of Rome Tor Vergata, Italy.

    The role of the L-arginine-NO pathway on the formation of PGE2 by cultured astroglial cells incubated with the HIV coating glycoprotein gp120 was investigated. Preincubation of human cultured T 67 astrocytoma cells with gp 120 (100-500 nM) produced a significant increase of nitrite (the breakdown product of NO) and PGE2 in cell supernatants. The effect of gp 120 on both nitrite and PGE2 production was antagonized by inhibition of NO synthase by L-NAME (20-300 microM). The inhibition of gp120-induced PGE2 production by L-NAME was reverted by addition of arachidonic acid (30 microM), an effect antagonized by the cyclo-oxygenase inhibitor indomethacin (10 microM). Methylen bleu, an inhibitor of the biological activity of NO acting at the guanylate cyclase level failed to affect gp 120-mediated PGE2 release showing that the increase of cGMP subsequent to NO production was not involved in the modulatory activity of NO on arachidonic acid cascade. On the basis of present experiments we conclude that gp-120-induced release of PGE2 by astroglial cells is driven by NO, thereby contributing in the involvement of glial cells in HIV-related cerebral disorders.

    Biochemical and biophysical research communications 1994;203;1;87-92

  • Expression of two types of nitric oxide synthase mRNA in human neuroblastoma cell lines.

    Fujisawa H, Ogura T, Kurashima Y, Yokoyama T, Yamashita J and Esumi H

    Biochemistry Division, National Cancer Center Research Institute, Tokyo, Japan.

    Expression of nitric oxide synthase (NOS) was studied in nine human neuroblastoma and two human glioblastoma cell lines. Neuronal NOS (n-NOS) mRNA of approximately 10 kb was detected in four of the nine neuroblastoma cell lines by northern blot analysis using human n-NOS cDNA as a probe. Expression of the n-NOS mRNA was also detected in another neuroblastoma cell line in a subsequent reverse transcriptase polymerase chain reaction (RT-PCR) study, but no n-NOS mRNA expression was observed in the other four neuroblastoma cell lines or in the glioblastoma cell lines. The level of NOS activity correlated well with that of n-NOS mRNA expression in neuroblastoma cell lines expressing n-NOS mRNA. Western blot analysis showed that the n-NOS expressed in neuroblastoma cells was a 160-kDa protein reacted with anti-n-NOS antibody. By using the RT-PCR method, a short n-NOS (n-NOS-2) mRNA with a 315-bp inframe deletion from the entire n-NOS (n-NOS-1) mRNA was detected in the human neuroblastoma cells. The structural diversity of human n-NOS mRNA was demonstrated for the first time.

    Journal of neurochemistry 1994;63;1;140-5

  • Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes.

    Geller DA, Lowenstein CJ, Shapiro RA, Nussler AK, Di Silvio M, Wang SC, Nakayama DK, Simmons RL, Snyder SH and Billiar TR

    Department of Surgery, University of Pittsburgh, PA 15261.

    Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon gamma. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon gamma, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, recognition sites for FMN, FAD, and NADPH are present, as well as a consensus calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is diminished by Ca2+ chelation and a calmodulin antagonist, reflecting a Ca2+ dependence not evident for mac-NOS. Northern blot analysis with hep-NOS cDNA reveals a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Human genomic Southern blots probed with human hep-NOS and human endothelial NOS cDNA clones display different genomic restriction enzyme fragments, suggesting distinct gene products for these NOS isoforms. hep-NOS appears to be an inducible form of NOS that is distinct from mac-NOS as well as brain and endothelial NOS isozymes.

    Funded by: NHLBI NIH HHS: K11 HL02451; NIGMS NIH HHS: GM-37753, GM-44100

    Proceedings of the National Academy of Sciences of the United States of America 1993;90;8;3491-5

  • Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.

    Nakane M, Schmidt HH, Pollock JS, Förstermann U and Murad F

    Vascular Biology, Abbott Laboratories, Abbott Park, Illinois 60064.

    Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co-factors. The cDNA transfected COS-1 cells showed significant NO synthase activity with the typical co-factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

    Funded by: NHLBI NIH HHS: HL 28474; NIDDK NIH HHS: DK 30787

    FEBS letters 1993;316;2;175-80

  • Regional localization of the gene coding for human brain nitric oxide synthase (NOS1) to 12q24.2-->24.31 by fluorescent in situ hybridization.

    Xu W, Gorman P, Sheer D, Bates G, Kishimoto J, Lizhi L and Emson P

    Department of Neurobiology, AFRC Babraham Institute, Cambridge, UK.

    The human gene for brain nitric oxide synthase (NOS1) was previously assigned to chromosome 12 by analysis of a panel of somatic cell hybrids. We report here the refinement of this localization to 12q24.2-->24.31 by fluorescent in situ hybridization.

    Funded by: Cancer Research UK: A3585

    Cytogenetics and cell genetics 1993;64;1;62-3

  • Localization of brain nitric oxide synthase (NOS) to human chromosome 12.

    Kishimoto J, Spurr N, Liao M, Lizhi L, Emson P and Xu W

    Department of Neurobiology, AFRC, Institute of Animal Physiology & Genetics Research, Babraham, Cambridge, United Kingdom.

    Recent research has shown that nitric oxide is a novel neuronal second messenger and transmitter that may be involved in neuronal cell death and damage in neurological illness. To map the chromosomal localization of this important brain enzyme, a rat cDNA probe was prepared by RNA PCR from rat cerebellum RNA. This rat cDNA was used to isolate a human nitric oxide synthase (NOS) cDNA from a human cerebellum cDNA library. The human cDNA clone containing 1.2 kb of brain NOS cDNA was hybridized to Southern blots containing DNAs obtained from human-rodent hybrid cell line panels using EcoRI and HindIII digestion to ascertain the location of the human NOS gene. These data showed that the human brain nitric oxide synthase mapped within 12q14-qter on human chromosome 12.

    Genomics 1992;14;3;802-4

  • Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme.

    Lowenstein CJ, Glatt CS, Bredt DS and Snyder SH

    Department of Neuroscience, Johns Hopkins Medical Institutions, Baltimore, MD 21205.

    Nitric oxide (NO) is a messenger molecule of macrophages, endothelial cells in blood vessels, and neurons. A neuronal form of NO synthase (NOS) has been previously cloned. We now report the molecular cloning of macrophage NOS. The macrophage enzyme displays 50% sequence identity to the neuronal enzyme. Like neuronal NOS, macrophage NOS has recognition sites for FAD, FMN, and NADPH and also has a consensus calmodulin binding site. Macrophage NOS mRNA is strikingly inducible; it is absent in quiescent macrophages or spleen but is prominent 2-6 hr after endotoxin treatment.

    Funded by: NHLBI NIH HHS: HL-02451; NIGMS NIH HHS: GM-07309; NIMH NIH HHS: MH-18501

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;15;6711-5

  • Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites.

    Bredt DS, Ferris CD and Snyder SH

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by approximately 77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

    Funded by: NIDA NIH HHS: DA-00074; NIGMS NIH HHS: GM-07309; NIMH NIH HHS: MH-18501; ...

    The Journal of biological chemistry 1992;267;16;10976-81

OMIM - other

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000032 G2C Homo sapiens Pocklington H1 Human orthologues of cluster 1 (mouse) from Pocklington et al (2006) 21
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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