Many of the complex cellular and biochemical events associated with inflammatory and allergic responses are now known to be regulated by a diverse network of mediators. Two lipid classes, platelet activating factor and leukotrienes, act as potent mediators of inflammatory processes. Each of these lipid mediator classes are produced by a variety of inflammatory cells and posses wide and often similar biological effects. The biochemistry of each of these lipid mediator classes in various inflammatory cells has proven to be extremely complex, probably reflecting the complexity of precursor pools and enzyme systems involved in the biosynthesis of these mediators. Recently, it has become increasingly apparent that the metabolism of these two lipid classes are interrelated in a number of inflammatory cells. In this proposal, we will examine the biosynthesis of both PAF and leukotrienes in the neutrophil and the mast cell in order to better understand the biochemical relationships between the two mediators. Since the precursor(s) of both leukotrienes and PAF are thought to be cellular phospholipids, initial studies would investigate, in detail, arachidonic acid-phosphoglyceride turnover in the resting cell. Specifically, these studies would examine the route of arachidonic acid through cellular phosphoglycerides and the subcellular localization of arachidonate-containing phosphoglyceride molecular species within the resting cell. The major hypothesis that this proposal examines is that leukotrienes and platelet activating factor are produced from a common precursor phosphoglyceride. Therefore, we will investigate in the activated neutrophil and mast cell how tightly PAF and eicosanoid biosynthesis are coupled by defining which phosphoglyceride molecular species are mobilized to release arachidonic acid and lyso PAF which forms eicosanoids and PAF, respectively. Initially, this will involve determining if there is selective mobilization of an individual phosphoglyceride molecular species by measuring the loss of potential precursors upon cell activation. In addition, this proposal will further examine this hypothesis by using a new strategy which involves prelabeling cells with lipid mediator precursors and measuring in the same experiment the specific activity of potential precursors as well as products produced during stimulation. Using this strategy, it may be possible to pinpoint an individual precursor pool or combination of pools which give rise to PAF and eiscosanoids as well as to document the rapid turnover of individual phosphoglyceride molecular species. In each of the experiments described in this proposal, high resolution chromatography techniques combined with GC/MS analysis will provide sensitive and selective assays for arachidonic acid, PAF, eicosanoids and phosphoglyceride precursors. By using an approach which examines the biochemistry of both PAF and leukotrienes and points at which their biochemistry intersects, it will be possible to better understand the complex lipid network by which the cells involved in inflammation communicate.

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National Institute of Allergy and Infectious Diseases (NIAID)
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Johns Hopkins University
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Winkler, J D; Sung, C M; Chabot-Flecher, M et al. (1998) Beta-lactams SB 212047 and SB 216754 are irreversible, time-dependent inhibitors of coenzyme A-independent transacylase. Mol Pharmacol 53:322-9
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