The kidney cytochrome P450-dependent arachidonic acid (AA) monooxygenase is now recognized as the third branch of the biomedically important AA cascade. The most extensively characterized of the P450-derived metabolites, the epoxy- and the 19/20-hydroxy AA, have been implicated in processes such as the control of systemic and renal vascular reactivity, ion fluxes, hormonal signaling and the pathophysiology of experimental hypertension. Thus, our studies and that of others indicate significant roles for this enzyme system in the control of kidney and body homeostasis. However, due to paucity of appropriate molecular tools and techniques, the physiological significance of renal P450 and the site and mode of action of its metabolites remains to be unequivocally defined. Project #2, in conjunction with the cell and organ physiology components of this Program Project, proposes to utilize molecular approaches to the development and use of cellular and/or animal models for the study of P450-gene specific functional phenotypes. We will apply a combination of recombinant DNA and biochemical techniques to the design and construction of high turnover, self contained, regio- and enantioselective AA monooxygenases, the expression of these enzymes in cultured cells, and the identification and mechanistic characterization of insert/cDNA-dependent cellular phenotypes. Targeted gene disruption will be employed for the whole animal integrated functional and biochemical analysis of the significance and the role(s) of specific kidney AA epoxygenase and omega/omega-1 hydrolases. The long term goals of this project are to provide a molecular understanding of renal P450 eicosanoid biological significance and mode of action. The answer to these important questions are needed for the development of meaningful approaches to: a) the unequivocal definition of their physiological and/or pathophysiological significance, and b) subsequent pharmacological and/or clinical interventions.
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