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 arteries, hormones such as acetylcholine, bradykinin and arachidonic acid (AA) and physiological stimuli such as flow-induced shear stress produce endothelium-dependent vasodilation that is inhibited by cytochrome P450 (CYP) inhibitors. Thus, CYP metabolite(s) of AA contribute to 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 CYP. EETs relax coronary arteries by opening calcium-activated potassium (KCa) channels and hyperpolarizing the smooth muscle. The proposed studies will test the hypothesis that endothelial metabolites of AA are involved in the regulation of coronary vascular tone. These studies will focus on the endothelium-derived EETs. We will test the hypothesis that EETs are EDHFs in the coronary vasculature by investigating the following specific aims: (1) We will identify and characterize analogs for the four EET regioisomers that act as selective EET agonists and antagonists. Regioisomer-specific antagonists will be developed. Stable agonists and antagonists that resist metabolism by vascular cells will be developed for use in vitro and importantly in vivo. These analogs will be used to define the role of EET isomers in regulating vascular tone and mediating dilation to agonists. (2) Vascular receptors for the EETs have not been characterized despite data suggesting their existence. We will determine if the EETs have specific binding sites/receptors on membranes from coronary arteries, ECs and smooth muscle cells using agonist analogs that incorporate iodine-125, biotin and photoaffinity groups and an agonist tethered to silica beads. 125I-14,15-EET analogs will be used as radioligands to characterize the receptor/binding site. Kinetics and ligand specificity of the receptor will be determined. The properties of EET binding proteins will be studied. (3) In response to agonists, ECs release mainly 14,15-EET with lesser amounts of 11,12- and 8,9-EET. EETs act stereospecifically in causing vascular relaxation. Thus, the total concentration of a released EET regioisomer may not reflect the concentration of the biologically active EET stereoisomer. The concentration and activity of the stereoisomers must be taken into account. We will determine the activity of the stereoisomers of the EET and DHET regioisomers. We will identify the stereoisomers of the EET regioisomers that are synthesized by ECs and coronary arteries and study the regulation of their release by agonists. These studies should indicate the major biologically active EET(s) released by coronary arteries. These studies will provide specific pharmacological tools to study the role of EETs as EDHFs and provide new insights into the vascular action of the EETs.
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