Endothelial cells (ECs) play an important role in the regulation of vascular tone, leukocyte function and platelet aggregation and accomplish these efforts through the release of several soluble mediators. Some of these mediators are metabolites of arachidonic acid such as prostaglandin (PG)12. Others include endothelial-dependent relaxing factor (EDRF), endothelium-dependent hyperpolarizing factor (EDHF), endothelin and endothelium-dependent contracting factor (EDCF). In coronary vessels, acetylcholine, bradykinin and arachidonic acid produce an endothelium- dependent vasodilation. Arachidonic acid metabolites, EDHF and EDRF contribute to the relaxations to bradykinin and acetylcholine. Studies with inhibitors indicate that equal contributions by a cyclooxygenase metabolite(s) and cytochrome P450 metabolite(s) account for the vasorelaxation to arachidonic acid. Coronary ECs synthesize vasodilators eicosanoids through both of these pathways: PGI2 by cyclooxygenase and epoxyeicosatrienoic acids (EETs) by cytochrome P450. The proposed studies will test the hypothesis that endothelial metabolites of arachidonic acid are involved in the regulation of coronary vascular tone and contribute to myocardial ischemia. We propose to test this hypothesis further by investigating the following specific aims: (l) We will further characterize the mechanism of action of the EETs on coronary vascular smooth muscle. Since the EETs appear to open potassium channels, we will determine if the EETs represent an endothelium-derived hyperpolarizing factor. Assays will be developed for the EETs and used to investigate the regulation of EET synthesis in ECs and coronary vessels. Other experiments will determine if EETs mediate the endothelium-dependent relaxation to vasoactive substances. (2) Cultured coronary ECs produce an unknown polar metabolite of arachidonic acid that causes vasodilation. We will isolate and identify this unknown polar metabolite by a combination of chemical modification, high pressure liquid chromatography and mass spectrometry. Biological activity will be determined in vitro. Assays will be developed for the unknown polar metabolite and used to determine factors that alter its synthesis. (3) Using a canine model of ischemic heart disease, we will determine the role of the polar metabolite and/or EETs in the regulation of vascular tone, platelet aggregation and leukocyte infiltration into the site of stenosis. Measurement of the metabolites, inhibitors of synthesis and infusions of the eicosanoids should also provide insights into the contribution of these compounds to experimental myocardial ischemia.
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