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 (^•O₂^-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and ^•O₂^- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca²⁺ ionophore A23187 triggered NO generation but no ^•O₂^- was seen. With cells in L-Arg-free medium, we observed ^•O₂^- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. ^•O₂^- 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 ^•O₂^- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving ^•O₂^-/NO-mediated cellular injury.