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

Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6770-4. doi: 10.1073/pnas.93.13.6770.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Arginine / analogs & derivatives
  • Arginine / metabolism*
  • Arginine / pharmacology
  • Cell Line
  • Enzyme Inhibitors / pharmacology
  • Humans
  • L-Lactate Dehydrogenase / metabolism
  • NG-Nitroarginine Methyl Ester
  • Nitrates / metabolism*
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Nitric Oxide Synthase / metabolism*
  • Superoxides / metabolism*

Substances

  • Enzyme Inhibitors
  • Nitrates
  • Superoxides
  • peroxynitric acid
  • Nitric Oxide
  • Arginine
  • L-Lactate Dehydrogenase
  • Nitric Oxide Synthase
  • NG-Nitroarginine Methyl Ester