SIMULATION OF NITRIC-OXIDE SYNTHASE DURING OXIDATIVE ENDOTHELIAL-CELLINJURY

The purpose of this study was to determine changes in nitric oxide syn thase (NOS) activity during the process of lethal oxidative cell injur y following H2O2 treatment of endothelial cells. NOS activity was dete rmined by measuring the conversion of [H-3]arginine ([H-3]Arg) to [3H] citrulline ([3H]Cit). Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Moreover, cell death an d changes in cytosolic free Ca2+ (Ca-i(2+)) were measured simultaneous ly using a confocal laser scanning system, and propidium iodide and fl uo-3 as fluorescent indicators, respectively. Treatment with H2O2 (125 -1000 mu M) concentration dependently increased L-Cit formation from L -Arg, and a peak was obtained at 90 min after the addition of 500 or 1 000 mu M H2O2. The H2O2-induced increase in L-Cit formation was blocke d 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 wa s evoked from 120 min after the addition of H2O2 (125-1000 mu M) in a concentration-dependent manner. Moreover, H2O2 increased Ca-i(2+) befo re cell death, and addition of Ca2+ chelator inhibited both the increa se in L-Cit formation and LDH release by H2O2. The H2O2-induced LDH re lease was reduced by L-NNA, but not by L-NMA. These results suggest th at H2O2 treatment of endothelial cells increases Ca-i(2+) before cell death, and stimulates NOS activity. The activation of NOS may be invol ved in oxidative endothelial cell death. (C) 1998 Elsevier Science Inc .