H₂O₂ has a dual effect on eNOS phosphorylation and activity. (A–C) Human endothelial cells were incubated with 0.1–500 µmol/liter H₂O₂ for 15 min. Thereafter, either (A) eNOS was immunoprecipitated (IP) and its phosphorylation on Tyr657 and Ser1177 were detected by Western blotting (IB), or (B and C) cyclic GMP levels were assessed by radioimmunoassay under basal conditions (B) or after stimulation with 1 µmol/liter bradykinin (BK) for 2 min or 100 nmol/liter ionomycin (iono) for 2 min (C). Some experiments were also performed in the presence of 300 µmol/liter N^ωnitro–L-arginine (L-NA) for 60 min. The graphs summarize data from 8–12 experiments. Data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus CTL or the corresponding solvent (Sol)-treated group. §§§, P < 0.001 versus solvent for Ser1177 phosphorylation.