In addition to the """"""""classic"""""""" neurotransmitters of the peripheral nervous system, acetylcholine and norepinephrine, roles in the regulation of exocrine gland function have been demonstrated in recent years for several other agents, including ATP and various neuropeptides, including vasoactive intestinal peptide (VIP). In salivary glands, much information has been obtained concerning neurotransmitter regulation of acinar cell function. However, due to the lack of suitable model systems, relatively little is known about neurotransmitter regulation of salivary gland duct cell function. Recently, morphological and biochemical data have been obtained which indicate that the HSG-PA cell line, of human submandibular gland ductal origin, possesses several important characteristics of normal duct cells and thus holds much promise as a model system for studying duct cell- specific functions and their regulation by neurotransmitters. Our studies to date have documented the presence in HSG-Pa cells of receptors for VIP, coupled to adenylyl cyclase, and for neurotensin and ATP, coupled to phospholipase C. Additional preliminary evidence demonstrates agonist- stimulated increases in K+ and C1- fluxes in response to one or more of these agents and alterations of these responses by osmotic challenge, in which exposure of cells to hypotonic medium results in a dramatic enhancement of neurotransmitter stimulated ion (K+) flux whereas hypertonic medium blocks the neurotransmitter-stimulated fluxes. This proposal focuses on neurotransmitter regulation of duct cell ion transport and the effects of osmotic changes on that regulation, using the HSG-PA cell line as a model. First, regulation by VIP, neurotensin and ATP of monovalent ion fluxes will be evaluated. Transcellular ion movements, studied in Ussing chambers, will measure agonist-induced changes in short circuit current in conjunction with radioisotope tracer and ion replacement experiments to identify the transported species. Receptor and non-receptor modulators of intracellular signaling pathways will be used to correlate changes in, and interactions of, second messengers with ion fluxes. Radioligand binding will be used to further characterize the receptors involved. The second area of investigation focuses on identification of the mechanisms by which changes in medium osmolarity modulate ion transport regulation by the neurotransmitters. Studies at discrete steps in the intracellular signaling processes (agonist binding to receptor; G protein involvement, second messenger production, activation of K+ flux) will be performed to define the points in the signaling pathway where osmotic challenges affect neurotransmitter regulation of K+ flux. Together, these studies promise to increase substantially our understanding of the roles of VIP, neurotensin and ATP in regulation of secretory processes in salivary duct cells and the role perturbations in the cells' osmotic environment have in regulation of transport by these neurotransmitters, thus helping to establish a foundation for evaluating the role of alterations in duct cell- specific functions and their regulation in various exocrine disease states.
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