Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (O-.(2)-) at lower L-Arg concentrations, By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and O-.(2)- formation in nNOS-transfected human kidney 293 cells, In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no O-.(2)- was seen, With cells in L-Arg-free medium, we observed O-.(2)- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. O-.(2)- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells, Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release, The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating O-.(2)- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving O-.(2)-/NO-mediated cellular injury.