Polarized secretory epithelial cells possess several sorting devices to effect vectorial flow of secretory and membrane proteins to their apical and basolateral poles. Our previous studies on pancreatic acinar cells have delineated the kinetics and routes of intracellular transpost of secretory proteins that enter a secretagogue activated (regulated) pathway leading to apical exocytosis while a separate secretory pathway results in basolateral, nonstimulated (constitutive) discharge of basement membrane proteins. Similarly, vectorial delivery of membrane proteins to the apical and basolateral poles of secretory cells is required to establish and maintain structural and functional poolarity. The goal of this project is to define the routes and control mechanisms for constitutive and regulated secretory pathways in pancreatic acinar and other epithelial cells and to characterize the pathways responsible for biogenesis of plasmalemmal polarity. To this end, we will use pancreatic acinar and other cell lines cultured in chambers that allow probing of apical and basolateral secretory compartments. Agents that perturb secretory protein sorting will be tested for their ability to affect differentially apical (e.g. amylase) or basolateral (e.g. basal lamina) secretory pathways. These will include secretagogues, acidothrophic agents, inhibitors of glycosylation and protein synthesis and drugs producing cytoskeletal disassembly. The effects of reduction in temperature and ATP levels will also be examined. Comparable experimental conditions will be used, in conjunction with immnoassays for polarity of apical (gamma-glutamyl transferase) and basolateral (insulin and laminin receptors) membrane protein delivery, to define factors regulating biogenesis of plasmalemmal polarity and to clarify relationships between secretory protein and membrane protein delivery pathways. We will also examine membrane targeting in MDCX cells transfected with cDNAs for gamma-glutamyl transferase, an apical and basolateral membrane protein in pancreatic acinar cells. Finally, we plan to study biogenesis of the regulated secretory pathway in developing pancreas as it acquires secretagogue responsiveness.
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