P450 enzymes metabolize not only exogenous chemicals but also participate in the disposition of key endogenous compounds, including fatty acids, prostaglandins, and leukotrienes. P450- mediated oxidation of these lipid-derived endobiotics gives rise, at least in animals, to products with potent biological effects, including vasoactivity and ion transport inhibition, or can result in their inactivation, as noted with prostanoids and proinflammatory leukotrienes. To that end, we will continue our characterization of two hepatic CYP4 gene family enzymes, namely CYP4A11 and CYP4F2, that we have isolated from human liver, and have thus far shown to be the principle catalysts of arachidonate metabolism to 20-HETE, a potent vasoconstrictor and Na+K+-ATPase inhibitor. Initially, we will examine the capacity of these P450s to omega-hydroxylate other long-chain fatty acids such as palmitate and stearate to products with potential physiological impact, and to oxidize prostaglandins A1 and E1 as well as leukotrienes B4 and E4, thereby inactivating these potent mediators of signal transduction processes. Immunochemical studies will be utilized to: a) reveal if CYP4A11 and/or CYP4F2 catalyze these same reactions in intact liver and kidney tissue; b) assess the distribution of these P450s within the hepatic acinus and kidney nephron and; c) to determine if the interindividual variations noted in fatty acid and eicosanoid metabolism by these tissues stem from differences in CYP4 enzyme levels. Cultures of human hepatocytes will then be employed to assess if therapeutic hypolipidemic agents that are capable of altering fatty acid homeostasis, such as clofibrate and gemfibrozil, are also capable of increasing CYP4A11 and/or CYP4F2 expression, and if ethanol, which increases lipid deposition in liver, induces these CYP4 proteins as it does CYP2E1.
Our aims are to define the role of human CYP4A11 and CYP4F2 in metabolism of important endogeous fatty acids and signal transduction agents in both liver and kidney, and to reveal if variations in endobiotic metabolism by the CYP4 enzymes can indeed perturb normal physiological processes, including the regulation of renal function, the control of inflammation, and cellular lipid flux.
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