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

Yong Xia, Valina Dawson, Ted M Dawson, Solomon H Snyder, Jay L. Zweier

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
StatePublished - Jun 25 1996

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Peroxynitrous Acid
Nitric Oxide Synthase
Superoxides
Arginine
Nitric Oxide
Wounds and Injuries
NG-Nitroarginine Methyl Ester
Calcimycin
Spin Trapping
Nitric Oxide Synthase Type I
Ionophores
Electron Spin Resonance Spectroscopy
L-Lactate Dehydrogenase
Oxidants
Superoxide Dismutase
Kidney

Keywords

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

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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title = "Nitric oxide synthase generates Superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury",
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.",
keywords = "Electron paramagnetic resonance, Free radicals, Nitrotyrosine, Spin trapping",
author = "Yong Xia and Valina Dawson and Dawson, {Ted M} and Snyder, {Solomon H} and Zweier, {Jay L.}",
year = "1996",
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language = "English (US)",
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TY - JOUR

T1 - Nitric oxide synthase generates Superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury

AU - Xia, Yong

AU - Dawson, Valina

AU - Dawson, Ted M

AU - Snyder, Solomon H

AU - Zweier, Jay L.

PY - 1996/6/25

Y1 - 1996/6/25

N2 - 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.

AB - 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.

KW - Electron paramagnetic resonance

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