FUNCTION AND REGULATION OF CYTOCHROME P450 4A ISOFORMS

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This is a proposal to investigate the mechanisms underlying the regulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis in vascular and tubular cells of the rat kidney. Our studies have established that 20- HETE, the omega-hydroxylation product of arachidonic acid (AA), is the principal AA metabolite formed in tubular and vascular structures of the rat renal cortex and outer medulla. We and others have demonstrated that 20 HETE is endowed with potent biological activities and have provided evidence that it contributes to the regulation of renal vascular and tubular functions and to the control of arterial pressures. The omega- hydroxylation of fatty acids, including AA, is catalyzed by enzymes of the CYP4A family including 4A1, 4A2, 4A3 and 4A8. Our studies indicated that despite the high homology, these isoforms display distinct catalytic properties including differences in kinetic parameters, product profile and inhibitor sensitivity. We hypothesize that the rat CYP4A enzymes, although sharing very high sequence homology and a common unique, i.e., hydroxylation of arachidonic acid at the omega carbon to form 20-HETE, exhibit different catalytic activities and substrate specificities, and display distinct patterns of localized and ,most likely, are under different regulatory mechanisms, thereby, distinct contributing to the endogenous level of 20-HETE, exhibit different catalytic activities and regulatory mechanisms, thereby distinctively contributing to the endogenous level of 20-HETE in a given tissue. To understand the regulatory mechanisms, thereby, distinctively contributing to the endogenous level of 20-HETE in a given tissue. To understand the regulatory mechanisms underlying the renal synthesis of this important eicosanoid, the proposed studies are designed to further identify differences among these isoforms at the biochemical and molecular levels with respect to catalytic activity and regulation of expression (Aim 1) and, secondly, to implicate such differences in terms of cell and renal function in vitro (Aim 2) and in vivo (Aim 3). These cellular, biochemical, and molecular studies of CYP4A-mediated, 20-HETE production will increase our understanding of the physiological function of 20-HETE and its possible involvement in the pathogenesis of hypertension and cardiovascular diseases.