AGONIST-INDUCED PEROXYNITRITE PRODUCTION FROM ENDOTHELIAL-CELLS

Nitric oxide reacts with superoxide to form peroxynitrite, a potential mediator of oxidant-induced cellular injury. The endothelium is a pri mary target of injury in many pathological states, including acute lun g injury, sepsis, multiple organ failure syndrome, and atherosclerosis , where enhanced production of nitric oxide and superoxide occurs simu ltaneously. It was hypothesized that stimulation of endothelial cell n itric oxide production would result in formation of peroxynitrite. Imm ediate oxidant production was detected by luminol- and lucigenin-enhan ced chemiluminescence from cultured bovine aortic endothelial cells ex posed to bradykinin or to the calcium ionophore A23187. Luminol-enhanc ed chemiluminescence was efficiently inhibited by the nitric oxide syn thase inhibitor nitro-L-arginine methyl ester and by superoxide dismut ase, implying dependence on the presence of both nitric oxide and supe roxide for oxidant production. Inhibition of luminol-enhanced chemilum inescence by nitro-L-arginine methyl ester was partially reversed by L -arginine, but not by D-arginine. Cysteine, methionine, and urate, kno wn inhibitors of peroxynitrite-mediated oxidation, inhibited luminol-e nhanced chemiluminescence, while the hydroxyl radical scavengers, mann itol and dimethylsulfoxide, and catalase did not. Bicarbonate increase d luminol-enhanced chemiluminescence in a concentration-dependent mann er. Superoxide production, detected by lucigenin-enhanced chemilumines cence, was slightly increased in the presence of nitro-l-arginine meth yl ester, suggesting that endothelial cell-produced superoxide was par tially metabolized by reaction with nitric oxide. These results are co nsistent with agonist-induced peroxynitrite production by endothelial cells and suggests that peroxynitrite may have an important role in ox idant-induced endothelial injury. (C) 1994 Academic Press, Inc.