Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages

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Abstract
Superoxide (O2⨪) and nitric oxide (NO) act to kill invading microbes in phagocytes. In macrophages NO is synthesized by inducible nitric oxide synthase (iNOS, NOS 2) froml-arginine (l-Arg) and oxygen; however, O2⨪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 O2⨪generation. It was observed that depletion of cytosolicl-Arg triggers O2⨪generation from iNOS. This iNOS-mediated O2⨪generation was blocked by the NOS inhibitorN-nitro-l-arginine methyl ester or byl-Arg, but not by the noninhibitory enantiomerN-nitro-d-arginine methyl ester. Inl-Arg-depleted macrophages iNOS generates both O2⨪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, orl-Arg. This iNOS-derived ONOO−resulted in nitrotyrosine formation, and this was inhibited by iNOS blockade. iNOS-mediated O2⨪and ONOO−increased the antibacterial activity of macrophages. Thus, with reducedl-Arg availability iNOS produces O2⨪and ONOO−that modulate macrophage function. Due to the existence ofl-Arg depletion in inflammation, iNOS-mediated O2⨪and ONOO−may occur and contribute to cytostatic/cytotoxic actions of macrophages.