This proposal seeks to develop a model system for mechanistic studies of HCO3 secretion by pancreatic duct cells. To achieve this goal, we will use the pancreatic adenocarcinoma cell line, CFPAC-l, and subclones that have been transduced to express the cystic fibrosis transmembrane conductance regulator (CFTR) in both its wild-type and delta F5O8 mutant forms. We will use a combination of electrophysiological and fluorescence-based transport assays, together with high resolution, morphological analysis, to identify the activities and cellular locations of CFTR, Cl/HCO3 exchange (AE), Na/H exchange (NHE) and K channels in these cells. Our preliminary data indicates that the transport components necessary for HCO3 secretion across pancreatic duct are expressed in CFPAC-1 cells. We will extend these studies to the use of antibodies and cDNA probes for NHE and AE and correlate their expression with transport assays that involve cell pH measurements. We will characterize the epithelial transport properties of electrically-resistive monolayers of these cells and study the regulation of HCO3 secretion by both cAMP- and Ca-dependent agonists. Membrane permeabilization techniques and fluorescence assays will be used to identify the membrane locations of the Cl and K conductance pathways and the NHE and AE mechanisms involved in HCO3 secretion. The use of both wild-type and delta F508-expressing CFPAC-l clones will permit assessment of the relation of the expression and regulation of HCO3 secretory mechanisms to CFTR expression. We will determine whether HCO3 secretory processes depend on cellular differentiation and identify the expression and regulation of the necessary transport components as cells polarize. We will localize CFTR and anion exchange to determine whether these proteins target to the apical membrane together and whether their apical targeting is influenced by cellular differentiation and agonist-dependent stimulation of HCO3 secretion. The results of these studies will have important consequences for our understanding of pancreatic duct cell electrolyte transport mechanisms, the relations of these processes to functional CF gene expression and their differentiation dependence. Analysis of the consequences of CFTR expression will provide a mechanistic explanation for derangements in HCO3 secretion in cystic fibrosis and other pancreatic diseases.
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