Bicarbonate and fluid secretion evoked by cholecystokinin, bombesin and acetylcholine in isolated guinea-pig pancreatic ducts

HCO3− secretion was investigated in interlobular duct segments isolated from guinea-pig pancreas using a semi-quantitative fluorometric method. Secretagogue-induced decreases in intracellular pH, following blockade of basolateral HCO3− uptake with a combination of amiloride and DIDS, were measured using the pH-sensitive fluoroprobe BCECF. Apparent secretory HCO3− fluxes were calculated from the initial rate of intracellular acidification.In the presence of HCO3−, stimulation with secretin (10 nm) or forskolin (5 μm) more than doubled the rate of intracellular acidification. This effect was abolished in the absence of HCO3−. It was also abolished in the presence of HCO3− when DIDS and NPPB were applied to the luminal membrane by microperfusion. We therefore conclude that the increase in acidification rate is a useful index of secretagogue-induced HCO3− secretion across the luminal membrane.Secretin, cholecystokinin (CCK) and bombesin each stimulated HCO3− secretion in a dose-dependent fashion. They evoked comparable maximal responses at about 10 nm and the EC50 values were 0.5 nm for secretin, 0.2 nm for CCK and 30 pm for bombesin. Acetylcholine (ACh) was also effective, with a maximum effect at 10 μm.The stimulatory effect of CCK was blocked completely by the CCK1 receptor antagonist devazepide but not by the CCK2 receptor antagonist L365,260. The CCK analogue JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-phenylethyl ester), which is an agonist of the high-affinity CCK1 receptor but an antagonist of the low-affinity receptor, also stimulated HCO3− secretion but with a smaller maximal effect than CCK. JMV-180 partially inhibited the response to a high concentration of CCK but not to a lower concentration, suggesting that both high- and low-affinity states of the CCK1 receptor evoke HCO3− secretion.The stimulatory effect of bombesin was blocked completely by the gastrin-releasing peptide (GRP) receptor antagonist d-Phe6-bombesin(6-13)-methyl ester (BME) but not by the neuromedin B (NMB) receptor antagonist d-Nal-cyclo[Cys-Tyr-d-Trp-Orn-Val-Cys]-Nal-NH2 (BIM-23127).Secretagogue-evoked fluid secretion was also examined using video microscopy to measure the rate of swelling of ducts whose ends had sealed during overnight culture. Secretin, CCK, bombesin and ACh all evoked fluid secretion with maximal rates of approximately 0.6 nl min−1 mm−2, and with concentration dependences similar to those obtained for HCO3− secretion.We conclude that CCK, bombesin and ACh stimulate the secretion of a HCO3−-rich fluid by direct actions on the interlobular ducts of the guinea-pig pancreas and that these responses are mediated by CCK1 receptors, GRP receptors and muscarinic cholinoceptors, respectively

Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO(3)(−) secretion: relevance to cystic fibrosis

Fluid and HCO(3)(−) secretion are vital functions of the pancreatic duct and other secretory epithelia. CFTR and Cl(−)/HCO(3)(−) exchange activity at the luminal membrane are required for these functions. The molecular identity of the Cl(−)/HCO(3)(−) exchangers and their relationship with CFTR in determining fluid and HCO(3)(−) secretion are not known. We show here that the Cl(−)/HCO(3)(−) exchanger slc26a6 controls CFTR activity and ductal fluid and HCO(3)(−) secretion. Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO(3)(−) secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTR(inh)-172, knock-down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6(−/−) duct cells revealed that deletion of slc26a6 resulted in dis-regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO(3)(−) secretion in vivo