As was previously shown , NO generated from nitric oxide synthase can result in a loss of recovery of contractile function after reperfusion. To evaluate the relative importance of enzyme-dependent vs. enzyme-independent NO generation in the pathogenesis of postischemic injury, hemodynamic studies were performed in hearts which were subjected to 30 min of global 37°C ischemia followed by 45 min of reperfusion with measurement of the recovery of contractile function. As reported previously , L-NAME-treated hearts exhibited significantly higher recovery of contractile function than untreated control hearts. Oxyhemoglobin-treated hearts (oxyhemoglobin is an efficient scavenger of NO), however, exhibited an even higher recovery of contractile function than the L-NAME-treated hearts. While, l-NAME only blocks NO formation from nitric oxide synthase, oxyhemoglobin scavenges NO formed from either pathway. Thus, this data suggests that enzyme independent NO formation contributes to the process of postischemic injury in the reperfused heart. To determine if further enhancing NO production from nitrite would reverse the protection afforded by l-NAME, hearts were also infused with 10 ^M nitrite during the 5 min immediately prior to ischemia. It was observed that this nitrite loading further increased postischemic injury and almost totally blocked the protective action of l-NAME (Fig. 7).
Amount of NO generated at the end of ischemia, measured using Fe-MGD, showed invert correlation with the final recovery of contractile function observed after reperfusion. Preischemic infusion with arginine did not significantly increase NO generation and did not alter the recovery of contractile function. This indicates that tissue arginine concentrations remained well above the Km of nitric oxide synthase even in the absence of arginine infusion. l-NAME pretreatment decreased nitric oxide generation by about 65% and resulted in approximately a twofold increase in the recovery of contractile function. With preischemic infusion of nitrite in addition to L-NAME, NO generation markedly increased and functional injury was restored. With preischemic loading of nitrite, NO generation was increased by 2-3-fold above levels seen in the untreated controls, and marked impairment of contractile function was seen; however, the function was only slightly lower than in the untreated controls. In both of these groups, very severe functional injury was present with more than a 90% loss of contractile function.
Because of the severe, near-complete, extent of this injury, further increases in NO generation from nitrite may only result in further small decreases in functional recovery which were seen. Alternatively, since NO-mediated injury can be due to its reaction with superoxide to form the potent oxidant peroxynitrite , it is possible that the magnitude of tissue injury could reach a maximum value when the concentrations of NO approach those of superoxide. With oxyhemoglobin treatment, NO was almost totally quenched with more than a 3-fold increase in the recovery of contractile function above the values observed in untreated controls. Thus, this data further confirms the presence and functional importance of enzyme-independent NO generation. In general, it is clear from these studies that both
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