SALT AND WATER TRANSPORT IN PANCREAS DUCT EPITHELIUM

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The exocrine pancreas secretes digestive enzymes and an isosmotic fluid. The digestive enzymes are packaged and stored in, and released from zymogen granules located in acinar cells. In most species the acinar cells also secrete a small volume of isosmotic NaCl solution into the lumen. In contrast, the ductal epithelial cells which comprise only approximately 5% of the mass of the pancreas secrete a large volume of an isosmotic solution. Pancreatic juice is alkaline and the [HCO3-] ranges from approximately 60 mM in rat to approximately 150 mM in pig. Little is known about the mechanisms of salt and water transport across pancreatic ductal epithelium. It is the goal of this research proposal to begin to understand at the cellular level the mechanisms of salt and water secretion by ductal epithelial cells. Transepithelial electrical and ion flux experiments, in conjunction with fluid transport measurements, will be done to determine baseline and stimulated transport properties of ductal cells. Intracellular microelectrode experiments will be done to determine at the single membrane level electrical driving forces and to identify electrodiffusive permeabilities. Intracellular ion activities will be measured with ion sensitive microelectrodes and/or by the use of fluorescent intracellular ion indicators. Such measurements will provide information, at the single membrane level, about the magnitude and direction of chemical driving forces. Furthermore, changes in intracellular ion activity in response to experimental perturbations will be used to identify electrically neutral transport mechanisms (e.g., exchangers, cotransporters). Patch clamp techniques will be used to identify and study in detail channels that are implicated by intracellular microelectrode studies as important components of transepithelial ion transport. A primary cell culture of main duct epithelial cells will be used because: 1) cell lines derived from pancreatic tumors are poorly differentiated and in general of uncertain origin (ductal vs. acinar), 2) the complex morphology of pancreas makes experiments with small ducts technically difficult, 3) cell culture will allow for amplification of biologic material for biochemical and molecular biological studies, and 4) the approaches and techniques developed for the study of bovine pancreas should be directly applicable to the study of salt and water secretion by human pancreas. The research outlined in this proposal will aid in our understanding of ion transport by mammalian pancreatic ductal epithelium and provide a model system for the study of regulation of HCO-3 secretion. The methods developed and the information obtained from these experiments will bc directly applicable to similar studies with normal and diseased human pancreas. This is particularly relevant to cystic fibrosis, a disease in which pancreatic salt and water secretion are severely compromised.