: Endothelial cells (ECs) regulate vascular tone, leukocyte function and platelet aggregation through the release of several soluble mediators including PGI2, nitric oxide and endothelium-dependent hyperpolarizing factor (EDHF). In coronary vessels, hormones such as acetylcholine, bradykinin and arachidonic acid and physiological stimuli such as shear stress and flow produce endothelium-dependent vasodilation that is inhibited by cytochrome P450 inhibitors. Thus, cytochrome P450 metabolite(s) of arachidonic acid may contribute to these agonist-induced relaxations. Coronary ECs synthesize vasodilator eicosanoids: PGI2 by cyclooxygenase and 14,15-, 11,12-, 8,9-and 5,6-epoxyeicosatrienoic acids (EETs) by cytochrome P450. Smooth muscle cells only synthesize PGI2. EETs relax coronary vessels by opening calcium-activated potassium (Kca) channels and hyperpolarizing the smooth muscle. Thus, EETs appear to represent the EDHFs in the coronary artery. The proposed studies will test the hypothesis that an endothelial metabolite or metabolites of arachidonic acid are involved in the regulation of coronary vascular tone. These studies will focus on the endothelium-derived EETs. We propose to test the hypothesis that EETs are EDHFs in the coronary vasculature by investigating the following specific aims: (1) We will identify and characterize EET analogs for each of the four regioisomers that act as BET antagonists. Preliminary studies have identified two 14,15-BET antagonists. These analogs will be characterized for their ability to antagonize the actions of the EETs and for their specificity. (2) We will investigate the role of EET regioisomers in agonist- and flow-induced relaxation and hyperpolarization using the specific EET antagonists. These studies will be performed in isolated vessels and with an EDHF bioassay. (3) We will study the regulation of EET release from ECs and coronary arteries. These studies will determine if EETs are release by agonists in a luminal and/or abluminal direction. The release of EETs that are esterified to endothelial phospholipids will also be examined. (4) We will confer cytochrome P450 activity on coronary smooth muscle cells using adenoviral vectors containing cytochrome P450s. We will determine if conferring cytochrome P450 epoxygenase activity on smooth muscle cells allows endogenously formed EETs to activate Kca channel activity and cause endothelium-independent relaxation.
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