Ischemia/reperfusion causes endothelial dysfunction leading to impaired endothelium-dependent vascular relaxation in hearts. Postischemic endothelial dysfunction has been attributed to a decrease in nitric oxide (NO) formation and increase in superoxide production. This unbalanced NO decrease/superoxide increase are the key initiating events in the pathogenesis of myocardial reperfusion injury. But the molecular mechanism underlying NO decrease/superoxide increase in postischemic endothelial cells remains poorly understood. In cardiovascular system, NO is primarily produced by endothelial NO synthase (eNOS). Besides synthesizing NO, the applicant and others found that eNOS also produces superoxide and this process is triggered by low levels of tetrahydrobiopterin. Superoxide generated by uncoupled eNOS has been implicated in various diseases. But the role of eNOS-catalyzed superoxide in postischemic endothelial dysfunction has not been explored yet. The preliminary studies in this proposal show that tetrahydrobiopterin is depleted in postischemic hearts and this may change eNOS function from NO to superoxide synthesis. Elevated methylarginines and protein phosphorylation appear to further enhance eNOS to produce superoxide in postischemic hearts. Thus, it is hypothesized that the NO decrease/superoxide increase in dysfunctional endothelial cells in postischemic hearts is caused by a switch of eNOS function from NO to superoxide generation. To test this hypothesis, the following specific aims are proposed: 1) To determine whether a tetrahydrobiopterin depletion-mediated switch of eNOS function from NO to superoxide synthesis renders NO decrease/superoxide increase in postischemic hearts;2) To define the roles of endogenous methylarginines in regulating superoxide generation from eNOS in postischemic hearts;3) To determine the effect of protein phosphorylation on superoxide generation from eNOS in postischemic hearts. For each of these aims, measurements of NO and superoxide will be combined with molecular, cellular, and physiological approaches to characterize the roles of eNOS-derived superoxide in postischemic endothelial injury. Results from these studies will provide fundamental mechanistic information regarding how unbalanced NO decrease/superoxide increase occurs in postischemic endothelial cells. This information may lead to new approaches to treat endothelial dysfunction and improve postischemic cardiac function.
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