Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages.
Superoxide (O-2) and nitric oxide (NO) act to kill invading microbes in phagocytes. In macrophages NO is synthesized by inducible nitric oxide synthase (iNOS, NOS 2) from L-arginine (L-Arg) and oxygen; however, O-2 was thought to be produced mainly by NADPH oxidase. Electron paramagnetic resonance (EPR) spin trapping experiments performed in murine macrophages demonstrate a novel pathway of O-2 generation. It was observed that depletion of cytosolic L-Arg triggers O-2 generation from iNOS. This iNOS-mediated O-2 generation was blocked by the NOS inhibitor N-nitro-L-arginine methyl ester or by L-Arg, but not by the noninhibitory enantiomer N-nitro-D-arginine methyl ester. In L-Arg-depleted macrophages iNOS generates both O-2 and NO that interact to form the potent oxidant peroxynitrite (ONOO-), which was detected by luminol luminescence and whose formation was blocked by superoxide dismutase, urate, or L-Arg. This iNOS-derived ONOO- resulted in nitrotyrosine formation, and this was inhibited by iNOS blockade. iNOS-mediated O-2 and ONOO- increased the antibacterial activity of macrophages. Thus, with reduced L-Arg availability iNOS produces O-2 and ONOO- that modulate macrophage function. Due to the existence of L-Arg depletion in inflammation, iNOS-mediated O-2 and ONOO- may occur and contribute to cytostatic/cytotoxic actions of macrophages.
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Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology, The Johns Hopkins University School of Medicine, Johns Hopkins Bayview Medical Center, Baltimore, MD 21224, USA.
Zweier
J L
JL
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D009566:Nitrates / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D009566:Nitrates; D013481:Superoxides; C039642:peroxynitric acid; D019001:Nitric Oxide Synthase