This is a proposal to 1) study the regulation of arachidonoyl-CoA synthesis in mammalian cells, and 2) attempt to identify the pool of arachidonoyl-CoA which is esterified into phospholipid and then released for production of icosanoids. We have recently demonstrated that icosanoid production in cultured cells can be controlled at a step prior to arachidonate release from phospholipid, namely, at the point of arachidonoyl-CoA formation. This represents a newly discovered option for the control of icosanoid formation. Icosanoids are implicated in a multitude of disease processes including atherosclerosis and thrombosis leading to myocardial infarction and stroke and autoimmune disease, produced by uncontrolled inflammation. We have recently shown, using the prostaglandin E2 producing cell line HSDM1C1 and several cell lines unique in various aspects of icosanoid precursor fatty acid metabolism, that arachidonoyl-CoA formation in mammalian cells involves three metabolic pathways: 1) the Delta6 desaturase/elongase/ Delta5 desaturase pathway, 2) the non-specific long chain acyl-CoA synthetase, and 3) arachidonoyl-CoA synthetase. Furthermore, we have been able to alter the flux through these pathways. Using our cultured cell lines, we propose to study the regulation of each of the three pathways leading to arachidonoyl-CoA formation. Specifically, we plan to study: 1) the induction of Delta6 desaturase and control of induction by fatty acid, 2) the identification of a factor(s) in an extract of horse serum which we have shown controls arachidonoyl-CoA synthetase activity, and 3) the induction of non-specific acyl-CoA synthetase activity by micromolar concentrations of various fatty acids. We will also investigate the capacity of arachidonate derived from its precursor fatty acids to enter the pool of arachidonoyl-CoA destined for prostaglandin E2 synthesis. The information from our studies on arachidonoyl-CoA synthesis, a new control point for icosanoid formation, could ultimately have a significant impact on icosanoid-mediated disease.
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