IMMORTALIZATION OF BOVINE PANCREATIC DUCT EPITHELIAL-CELLS

Pancreatic duct cell lines have been isolated from a number of animal and human tumors, but none appear to express ion transport properties expected for differentiated pancreatic duct epithelial cells. We sough t to generate an immortalized ductal cell line from well-differentiate d primary cultures of bovine pancreatic duct epithelium. Epithelial ce lls from the main duct of the bovine pancreas were isolated and immort alized by transfection with a DNA construct encoding simian virus 40 l arge T antigen. A single clone (BPD1) survived negative selection and was maintained in culture for >100 passages over 2 yr. The cells grow readily in culture as monolayers and express several properties charac teristic of differentiated pancreatic ductal epithelium. The cells do not appear to form a functional tight junction complex, since the tran sepithelial resistance of the monolayer cultures grown on a permeable support is <10 Omega . cm(2). Northern blot analysis revealed that the cells continue to express simian virus 40 large T antigen and contain significant levels of mRNA for proteins thought to be important in tr ansepithelial bicarbonate secretion [carbonic anhydrase II, Cl-/HCO3- exchanger, Na+/H+ exchanger, and cystic fibrosis transmembrane conduct ance regulator (CFTR)]. In vivo pancreatic ductal secretion is stimula ted by the peptide hormone secretin. The secretin receptor is expresse d and functionally coupled to adenylate cyclase in the immortalized ce lls, since secretin caused a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP; similar to 20-fold increase over bas al levels) with a mean effective concentration of 15 nM. Elevation of intracellular cAMP by exposure of the cells to forskolin (10 mu M) or secretin (0.1 mu M) increased plasma membrane Cl- permeability, most l ikely mediated by activation of CFTR. The results of these studies dem onstrate that the pancreatic duct cell line (BPD1) retains several pro perties exhibited by the secretory epithelial cells that line the panc reatic ductal tree. This cell line should prove useful for studies of expression, function, and regulation of pancreatic duct cell proteins.