The long-term objective of this project is to elucidate the mechanisms of water and electrolyte secretion in salivary acinar cells. This secretory response is associated with a signal transduction pathway that involves the turnover of membrane phosphoinositides, the mobilization of Ca2+ from intracellular and extracellular compartments and the activation by Ca2+ of monovalent ion fluxes (K, C1) that are critical for secretion. Our studies during the current grant period and those of others have demonstrated complex functional links in this pathway, some of which are only partially understood at present and may or may not operate with all stimuli that enhance salivary fluid and electrolyte secretion. Some elements of signal transduction may show, furthermore, unique features in salivary cells. Our general goal for the new grant period is, therefore, to explore in greater detail these functional associations in salivary cell signaling.
Our specific aims are: 1) to investigate further the coupling between three types of receptors (cholinergic, alpha-adrenergic and substance P receptors and phosphoinositide turnover. This coupling occurs by GTP binding proteins in many cells but little information is available about G protein coupling in salivary cells. We will use immunoprecipitation, competitive radioligand binding and biochemical methods to identify the specific G proteins associated with each one of these receptors, 2) to characterize further the functional properties of Ca2+ storage sties and Ca2+ entry mechanisms and the manner in which they are affected by each type of stimulus. We will use spectrofluorimetric, imaging, isotopic and x-ray microprobe analysis techniques to evaluate changes in cell Ca2+ in the absence and presence of agonists and of substances that can modify the mobilization of internal or external Ca2+, 3) to explore further the functional link between K and C1 conductances and possible regulatory factors, including [Ca2+]i, G proteins, protein kinases and cADP ribose. We will use imaging, spectrofluorimetric, isotopic and x-ray diffraction techniques to measure changes in the content and transmembrane fluxes of K and C1 after exposure to the various agonists and of the substances listed above. We propose to compare these various parameters in control salivary acinar cells an in cells derived from rats treated chronically with drugs (reserpine, atropine) that modify receptor functions and, s shown in preliminary findings, downstream elements in the signaling pathway. These drug treatments cause reduced salivary secretion of fluid and are useful models for the study of salivary hypofunction and its possible relation to altered elements of the signaling pathway. A major cause of xerostomia in humans is the use of drugs acting as receptor antagonist and our studies would contribute to our understanding or normal salivary signaling and of how it may be affected by therapeutic agents.
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