Variation of nitric oxide synthase activity in encothelial cells and effects of the variation on the cell injury

一氧化氮合酶在血管内皮细胞中存在钙调素、NADPH和四氢生物蝶呤(BH4)的情况下，由L精氨酸产生NO。然而，在低浓度的L-精氨酸或BH_4的作用下，一氧化氮合酶也能产生活性氧自由基(ROS)。我们先前报道一氧化氮合酶的抑制剂L-硝基-L精氨酸(NNA)，而不是N^G-甲基-L-精氨酸(L-NMA)能减轻H_2O_2诱导的内皮细胞损伤。L-NNA可阻断底物非依赖性ROS的产生，而L-NMA对该反应无影响。因此，我们推测L-NNA可阻断氧化应激过程中一氧化氮合酶非依赖底物产生ROS，从而减轻H_2OS_2诱导的内皮细胞损伤。在本研究中，L-NNA不仅减轻了H_2O_2诱导的内皮细胞损伤，而且还减轻了细胞内氧化应激(谷胱甘肽耗竭)诱导的内皮细胞损伤。而在缺乏NO的RFL-6细胞中，L-NNA对H_2O_2或谷胱甘肽耗竭所致的细胞损伤无明显影响。提示L-NNA的保护作用可能与NO有关。此外，我们发现H_2O_2对内皮细胞的保护作用可能与细胞死亡前细胞内Ca~(2+)和Gt~(2+)的升高有关，并能刺激一氧化氮合酶的活性。这些结果有力地支持了我们的假设，即L-NNA在氧化应激过程中阻止一氧化氮合酶产生ROS，从而减轻H_2O_2诱导的内皮细胞损伤。此外，在本研究中，我们还发展了一种利用非灵敏电极直接测定NO的方法。在未来，我们将尝试开发新型的NO敏感但ROS不敏感的电极，并测量氧化应激过程中NO的释放。

Nitric oxide synthase (NOS) generates NO from L-arginine in the presence of Ca^<2+>/calmodulin, NADPH,and tetrahydrobiopterin (BH_4) in vascular endothelial cells. However, NOS has been also shown to generate reactive oxygen species (ROS) at low concentrations of L-arginine or BH_4. We previously reported that N^G-nitro-L-arginine (L-NNA), an inhibitor of NOS,but not N^G-methyl-L-arginine (L-NMA), reduced H_2O_2-induced endothelial cell injury. L-NNA has been shown to block the substrate-independent generation of ROS,whereas L-NMA has not effect on this reaction. Therefore, we speclated that L-NNA blocked the substrate-independent generation of ROS by NOS during oxidative stress and consequently reduced H_2OS_2-induced endothelial cell injury. In the present study, L-NNA reduced not only H_2O_2-induced endothelial cell injury but also intracellular oxidative stress (glutathione depletion) -induced endothelial cell injury. On the other hand, L-NNA did not affect H_2O_2-induced or glutathione depletion-induced cell injury in RFL-6 cells which lack NOS.These results suggested the protective effect of L-NNA is likely to be related to NOS.Moreover, we fund that H_2O_2 treatment of endothelial cells increases intracellular Ca^<2+> before cell death, and stimulates NOS activity. These results strongly supporte that our hypothesis that L-NNA blocks the generation of ROS by NOS during oxidative stress and consequently reduces H_2O_2-induced endothelial cell injury. Moreover, in the present study, we developed a method of direct measurement of NO using NO-sensitive electrode. In the future, we will attempt to develop the new NO-sensitive electrode but ROS-insensitive and measure the NO release during oxydative stress.