Endothelial cells (Ecs) play an important role in the regulation of vascular tone through the release of soluble mediators. Some of these mediators are prostaglandin (PG)I2, nitric oxide (NO) and endothelium- derived hyperpolarizing factor (EDHF). In coronary vessels, acetylcholine, bradykinin and arachidonic acid produce an endothelium- dependent vasodilation. Arachidonic acid metabolites, EDHF and NO contribute to the relaxations to bradykinin and acetylcholine. Studies with inhibitors indicate that equal contributions by a cyclooxygenase metabolite, PGI2, and cytochrome P450 metabolite(s), epoxyeicosatrienoic acids (EETs), account for the vasorelaxation to arachidonic acid. Our studies indicate that EETs relax and hyperpolarize smooth muscle and open calcium-activated potassium (KCa) channels. They may represent the EDHF released by acetylcholine. The proposed studies will test the hypothesis that endothelial metabolites of arachidonic acid are involved in the regulation of coronary vascular tone. These studies will focus on PGI2 and EETs. We further hypothesize that EETs represent EDHFs in the coronary vasculature. We propose to test this hypothesis further by investigation the following specific aims: (1) We will determine the regulation of EET and PGI2 release form ECs and coronary arteries. The ability of vasoactive substances to stimulate the release of these eicosanoids will be studied, and the effects of drugs that inhibit specific enzymatic pathways will be characterized and investigated. (2) We will compare the regulation of EDHF release with EET release. Using a new bioassay for EDHF, we will characterize the agents that stimulate and inhibit the release with EDHF. The agents that influence EDHF release will be compared to those that affect EET release. These studies should indicate if the EETs are the EDHF release by agonists as bradykinin and acetylcholine in coronary arteries. (3) We will investigate the mechanism of action of the EETs on coronary vascular smooth muscle. These studies will determine how EETs open Kca channels and hyperpolarize smooth muscle. These studies will focus on the role of guanine nucleotide binding proteins and the contribution of endogenous ADP-ribosylation to the regulation of K channels. (4) Using a canine model, we will determine the role of EETs and EDHF in the regulation of coronary microvascular tone. Vascular diameters will be measured in arteries and arterioles in the beating heart. Measurement of the metabolites, inhibitors of eicosanoid synthesis and K channels and infusions of the eicosanoids should provide insights into the contribution of the EETs to coronary microvascular tone.
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