Several renal cytochrome P-450 (CP-450) isoenzymes have been shown to catalyze the oxidative metabolism of arachidonic acid to epoxyeicosatrienoic acids (EETs), and omega and omega -1 alcohols (19- and 20- OH- AA). there is a rapidly growing interest in defining the biological significance of this pathway of arachidonate metabolism. An impressively increasing number of published studies demonstrate that metabolites from this pathway exhibit numerous interesting actions on cellular functions as diverse as peptide hormone secretion by cells from endocrine organs, sodium transport by various epithelial cells, contraction or relaxation by smooth muscle cells, and even proliferation of several cell types. A significant but smaller number of studies have addressed whole organ physiology, predominantly the kidney, and demonstrated that compounds from this pathway have important regulatory effects on renal blood flow, glomerular filtration rate, and renal sodium excretion. A few studies have even suggested some link between CP-450 arachidonate metabolism in the kidney and hypertension in two popular genetic models of hypertension -- the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive rat. The six scientific projects and two scientific cores of this proposal represent a multi-discipline approach employing a range of techniques from in vivo studies to molecular biology designed to clearly define the functional significance of the CP-450 arachidonate metabolites in the kidney. Project #1 will utilize in vitro microperfusion and focus on regulation of sodium transport in the thick ascending limb and collecting duct. The biologic significance of cross metabolites (CP-450 and cyclooxygenase) will be examined. Project #2 will focus on the biochemical and chemical characterization of the renal cytochrome P-450 arachidonate monooxygenase isoforms and investigate the regulation of these enzymes by physiologically important manipulations of whole animals (rats); Project #3 will focus on the interesting observation that the biologic activities of certain P-450 arachidonate metabolites are cyclooxygenase dependent. These cross-metabolites and the role of transcellular metabolism in their generation will be characterized; Project #4 will clone the normal and dietary salt-induced epoxygenase genes from rat kidney and pursue the molecular basis for the absence of salt regulation of the epoxygenase in Dahl salt-sensitive rats; Project #5 will explore the regulation of vascular tone and proximal tubule sodium reabsorption by CP-450 arachidonate metabolites in response to acute and chronic volume and/or pressure overload. A powerful combination of in vivo and in vitro models are proposed; Project #6 will develop synthesis strategies for producing the important eicosanoids from this pathway and will develop and prepare selective inhibitors of the important CP-450 arachidonate metabolizing enzymes. Attempts will also be made to synthesize eicosanoid analogs with modified activity or stability. These six projects will be supported by two scientific cores which can be briefly described as the analytic core (Core B) and the chemistry core (Core C). Core B will apply standard methods of eicosanoid extraction, purification, HPLC, GC/MS, protein immunoblotting, and cDNA purification to characterize and quantify the enzymes and the metabolites of the CP-450 pathway. Core C will provide labeled and unlabeled eicosanoids, standards, metabolite analogs, and inhibitors for biologic studies, as assay standards, for structural elucidation and for methods development. The combined efforts of the scientists involved in this program will define the biologic relevance of the cytochrome P-450 arachidonate pathway and its role in both normal physiology and, possibly, the important clinical disorder of hypertension.
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