Changes in nitric oxide synthase activity during exposure to hydrogen peroxide in cultured endothelial cells

This study clarified the changes in maximum nitric oxide synthase (NOS) activity in cultured bovine aortic endothelial cells during exposure to hydrogen peroxide (H2O2). NOS activity was determined by measuring the conversion of [3H]arginine to [3H]citrulline. Ionomycin, a Ca2+ ionophore, was used to stimulate NOS activity. Addition of ionomycin (10(-5) M) increased the level of L-citrulline formation, and the ionomycin-induced increase in L-citrulline formation was stimulated by H2O2 pretreatment (15-90 min). Longer exposure to H2O2 (> or = 180 min) markedly inhibited the ionomycin-induced L-citrulline formation. The stimulation of ionomycin-induced L-citrulline formation by H2O2 was completely inhibited by N(G)-nitro-L-arginine (L-NNA) or N(G)-methyl-L-arginine (L-NMA), both blockers of NOS. Cell death which was determined by lactate dehydrogenase (LDH) release was induced from 120 min after the addition of H2O2. These findings suggest that the maximum L-citrulline formation from L-arginine, coupled with NOS activity, was increased by H2O2 treatment before cell death. The stimulation of NOS activity may be implicated in H2O2-induced endothelial cell death.