The two major pathways of cytochrome P450 (CYP)-catalyzed arachidonic acid (AA) metabolism are omega-hydroxylation to form 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxidation which produces regio- and stereoisomeric epoxyeicosatrienoic acids (EETs). The EETs are further metabolized by epoxide hydrolases to the corresponding dihydroxyeicosatrienoic acids (DHETs). These CYP-derived eicosanoids are of interest since they are endogenous constituents of numerous tissues and posses a wide array of biological effects. The cell specific pattern of expression, relative abundance, and activity of the enzymes catalyzing these reactions will be a major determinant of the intracellular effect of these eicosanoids. In the kidney, CYP-derived eicosanoids have potent effects on renal vascular tone and tubular ion transport and have been implicated in the regulation of blood pressure. The overall goal of the proposed studies is to understand the molecular mechanisms controlling CYP-catalyzed AA metabolism in the rat kidney and the importance of renal CYP eicosanoid levels in blood pressure regulation.
The specific aims of the proposed studies are (1) to examine the mechanistic basis of the antihypertensive effect of inhibition of AA omega-hydroxylase activity. Specifically, we will determine the isoform-specificity and potency of mechanism-based inhibitors of CYP AA omega-hydroxylases and evaluate their effect on blood pressure and vascular tone; (2) to determine the contribution of CYP4E isoforms to renal AA metabolism. CYP4F expression will be localized in the rat kidney and AA metabolism by the CYP4F isoforms will be characterized; (3) to isolate the major CYP2J epoxygenase expressed in the rat kidney. The cDNA encoding the CYP2J2 immunoreactive protein overexpressed in the spontaneously hypertensive rat kidney will be identified by expression cloning and functionally characterized; and (4) to examine the regulation of EET hydrolysis in the rat kidney. Specifically, we will determine the biochemical basis of EET hydrolysis in rat renal microsomes and the effect of soluble epoxide hydrolase inhibition on blood pressure and renal eicosanoid formation. The findings from these studies will lead to a comprehensive understanding of the expression and regulation of the major CYP and epoxide hydrolase enzymes involved in AA metabolism. A long term goal of these studies is to develop novel targeted therapeutics for the regulation of renal CYP eicosanoid production and blood pressure. Application of these principles for regulating CYP eicosanoid formation in other tissues is anticipated.
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