Research Center of Biochemistry and Molecular Biology; Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu, People's Republic of China.
PLoS One. 2012;7(12):e52788. doi: 10.1371/journal.pone.0052788. Epub 2012 Dec 28.
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.
我们的实验室曾报道，在脑缺血再灌注期间，大鼠海马神经元型一氧化氮合酶（nNOS）的 S-亚硝基化作用降低，但具体机制尚不清楚。在这项研究中，我们发现 nNOS 活性受到 S-亚硝基化作用的动态调节。我们发现，过表达的 nNOS 在人胚肾 293 细胞（HEK293 细胞）中可被外源性一氧化氮供体 GSNO 进行 S-亚硝基化，这与酶活性降低有关。Cys（331），锌四硫代半胱氨酸之一，被鉴定为 nNOS S-亚硝基化的关键位点。此外，我们还发现，静息状态下的大鼠海马神经元中 nNOS 高度 S-亚硝基化，并且在大鼠脑缺血/再灌注过程中，该酶发生脱亚硝基化。有趣的是，nNOS 脱亚硝基化的过程与 nNOS 在 Ser（847）位点的磷酸化程度降低有关，该位点与 nNOS 的激活有关。此外，我们还记录到，用 NMDA 受体拮抗剂 MK801、GSNO、EGTA、BAPTA、钙调蛋白抑制剂 W-7 以及 TrxR1 反义寡核苷酸（AS-ODN）预处理神经元可以抑制 nNOS 脱亚硝基化。综上所述，我们的数据表明，在缺血/再灌注的早期阶段，钙内流诱导的 nNOS 脱亚硝基化是一种 NMDA 受体依赖性过程，主要由硫氧还蛋白-1（Trx1）系统介导。nNOS 的去磷酸化可能是由酶脱亚硝基化诱导的，这表明 nNOS 的 S-亚硝基化/脱亚硝基化可能是调节酶活性的重要机制。
