Endogenous peroxynitrite is involved in the inhibition of mitochondrial respiration in immuno-stimulated J774.2 macrophages.
The free radicals nitric oxide (NO) and superoxide (O2-) are known to react to form peroxynitrite (ONOO-), a potentially more injurious species. Here we compared the inhibitory effects of ONOO- and NO on mitochondrial respiration in J774.2 macrophages. In addition, using uric acid, a potent scavenger of ONOO-, we investigated the potential involvement of endogenous ONOO- in the inhibitory effects of bacterial lipopolysaccharide (LPS) and gamma-interferon (IFN) on mitochondrial respiration. The NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP, 1 mM) or diethylamine NONOate (DN, 1 mM) inhibited cellular respiration by approximately 30% over 24h. Equimolar amounts of ONOO- caused a more pronounced inhibition of cell respiration. There was a synergistic effect between the O2- generator pyrogallol (10 microM-1 mM) and the NO donor SNAP (1 mM) in inhibiting mitochondrial respiration. The ONOO- scavenger uric acid (UA, 1 mM) did not prevent the decrease in viability in response to SNAP, DN or pyrogallol, but significantly prevented the decrease in cell viability in response to ONOO-, to the combination of SNAP and pyrogallol, and to SIN-1, a compound that simultaneously generates NO and O2-. The decrease in mitochondrial respiration in response to LPS and IFN was also inhibited by UA as well as by NG-methyl-arginine, an inhibitor of NOS. Thus, ONOO- is a more potent suppressant of mitochondrial respiration than NO and endogenous formation of ONOO- appears to be involved in the cytotoxicity associated with immune stimulation.
Szabó
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Children's Hospital Medical Center, Division of Critical Care, Cincinnati, Ohio 45229, USA.
Salzman
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D000818:Animals; D002460:Cell Line; D066298:In Vitro Techniques; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009566:Nitrates / Q000633:toxicity*; D009569:Nitric Oxide / Q000633:toxicity*; D010101:Oxygen Consumption / Q000187:drug effects*; D011748:Pyrogallol / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism
D009566:Nitrates; D011748:Pyrogallol; D013481:Superoxides; C039642:peroxynitric acid; D009569:Nitric Oxide