We propose to dissect the molecular topology of membrane signalling, and each aim will exploit novel findings made in years 15-20:
SPECIFIC AIM 1 : In neutrophils and their organelle-free cytoplasts, we will determine the disposition and function of phosphatidic acid (PA) and diacylglycerol (DAG), formed within 5 sec of ligand/receptor interactions. We will determine if and when PA is available at the outer face of the plasmalemma (phospholipase D), if and when PA forms a hexagonal II lipid phase with Ca, by means of phase-specific monoclonal antibodies and whether it is the precursor or product of DAG which is formed far in excess of the amounts of phosphoiositides (PI, PIP, PIP2) broken down.
SPECIFIC AIM 2 : To determine the kinetics of phosphorylation, acetylation and acylation of neutrophil proteins. Since intact microtubules are required for full expression of the 5 lipoxygenase, we will determine if microtubule integrity is required for traffic of di acylglycerol and """"""""site-directed"""""""" translocation of protein kinase C. We will test the novel hypothesis that protein acetylation and acylation are linked to the metabolism of alkylarachidonyl glycerophospholipids.
SPECIFIC AIM 3 : To resolve the """"""""leukotriene B4 paradox"""""""", that neutrophils will make LTB4-the most potent inflammatory eicosanoid - only with greater than 10uM exogenous arachidonic acid (AA). We will determine if AA or a metabolite is required for activation of 5- lipoxygenase, if AA or a metabolite is provided by other cells and if triglycerides derived from cells or plasma lipoproteins (VLDL, LDL via the scavenger pathway) provide AA for lipoxygen ation, using a sensitive lipase assay with Fura II in liposomes.
SPECIFIC AIM 4 : We will test the hypothesis that aspirin-like drugs interfere with neutrophil activation by interfering with signal transduction at the plasmalemma by uncoupling G-protein from receptors (e.g. F-met-leu-phe). Having satisfied four of the criteria of Stryer and Bourne for G-protein transduction, we will test this hypothesis against the remaining two.
SPECIFIC AIM 5 : We will use marine sponge cells which lack cyclooxygenase or lipoxygenase activity as """"""""phylogenetic controls."""""""" Sponges (dating 109 yrs) respond to ligands simply by diverting fre fatty acids from de novo synthesis of triglycerides into diglycerides and phospholipids. Since their activation is inhibited by NSAIDS and elicited by phorbol esters with ionomycin, we will test the roles of C-kinase and G protein in the absence of enzymes which oxidize polyenoic fatty acids (eg 20:4, 22:6).
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