Angiotensin II evokes calcium-mediated signaling events in isolated dog pancreatic epithelial cells

Abstract

Introduction: Calcium-activated chloride conductance has been identified in normal pancreatic duct cells. Recent in vitro evidence suggests that angiotensin II (AngII) stimulates pancreatic secretion in both cystic fibrosis (CFPAC) and transformed pancreatic cells. Aims: To investigate calcium-mediated stimulatory effects of AngII in both nontransformed dog pancreatic duct epithelial (DPDE) and CFPAC cells. Methods: Western blots were performed in both cells seeking AngII receptors. In additional studies, DPDE and CFPAC cells were grown on vitrogen-coated glass cover slips and loaded with Indo-1-AM dye. Cells were placed in a confocal microscope's perfusion chamber and perfused with 100 μM AngII or ATP (control). Cells were excited with UV light, and intracellular calcium ([Ca+2]i) was read using fluorescence emission at 405 and 530 nm. Finally, single channels in the DPDE cells were examined using cell-attached patch clamps. Current amplitude histograms provided estimates of the conductance and open probability of channels. Results: Western blots demonstrated presence of both AT1 and AT2 AngII receptors in DPDE and CFPAC cells; the density of AT1 receptors appeared lower than that of AT2 receptors. Basal intracellular calcium concentrations did not differ between DPDE (109 ± 11 nM) and CFPAC (103 ± 8 nM) cells. AngII significantly increased measured intracellular calcium concentrations in both DPDE (909 ± 98 nM) and CFPAC (879 ± 207 nM) cells, as did ATP (DPDE = 1722 ± 228 nM; CFPAC = 1522 ± 245 nM). In the patch clamp studies, a variety of different channels were observed; they appeared to be an 11pS nonselective cation (NSC) channel, a 4.6pS Na+ channel, a 3pS anion channel, and an 8pS chloride channel. The latter channel had characteristics similar to cystic fibrosis transmembrane conductance regulator (CFTR). Apical or basolateral application of AngII activated both the 11pS NSC and the 3pS channels. Conclusion: In nontransformed DPDE and CFPAC cells, specific AngII receptors mediate increases in [Ca+2]i. The latter effect of AngII may elicit activation of calcium-mediated chloride channels, suggesting a role for AngII as an alternative mediator of pancreatic ductal secretion.