Platelet activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3- phosphocholine; 1-0-alkyl-2-acetyl-GPC) is a potent biologically active ether-linked phospholipid. It causes human platelets and neutrophils to degranulate and aggregate at 10-10 to 10-9M concentrations, and appears to be even more active in the presence of other mediators such as 5-hydroxyeicosatetraenoic acid (5-HETE). When injected intravenously into rabbits, baboons or guinea pigs, it causes severe anaphylactoid reactions; in rats it is hypotensive. PAF has been shown to have numerous other activities including injurious effects on the cardiovascular system. It is synthesized by a number of cells including neutrophils, macrophages, endothelial cells, and platelets. In stimulated PMN, PAF is synthesized from membrane-bound 1-0-alkyl-acetyl-GPC by a phospholipase A2 reaction followed by acetylation of the lysoPAF released. The studies outlined are designed to elucidate further the biochemical pathways responsible for the metabolism of PAF and to explore how the enzymatic reactions are regulated; they will focus on the mechanism of action of PAF. Studies of the previous grant period have revealed that 1-0-alkyl-2-arachidonoyl-GPC can serve as a precursor of both PAF and bioactive arachidonic acid (AA) metabolites. Our hypothesis is that the AA-linked species are obligate precursors, and that the lipoxygenase products formed act on concert with PAF to elicit activity. This hypothesis will be tested by measuring the molecular species of 1-0-alkyl-2-acyl-GPC before and after stimulation using a newly developed HPLC procedure to determine if the AA-linked species is selectively hydrolyzed. The subcellular distribution of the PAF precursor will be determined, and the subcellular origin of PAF investigated. emphasis will be placed on determining the mechanisms by which the phospholipase A2, acetyl transferase and acetyl hydrolase responsible for PAF metabolism are regulated. The role of diglycerides acid protein kinases will be examined in intact cells and cell-free systems. Considerable evidence for the existence of a specific PAF receptor has been obtained by the investigators and others. The receptor is to be further characterized and attempts will be made to purify it. A novel ethanolamine plasmalogen analog of PAF was recently identified by us. Its biochemistry and bioactivity will be explored. In recent studies of PMN from monkeys maintained on a fish-oil-enriched diet, we found a major shift from AA to eicosapentaenoic acid in the acylation of lysoPAF. Effects of the diet on PAF synthesis and on responses to PAF will be investigated.
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