It is now evident that P450 enzymes metabolize key cellular constituents, including long-chain fatty acid such as arachidonic acid (AA), leukotrienes (LT) such as LTB4, and other eicosanoids. P450-mediated oxidation of these lipid-derived endobiotics results in products with potent effects on ion transport and vasomotion, or results in their inactivation, as observed with the proinflammatory LTs and prostanoids. Yet, the contribution of specific P450s to the formation and/or catabolism of fatty acids and eicosanoids remain unclear, as does the regulation of these endobiotic-metabolizing enzymes. In this competitive renewal proposal, we will continue our studies on P450 enzymes comprising the CYP4A and CYP4F gene subfamilies (i.e., CYP4F/A P450s) which, in human liver and kidney, function as the principle fatty acid and eicosanoid omega-hydroxylases. We will extend our studies on arachidonic acid (AA) and leukotriene B4 (LTB4) omega-hydroxylation by CYP4F2 and CYP4A11 to now include CYP4F3b, CYP4F11, and CYP4F12, as the latter CYP4F gene products, which have recently been cloned, can catalyze the same eicosanoid-metabolizing reactions, at least in heterologous expression systems. We will clarify the discrepancies described in substrate specificities between native and recombinant human CYP4F/A P450s, especially with regards to 20-HETE formations, in light of this oxygenated AA product's physiological importance. We will determine whether the CYP4F omega-hydroxylases embody an important control point in hepatic inflammatory processes and/or disease states by gauging their capacity to convert LTB4 to an oxygenated metabolite which, unlike its precursors, is devoid of all inflammatory properties. The molecular basis for CYP4A11 catalysis of AA omega-hydroxylation in kidney but not in liver, despite its expression in both tissues, will be pursued. RNA and protein quantitation studies will be performed to a) determine expression of the diverse CYP4F/A proteins in liver and kidney and; b) to assess whether these P450s are localized in kidney nephronic regions implicated in blood pressure control and/or ion transport. Primary cultures of human hepatocytes will be employed to assess if fatty acids or hypolipidemic agents capable of altering circulating fatty acid levels are also capable of influencing expression of hepatic CYP4F/A P450s, and if ethanol, which alters the AA content in cellular membranes, induces these CYP4F/A proteins as it does CYP2E1.
Our aims are to define the role of the human CYP4F/A P450 enzymes proteins in the disposition of endogenous fatty acids and bioactive eicosanoids and, ultimately, to reveal if variations in the metabolism of such agents by members of the CYP4 gene family could perturb normal physiological processes in the liver or kidney, such as blood flow, ion transport, the control of inflammation, and the intracellular flux of fatty acids.
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