Nitric Oxide and Peroxynitrite in Postischemic Myocardium

Abstract

Alterations in the production of nitric oxide (NO^•) are a critical factor in the injury that occurs in ischemic and reperfused myocardium; however, controversy remains regarding the alterations in NO^• that occur and how these alterations cause tissue injury. As superoxide generation occurs during the early period of reperfusion, the cytotoxic oxidant peroxynitrite (ONOO^-) could be formed; however, questions remain regarding ONOO^- formation and its role in postischemic injury. Electron paramagnetic resonance spin trapping studies, using the NO^• trap Fe²⁺-N-methyl-D-glucamine dithiocarbamate (Fe-MGD), and chemiluminescence studies, using the enhancer luminol, have been performed to measure the magnitude and time course of NO^• and ONOO^- formation in the normal and postischemic heart. Isolated rat hearts were subjected to control perfusion, or ischemia followed by reperfusion in the presence of Fe-MGD with electron paramagnetic resonance measurements performed on the effluent from these hearts. Whereas only trace signals were present prior to ischemia, prominent NO^• adduct signals were seen during the first 2 min of reflow. The reperfusion associated increase in these NO^• signals was abolished by nitric oxide synthase inhibition. In hearts perfused with luminol to detect ONOO^- formation, a similar marked increase was seen during the first 2 min of reperfusion that was blocked by nitric oxide synthase inhibitors and by superoxide dismutase. Either N ^G-nitro-L-arginine methyl ester or superoxide dismutase treatment resulted in more than twofold higher recovery of contractile function than in untreated hearts. Immunohistology studies demonstrated that the ONOO^--mediated nitration product nitrotyrosine was formed in postischemic hearts, but not in normally perfused controls. Thus, NO^• formation is increased during the early period of reperfusion and reacts with superoxide to form ONOO^-, which results in protein nitration and myocardial injury.