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.
Xia
Y
Y
Molecular and Cellular Biophysics Laboratories, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, MD 21224, USA.
Dawson
V L
VL
Dawson
T M
TM
Snyder
S H
SH
Zweier
J L
JL
D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009566:Nitrates; D013481:Superoxides; C039642:peroxynitric acid; D009569:Nitric Oxide; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester