Stimulation of nitric oxide synthase during oxidative endothelial cell injury.
The purpose of this study was to determine changes in nitric oxide synthase (NOS) activity during the process of lethal oxidative cell injury following H2O2 treatment of endothelial cells. NOS activity was determined by measuring the conversion of [3H]arginine ([3H]Arg) to [3H]citrulline ([3H]Cit). Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Moreover, cell death and changes in cytosolic free Ca2+ (Ca(i)2+) were measured simultaneously using a confocal laser scanning system, and propidium iodide and fluo-3 as fluorescent indicators, respectively. Treatment with H2O2 (125-1000 microM) concentration dependently increased L-Cit formation from L-Arg, and a peak was obtained at 90 min after the addition of 500 or 1000 microM H2O2. The H2O2-induced increase in L-Cit formation was blocked completely by N(G)-nitro-L-arginine (L-NNA) or N(G)-methyl-L-arginine (L-NMA), both inhibitors of NOS. LDH release from endothelial cells was evoked from 120 min after the addition of H2O2 (125-1000 microM) in a concentration-dependent manner. Moreover, H2O2 increased Ca(i)2+ before cell death, and addition of Ca2+ chelator inhibited both the increase in L-Cit formation and LDH release by H2O2. The H2O2-induced LDH release was reduced by L-NNA, but not by L-NMA. These results suggest that H2O2 treatment of endothelial cells increases Ca(i)2+ before cell death, and stimulates NOS activity. The activation of NOS may be involved in oxidative endothelial cell death.