Stimulated polymorphonuclear neutrophils (PMN) metabolize their arachidonate and phospholipid stores into a series of bioactive products: leukotriene (LT)B4, 5-hydroxyicosatetraenoate (5-HETE), prostaglandins (PG), platelet-activating factor (PAF), and diacylglycerol (DAG). We postulate that these products interact to influence functional responses of various tissues as follows. LTB4, PAF, and 5-HETE excite cells by binding to their respective plasma membrane receptors. Receptors for LTB4 and PAF operate by raising cytosolic Ca and activating protein kinase C (PKC) through a linkage cascade involving GTP-binding proteins, phospholipase C, and cleavage of phosphatidylinositol diphosphate into inositol triphosphate and DAG. Inositol triphosphate releases Ca2+ from subcellular pools to cytosol whereas DAG directly activates PKC by a Ca2+-enhanced reaction. Receptors for 5-HETE operate by a different mechanism, (which, we postulate, is synergistic with that used by PAF and LTB4), to raise cytosolic Ca2+ and promote PKC activation. The elevated Ca2+ and activated PKC then initiate function. However, PG and, under certain conditions, activated PKC inhibit function; they may act by down-regulating receptors for LTB4, PAF, and 5-HETE or by interfering with these receptors' linkages to GTP-binding proteins. Our concepts will be tested on human PMN, PMIN cytoplasts, and PMIN isolated organelles in studies that: a) define the subcellular distributions and binding parameters of receptors for LTB4, PAF, 5-HETE, and PG; b) examine these receptors' specific linkages to, and effects upon, Ca2+ fluxes and PKC activation: c) determine the influences of PG and activated PKC on these receptors' binding of ligands and functional linkages to GTP-binding proteins; and d) evaluate the roles of LTB4. 5-HETE, PG, PAF, DAG, Ca2+ and PKC in PMN responses to exogenous stimuli (e.g., chemotactic peptides and calcium ionophores) using pharmacologic agents and other methods that selective abrogate PMN synthesis of or responses to the lipid products. Our studies rely heavily upon the use of PMN under physiological conditions and the reconstruction of relevant cell- free models comprised of purified PMN organelles, PKC, and GTP- binding proteins. Ultimately, we aim to define how the lipid products stimulate diverse cell types and to implicate the products in stimulus-response coupling events. Since these products and their cells of origin appear involved in inflammatory, allergic, anaphylactic, and bronchospastic reactions, this proposal is relevant to the general fields of cellular biology, host defense mechanisms, pulmonary physiology, pharmacology, and clinical medicine.
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