NADPH-DEPENDENT METABOLISM OF ARACHIDONIC ACID

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The role of lipids as important mediators of cell function and physiology has become increasingly evident in the last few years. Arachidonic acid is metabolized by mammalian cells through the "arachidonate cascade" to a variety of products with important biological activities and which have been implicated in the pathophysiology of many disease states. Microsomal cytochrome P- 450 is an active catalyst for the oxygenated metabolism of arachidonic acid, and studies from several groups have suggested a role for this important enzyme system in the bioactivation of arachidonic acid. The NADPH-dependent epoxygenase reaction catalyzes the stereospecific formation of four regioisomeric epoxyeicosatrienoic acids (EETs). The EETs have been documented as endogenous constituents of several mammalian organs as well as of human urine. In addition to their in vitro vasoactive properties and effects in renal physiology, the EETs are potent in vitro stimuli for the release of several peptide hormones. Significantly, a link between EET stimulated hormone release and changes in cell calcium metabolism has been documented. The presence in rat liver of two novel pools of phosphatidylinositol (EET-PI) and ethanolamine (EET-PE) containing an esterified EET moiety has been described. Quantitative analysis showed that the mole fraction of EET-PI is 7-8 times that of EET-PE. We believe that the importance of this finding must be analyzed in terms of the pivotal role of inositol lipids in receptor mediated signal transduction, calcium mobilization and generation of second messengers, the described biological activities of the EETs as well as the possible role of the epoxygenase reaction in the generation of unique pools of bioactive lipids. By the use of stable and radioisotopes and established extraction, HPLC and GSH procedures, we plan to 1) characterize the molecular structure of the glycerolipids present in rat liver, 2) study the enzymology of epoxidation of arachidonylglycerolipids, 3) study the enzymology of esterification of synthetic EETs, 4) study the hydrolysis of EET-PI by phospholipase C, and 5) utilize rat hepatocytes as an integrated model of the above reactions in order to begin to understand their possible role in cell function.