Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages

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) from L-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 cytosolic L-Arg triggers O₂/^-· generation from iNOS. This iNOS-mediated O₂/^-· 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 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, or L-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 reduced L-Arg availability iNOS produces O₂/^-· and ONOO^- that modulate macrophage function. Due to the existence of L-Arg depletion in inflammation, iNOS-mediated O₂/^-· and ONOO^- may occur and contribute to cytostatic/cytotoxic actions of macrophages.