Many of the complex cellular biochemical events associated with inflammatory and allergic responses are now known to be regulated by a diverse network of mediators. Two newly-formed lipid classes, platelet activating factor and arachidonic acid metabolites, have been recognized as potent immunoregulating mediators. Each of these lipid mediator classes possess a wide and often similar range of 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 biosynthetic enzymes. In addition, 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 many of these biochemical interrelationships in the human neutrophil. 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 neutrophil. Specifically, these studies would examine the route of arachidonic acid through neutrophil phosphoglycerides, the biosynthetic pathway and role(s) for newly-found phosphoglyceride such as 1,2-diarachidonoyl-sn-glycero-3phosphocholine and the subcellular localization of arachidonate-containing phosphoglyceride molecular species within the neutrophil. Furthermore, we propose to investigate in the activated human neutrophil 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. The experiments described here will approach this problem by using a new strategy which involves prelabeling neutrophils 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 eicosanoids. In examining each of these proposals, 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 all lipid mediators 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.
