""""""""Priming"""""""" of granulocytes (PMNL) signifies an increased responsiveness to stimulation in vitro. The respiratory burst is important to the bactericidal activity of PMNL and priming of the burst may be an important aspect of host defense in such patients. Normal PMNL can be primed in vitro by preincubation with certain agonists, followed by stimulation with a second agonist. We hypothesize that priming involves synergistic convergence of separate, collateral stimulus-response coupling mechanisms. Since it is effective for over 10 minutes, priming can be used to dissect transductional mechanisms into sequential events. This facilitates analysis of causal relationships among the many """"""""early events"""""""" occurring after stimulation of membrane receptors. 1,2-diacylglycerols prime both initiation (lag time, initial rate) and duration of the burst stimulated by formyl-met-leu-phe or by phagocytosis. 1-0-alkyl-2-acylglycerols prime only initiation. Thus, initial activation of the NADPH oxidase and the maintenance of ongoing oxidase activation are separable and will be probed with this model system. Priming and primed stimulation are hypothesized to involve interactions between 1) diglycerides, 2) arachidonic acid (AA) release and metabolism, 3) altered membrane structure, 4) synergy between DGs and/or AA with Ca++ in activation of Ca++ -dependent enzymes. These synergistic mechanisms are proposed to cause 5) structural modification (e.g., protein phosphorylation) and 6) translocation to the plasma membrane of intracellular components with assembly and activation of the NADPH oxidase. The project is therefore divided into 6 specific aims as follows: 1) Using synthetic diglyceride analogs, define the molecular specificity for priming of initiation and prolongation of the burst. Use these models in aims 2-6. 2) Determine the role of PLA2 activation by direct measurement of AA release and metabolism; determine that primed-stimulation also involves increased sensitivity to AA-mediated events. 3) Determine physical membrane changes induced by priming and primed stimulation using several fluorescent and resonance methods. 4) Determine changes in cytosolic (Ca++), 45Ca fluxes, and the effects of chelation of intracellular Ca on each of the parameters. 5) Define protein phosphorylation occurring with priming and primed stimulation. 6) Define translocation of oxidase components, oxidase kinetics and changes in concentration of the oxidase substrate, NADPH, in each model system.
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