Role of Ion Channels in Mononuclear Phagocyte Activation

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DESCRIPTION (provided by applicant): The macrophage is the primary differentiated cell of the mononuclear phagocyte system and plays a major role in host defense against microbial infections. Phagocytes possess a diversity of plasma membrane receptors which recognize and bind both particulate and soluble stimuli found in body fluids. The activated macrophage secretes a large array of small and macromolecular products including, cytokines, chemokines, growth factors, and oxidative metabolites. While it is well known that receptor-ligand binding is the initial step in the intracellular signal transduction cascades that eventuate secretion and/or phagocytosis, the calcium and G protein dependence of the pathway(s) which links receptor binding and cross-linking to vesicle fusion is still unclear. Studies in this application are directed at the development of a model for secretion in phagocytic cells following stimulation with both soluble and particulate stimuli. The regulation of the secretory process, the proteins involved, and the identity of the vesicles themselves remain poorly understood. We will examine the regulation of the secretory process by independent manipulation of internal calcium concentration and GTP-gamma-S. Preliminary studies strongly support a model in which Ca and G proteins act independently of one another in the regulation of granulocyte secretion. It is our goal in this application to determine the complementary roles of G proteins and cytosolic calcium in the modulation of secretion induced by Fe-receptor cross linking. We will use a combination of single cell capacitance measurements coupled with amperometry and fluorimetry to determine the contribution of the two pathways and determine a possible mechanism of action. It is our hypothesis that cytosolic Ca helps mobilize vesicles from a reserve pool and promotes docking at the plasma membrane while G proteins trigger the actual fusion step. In the development of a comprehensive model, we will (1) examine the complementary roles of cytosolic Ca and G proteins in the mobilization and docking steps of the secretory process, (2) investigate the identity of the Ca receptor in the secretory response, and (3) identify the G proteins involved in the vesicle fusion process.