Neuronal nitric oxide synthase (nNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: Implication for oxidative stress
Neuronal nitric oxide synthase (nNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: Implication for oxidative stress
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DOI：
10.1016/j.freeradbiomed.2004.04.023
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发表时间：
2004-08-01
影响因子：
7.4
通讯作者：
Kunimoto, M
中科院分区：
文献类型：
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作者：
Kumagai, Y;Kikushima, M;Kunimoto, M
为了确定 2,4,6-三硝基甲苯 (TNT) 诱导的涉及神经元一氧化氮合酶 (nNOS) 的氧化应激的机制，我们使用酶制剂和大鼠小脑原代神经元细胞检查了 TNT 对 nNOS 酶活性和基因表达的改变。根据 20,000g 小脑上清液制剂中瓜氨酸形成的评估结果，TNT 抑制一氧化氮形成（IC50 = 12.4 muM）。动力学研究表明，TNT 是 NADPH 的竞争性抑制剂，而 L-精氨酸是非竞争性抑制剂。结果发现，纯化的nNOS能够还原TNT，氧化NADPH的比活性为3900 nmol/mg/min，但该反应需要CaCl2/钙调蛋白(CaM)。电子自旋共振 (ESR) 研究表明，nNOS 还原 TNT 时会产生超氧化物 (O-2(.-))。将大鼠小脑原代神经元细胞暴露于 TNT (25 muM) 会导致细胞内产生 H2O2，同时 nNOS mRNA 水平显着增加。这些结果表明 TNT 的 CaM 依赖性单电子还原是由 nNOS 催化的，导致 NO 形成减少和 O-2(.-) 衍生的 H2O2 生成。因此，nNOS 的上调可能代表了对 TNT 暴露期间氧化应激增加的急性适应。 (C) 2004 Elsevier Inc. 保留所有权利。
To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC50 = 12.4 muM) as evaluted by citrulline formation in a 20,000g cerebellar supernatant preparation. A kinetic study revealed that TNT was a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. It was found that purified nNOS was capable of reducing TNT, with a specific activity of 3900 nmol of NADPH oxidized/mg/min, but this reaction required CaCl2/calmodulin (CaM). An electron spin resonance (ESR) study indicated that superoxide (O-2(.-)) was generated during reduction of TNT by nNOS. Exposure of rat cerebellum primary neuronal cells to TNT (25 muM) caused an intracellular generation of H2O2, accompanied by a significant increase in nNOS mRNA levels. These results indicate that CaM-dependent one-electron reduction of TNT is catalyzed by nNOS, leading to a reduction in NO formation and generation of H2O2 derived from O-2(.-). Thus, it is suggested that upregulation of nNOS may represent an acute adaptation to an increase in oxidative stress during exposure to TNT. (C) 2004 Elsevier Inc. All rights reserved.