NADPH-DEPENDENT METABOLISM OF ARACHIDONIC ACID

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The cytochrome P450 (P450)-dependent arachidonic acid (AA) monooxygenases, are now recognized as the third branch of the bio-medically important AA cascade. The most extensively characterized of the P450 eicosanoids, the epoxy- and the 19/20-hydroxy arachidonic acids, have been implicated in the control of vascular reactivity, ion fluxes, hormonal signaling and the pathophysiology of experimental hypertension. However, due to paucity of appropriate molecular tools, the mechanisms of action and physiological significance of these metabolites remains to be established. The hypotheses are that: A) the intracellular biosynthesis of specific P450 eicosanoids provides important lipid mediators of cellular and organ function, B) oxidative metabolism plays an important role in the control of cell/organ P450 eicosanoid levels and thus, biological function and potency, and C) the ligand activated peroxisomal proliferator activated receptor alpha (PPARalpha) participates in the coordinated regulation of P450 eicosanoid and fatty acid oxidative metabolism. Inasmuch as eicosanoid informational content, signaling properties, and biological role are controlled by enzymatic, isoform specific, regio-, and stereoselective oxygenations of the AA molecular template, this project will utilize combinations of recombinant DNA, biochemical, and bioanalytical techniques for: a) the design and construction of regioselective AA monooxygenases and their expression in cultured cells, b) the documentation of cellular P450 eicosanoid biosynthesis and metabolism, c) the study of cDNA-dependent cellular phenotypes, and d) the functional and biochemical analysis of the role(s) of peroxisomal beta-oxidation and microsomal omega/omega-1 hydroxylation in P450-eicosanoid metabolism and function. The overall goals are to provide a biochemical and molecular description of the role(s) of this pathway in organ/body physiology and/or pathophysiology. The documented cellular and systemic effects of the P450 eicosanoids and their potential biomedical significance continue to provide the rationale for the studies of this metabolic pathway. The experimental answers to the questions posed by these hypotheses are needed for: a) the unequivocal definition of the physiological and/or pathophysiological significance of the P450 AA monooxygenases, and b) the formulation of effective strategies for future pharmacological and/or clinical intervention.