PHARMACOLOGICAL EFFECTS OF CYTOCHROME P450 4A METABOLISM

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DESCRIPTION: (Adapted from the Applicant's Abstract) Renal cytochrome P450-mediated eicosanoid production is an important determinant of integrated kidney function and renal vascular tone. In the spontaneously hypertensive rat (SHR), alterations in the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) by enzymes of the cytochrome P450 4A (CYP4A) subfamily have been associated with increases in blood pressure and changes in renal function. In the proposed studies the spontaneously hypertensive rat will be used to test the hypothesis that CYP4A-mediated hydroxylation of arachidonic acid is involved in the development of hypertension. Based on differences in renal arachidonic acid and omega-1 hydroxylase activity, Dr. Kroetz and her colleagues hypothesize that one or more of the CYP4A genes are differentially expressed in the hypertensive rat kidney as compared to its normotensive control. Gene-specific ribonuclease protection assays will be used to distinguish between the mRNA levels of CYP4A1, CYP4A2, CYP4A3 and CYP4A8 in the renal cortex and medulla, and to localize their expression to specific segments of the nephron. Expression patterns will be correlated with 20-HETE formation rates to assess the relative contribution of each CYP4A isoform in the generation of this prohypertensive eicosanoid. The specific aims of this proposal are: 1) to identify differences between hypertensive and normotensive rats in the developmental pattern of expression and the intrarenal distribution of mRNAs encoding cytochrome P450 4A enzymes in the renal cortex and medulla; 2) to establish the relationship between blood pressure and CYP4A expression by manipulating arachidonic acid omega-hydroxylase activity through induction of the CYP4A genes or mechanism-based inactivation of the CYP4A proteins; and 3) to characterize the metabolic profile for arachidonic acid oxidation by each of the renally expressed CYP4A proteins using an in vitro protein expression system. Knowledge of the renal CYP4A mRNA expression levels and distribution patterns in hypertensive and normotensive rats, the effect of modulation of CYP4A expression on blood pressure, and information about whether a given CYP4A gene product metabolizes physiological concentrations of arachidonic acid to prohypertensive metabolites will provide evidence for the involvement of the CYP4A family in the regulation of blood pressure. These data will provide a basis for investigating a similar mechanism for the pathophysiological changes associated with essential hypertension in humans. The long term goal of this program is to use this knowledge to develop therapies for targeted modification of blood pressure.