Platelet-activating factor (PAF), 1-alkyl-2-acetyl-sn-glycero-3- phosphocholine (alkylacetyl-GPC), is one of the most potent lipid agonists known. It initiates a variety of both pathological and physiological responses including activation of various blood cells and tissues (e.g., liver. lung, and intestines, etc.), bronchoconstriction, systemic hypotension, inflammatory and allergic reactions, implantation and parturition. Some of these biological effects are shown to be mediated through surface or intracellular receptor(s). We have recently demonstrated CoA-independent transacetylases associated with membranes of HL-60 cells that can transfer the acetate group from PAF to a variety of lipid acceptors. Some of the acetylated analogs synthesized by the PAF-dependent transacetylase, namely, the acetylated analogs of ethanolamine plasmalogens and acyl PAF appear to be biologically less active than PAF, while the biological activities of the other products formed differ from PAF. For example, C2-ceramide (N- acetylsphingosine) can induce cell apoptosis and stimulate protein kinases and phosphatase 2A activity. Therefore, the specific objective of this application is to test the hypothesis that the PAF-dependent (CoA- independent) transacetylases play a central role in mediating some of the PAF biological effects through generating a myriad of intracellular lipid mediators that participate in and interact with various signal transduction pathways. The projects that are planned to achieve this aim are as follows: 1) to produce antibodies and molecular probes for the biochemical, mechanistic, and regulation studies of the transacetylases and to delineate whether one or two isoforms of transacetylases catalyzed the synthesis of acetylated phospholipids and C2-ceramide (also a prerequisite for future understanding of the structural requirements of the enzyme in relationship to its functions), 2) to establish the subcellular localization of the transacetylases so that the biological functions of the transacetylases can be clearly defined, 3) to determine the contribution of CoA-independent transacetylase in the level of acyl analogs of PAF, and 4) to characterize the function of the transacetylase in the biosynthesis of C2-ceramide in intact cells and to demonstrate that C2-ceramide is a naturally occurring lipid mediator. The broad, long-term goal of this proposal is to elucidate the mode of actions of PAF and complex interaction or """"""""networking"""""""" of PAF via unique transacetylases with various signal transduction pathways. Obtaining such information could ultimately be used to modify some of the adverse biological consequences elicited by PAF.
