NADPH-DEPENDENT METABOLISM OF ARACHIDONIC ACID

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The oxygenated metabolism of arachidonic acid is characterized by the increasing complexity of its metabolic pathways and products and, by the diversity of functional properties attributed to them. While the metabolic versatility of the arachidonate molecular template allows for the storage and transfer of an exceptional degree of functionally meaningful chemical information, the integration of these multiple reactions and of their associated products and biological activities to cell, tissue or body physiology is an important task which, although difficult and complex, is greatly facilitated by the understanding of the biochemistry of the reactions, the structure of metabolites and the molecular properties of the enzymes involved. The microsomal cytochrome P-450 arachidonic acid epoxygenase catalyzes the enantioselective formation of four regioisomeric epoxyeicosatrienoic acids (EETs). The asymmetric nature of the EETs present, endogenously, in several tissues demonstrated their enzymatic origin and confirmed cytochrome P450 as the in vivo arachidonic acid epoxygenase and, as a formal member of the fatty acid metabolic cascade. The last few years have seen a renewed interest in the delineation of the role of cytochrome P-450 in the bioactivation of arachidonic acid and, therefore, in its significance for cell and organ physiology. Many of these efforts have been stimulated by the potent biological activities of the EETs as mediators for peptide hormone release, vasoactive molecules and modulators of membrane ion fluxes. More recently, several lines of evidence suggest that this pathway may be involved, in yet undefined ways, in the pathophysiology of hypertension. The potential physiological and clinical implication of this proposal are of pivotal importance and area therefore, stimulating extensive research. During the last 10 years, this project completed a detailed characterization of the enzymology and the structural properties of the epoxygenase metabolites, those studies contributed substantially to our present understanding of the biochemical and functional significance of this pathway. Further progress will require a detailed molecular knowledge of the enzymes involved, as well as the development of the biospecific probes needed for studies of the regulation of this pathway at the protein and gene level. We propose to utilize modern molecular biology techniques in order to identify and characterize the cytochrome P-450 isoforms involved in the generation of endogenous EETs. We propose to clone the relevant gene(s), to express the cloned cDNA to characterize the recombinant protein(s), to generate immunospecific probes and to study the role of recombinant proteins in cell EET and calcium metabolism.