Endogenous peroxynitrite is involved in the inhibition of mitochondrial respiration in immuno-stimulated J774.2 macrophages

The free radicals nitric oxide (NO) and surperoxide (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 γ-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 μM-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 N^G-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.