ISOLATION AND CULTURE OF BOVINE PANCREATIC DUCT EPITHELIAL-CELLS

We describe a method to isolate and culture epithelial cells from the main duct of the bovine pancreas. In primary cultures, secretin caused a dose-dependent increase in intracellular adenosine 3',5'-cyclic mon ophosphate (cAMP) and stimulated electrogenic transepithelial ion tran sport. Elevation of intracellular cAMP increased the rate coefficient for Cl-36(-) efflux from 0.14 +/- 0.03 to 0.47 +/- 0.12 min(-1), and p lasma membrane conductance, measured by the whole cell patch-clamp tec hnique, was increased from 0.7 +/- 0.1 to 6.9 +/- 0.8 nS. The cAMP-act ivated anion currents had properties similar to those mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Ce lls grown on permeable supports formed confluent monolayers with high transepithelial electrical resistance (1,004 +/- 96 Omega.cm(2)) and g enerated a lumen negative transepithelial voltage difference (-2.5 +/- 0.6 mV). The short-circuit current (I-sc) was increased by forskolin or secretin and was inhibited 87 +/- 4% by addition of ouabain (100 mu M) to the basolateral bathing solution. Replacement of bathing soluti on Cl- by cyclamate reduced the forskolin-induced steady-state increas e in I-sc from 5.3 +/- 0.5 to 0.2 +/- 0.2 mu A/cm(2), suggesting that the stimulated current is due to anion secretion. The results of these studies demonstrate that large numbers of pancreatic ductal cells can be isolated and grown in primary cell culture. The monolayers express differentiated functions and will be useful for studies of acute and chronic regulation of ion transport in pancreatic duct epithelial cell s.