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

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)6770-6774
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number13
DOIs
StatePublished - Jun 25 1996

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Keywords

  • Electron paramagnetic resonance
  • Free radicals
  • Nitrotyrosine
  • Spin trapping

ASJC Scopus subject areas

  • General

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