Human polymorphonuclear neutrophils (PMN) produce and inactivate platelet-activating factor, leukotriene B 4, and 5- hydroxyicosatetraenoate. These lipid mediators stimulate cells by conventional transduction pathways, i.e., by binding to their respective, N-protein coupled, plasma membrane receptors. Compared to other similarly acting stimuli (e.g., peptide hormones), however, the mediators have radically different physiochemical properties and in vivo toxicities. Thus, cells may process and respond to the mediators using novel mechanisms. Our progress supports this notion and suggests the following models. First, the mediators bypass their cell surface receptors to penetrate plasmalemma and reach their intracellular sites of metabolism. Their passage through cytosol may involve soluble transfer proteins. Second, the mediators stimulate their own synthesis while inhibiting each others metabolic inactivation. These effects promote mediator accumulation and bioaction. Concurrently, the mediators rapidly promote mediator accumulation and bioaction. Concurrently, the mediators rapidly (<15 sec) inactivate their receptors. This effect limits the cell's response. The two models make a series of predictions which we will test in PMN and HL-60 cells. Proposed experiments employ chemical, biochemical, and enzymatic techniques to prepare and measure production of diverse cellular phospholipids and arachidonate metabolites; density gradient techniques to isolate PMN organelles; binding assays to measure mediator receptors; and several bioassays to evaluate mediator actions and receptor function. Experiments will examine: mediator/mediator receptor internalization and other movements; binding and transport of mediators by cytosolic carrier proteins; mediator metabolism; subcellular locations, substrate specificities, and activities of enzymes inactivating the mediators; the bioactions of recently defined mediators (e.g., alkenyl acetyl glycerophosphoethanolamine); and function of the receptors as they bind their ligand and their patent cells become mediator-unresponsive. Our studies may expand existing concepts on receptors, stimulus transduction, and ligand processing. Furthermore, the mediators as well as PMN are crucial for host defenses but also contributors to inflammatory, allergic, bronchospastic, anaphylactic, and shock reactions. This proposal, then, is relevant to cellular physiology, pharmacology, hematology, pulmonary diseases, immunology, and clinical medicine.
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