Arachidonic acid (AA) is the precursor of an array of potent oxidation products (eicosanoids) whose role in inflammation and hypersensitivity reactions is widely recognized. The AA is bound in the 2-position of membrane phospholipids and must be released before conversion to eicosanoids. In PMN phospholipase A2 appears to be the major enzyme responsible for release of the AA and the choline-containing phosphoglycerides (PC) appear to be a major donor of AA. Earlier studies revealed that the PC of human PMN is comprised of 46% alkyl ether-linked species enriched in AA. Further studies demonstrated that l-0-alkyl-2-AA-sn-glycero-3- phosphocholine (l-0-alkyl-2-AA-GPC) can serve as a precursor of both eicosanoids and platelet activating factor (PAF), another powerful mediator of PMN function. A series of studies have indicated that the eicosanoids and PAF act in concert to elicit activity. The PC was also recently shown to yield alkyl-ether- linked diglycerides (DG) upon stimulation, possibly providing an alternate route to the phosphatidylinositol cycle for production of second messenger DG. The alkyl-DG was found to potentiate the activity of other agonists in the activation of PMN but differed in its effects from those of diacyl-DG. The alkyl-DG may be able to terminate responses elicited through protein kinase C activation. The formation of products from l-0-alkyl-2-AA-GPC will be investigated in PMN by prelabeling the membrane lipids with labeled l-0-alkyl-2-AA-GPC and studying the conversion to products in both whole cells and cell-free systems. A goal of the study is to characterize the phospholipase A2 responsible for the release of AA, and the phospholipase responsible for the formation of DG from PC. Another goal is to determine the subcellular distribution of AA and the molecular species distribution of AA in various subcellular organelles and to identify the specific donor(s) of AA in stimulated PMN. A highly specific CoA-independent transacylase acylates l-0-alkyl-2-lyso-GPC with AA. This transacylase is to be further characterized. In PMN obtained from monkeys maintained on a fish oil-enriched diet, a dramatic shift in the pattern of acylation of l-0-alkyl-2-lyso-GPC was observed; the major product formed contained eicosapentaenoic acid (EPA) rather than AA. Beneficial effects on cardiovascular and inflammatory disorders have been attributed to EPA. In the proposed studies the possible influence of EPA-containing phospholipids on the interrelationships of AA and PAF metabolism will be explored.
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