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

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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.