title: N-methyl-D-aspartate receptor-dependent denitrosylation of neuronal nitric oxide synthase increase the enzyme activity.
abstract: Our laboratory once reported that neuronal nitric oxide synthase (nNOS) S-nitrosylation was decreased in rat hippocampus during cerebral ischemia-reperfusion, but the underlying mechanism was unclear. In this study, we show that nNOS activity is dynamically regulated by S-nitrosylation. We found that overexpressed nNOS in HEK293 (human embryonic kidney) cells could be S-nitrosylated by exogenous NO donor GSNO and which is associated with the enzyme activity decrease. Cys(331), one of the zinc-tetrathiolate cysteines, was identified as the key site of nNOS S-nitrosylation. In addition, we also found that nNOS is highly S-nitrosylated in resting rat hippocampal neurons and the enzyme undergos denitrosylation during the process of rat brain ischemia/reperfusion. Intrestingly, the process of nNOS denitrosylation is coupling with the decrease of nNOS phosphorylation at Ser(847), a site associated with nNOS activation. Further more, we document that nNOS denitrosylation could be suppressed by pretreatment of neurons with MK801, an antagonist of NMDAR, GSNO, EGTA, BAPTA, W-7, an inhibitor of calmodulin as well as TrxR1 antisense oligonucleotide (AS-ODN) respectively. Taken together, our data demonstrate that the denitrosylation of nNOS induced by calcium ion influx is a NMDAR-dependent process during the early stage of ischemia/reperfusion, which is majorly mediated by thioredoxin-1 (Trx1) system. nNOS dephosphorylation may be induced by the enzyme denitrosylation, which suggest that S-nitrosylation/denitrosylation of nNOS may be an important mechanism in regulating the enzyme activity.
authors.affiliation: Research Center of Biochemistry and Molecular Biology; Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China.
authors.forename: Zhong-Wei
authors.initials: ZW
authors.lastname: Qu
authors.forename: Wan-Ying
authors.initials: WY
authors.lastname: Miao
authors.forename: Shu-Qun
authors.initials: SQ
authors.lastname: Hu
authors.forename: Chong
authors.initials: C
authors.lastname: Li
authors.forename: Xing-Li
authors.initials: XL
authors.lastname: Zhuo
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Zong
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Wu
authors.forename: Guang-Yi
authors.initials: GY
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis; D002545:Brain Ischemia / Q000378:metabolism; D056547:CA1 Region, Hippocampal / Q000378:metabolism / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D003553:Cystine / Q000378:metabolism; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D009474:Neurons / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D015427:Reperfusion Injury / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D003553:Cystine; D013879:Thioredoxins; D052248:Nitric Oxide Synthase Type I; D002118:Calcium