Effects Of Slc26a6 Deletion And CFTR Inhibition On HCO₃^- Secretion By Mouse Pancreatic Duct

Pancreatic juice is very important for digestion and is secreted by pancreatic duct cells and acinar cells. The fluid is colorless and ordorless and the pH value is 7.8～8.4. The pancreatic duct system of humans produces approximately 2.5 liters per day of isotonic fluid containing～140 mM HCO3-. CO2 diffuses into the duct cells across the basolateral membrane and is hydrated through the action of intracellular carbonic anhydrase (CA). HCO3- is derived from CO2 through the action of CA. HCO3- is very important for human body. It acts both as a vehicle for the pancreatic enzymes and as a buffer for duodenal acidity. When the secretion of HCO3- and fluid is impaired, the protein-rich fluid secreted by the acinar cells is not washed away effectively. This may result in the formation of protein plugs, and subsequently intraductal stones, which prevent the smooth flow of the pancreatic juice. Enzymes activated by the low pH in the duct lumen and the increase of intraluminal pressure may then destroy the parenchyma, as occurs in the pathogenesis of cystic fibrosis and in certain cases of idiopathic chronic pancreatitis. In a word, the normal level of HCO3- in human pancreatic ducts is very important for physiology. HCO3- secretion is dependent on cystic fibrosis transmembrane conductance regulator (CFTR) and the apical membrane includes Cl-/HCO3-exchange by an SLC26 anion transprter. The HCO3--rich ductal secretion acts as a vehicle for acini-derived digestive enzymes and as a buffer for duodenal acidity. As the exchangers and channels of pancreatic duct cells were discovered, the mechanisms of HCO3- secretion is now understood further. Nevertheless, there are so many discrepancies between laboratories, for example, SLC26A6 and/or SLC26A3 is expressed on the apical membrane? induce Cl-/HCO3- exchange? the stoichiometry of Cl-/HCO3- exchange are 1:2 or 2:1? the relationships of CFTR and SLC26 in the HCO3- secretion? These questions are focused on and still not very clear.Objective:In this study we examined the relative contribution and interaction of SLC26A6 and CFTR in apical HCO3- transport and define the stoichiometry and molecular identity of apical Cl--HCO3- exchange in pancreatic duct. We analysed the mechanisms of HCO3- secretion on apical membrane of pancreatic duct, so that the high concentration of HCO3- in human pancreatic ducts was explored further more.Methods:slc26a6 knockout (KO) mice and their WT littermates,ΔF mice and their WT littermates were used for the experiments. Interlobular pancreatic ducts (diameter～100μm) were isolated. Usually 10-15 interlobular duct segments of 300-400μm in length were isolated from one pancreas. The ducts were cultured overnight at 37℃. The lumen of the interlobular duct segment was microperfused. The bath and luminal solutions were modified separately. Intracellular pH (pHi) in the duct cells was estimated by microfluorometry as described previously using the pH-sensitive fluoroprobe BCECF. Transepithelial fluxes of HCO3- and Cl- were estimated under the anion gradients favoring rapid exchange of intracellular HCO3- with luminal Cl- in forskolin-stimulated ducts. Real-Time PCR was used to measure the expression of slc26a3 and CFTR. Their expression was normalized by house-keeping geneβ-actin. Comparative 2-(ΔΔCt) method was used to calculate the relative quantification of the target genes. The membrane potential was measured by impaling the basolateral membrane of the microperfused ducts with glass microelectrodes.Results:①When the lumen of wild-type ducts was microperfused with the standard HCO3--buffered solution containing 25 mM HCO3- and 124 mM Cl-, the rate of pHi decrease was 0.046±0.009 pH unit min-1. Luminal application of the specific inhibitor of CFTR, CFTRinh-172 (10μM) significantly (p<0.05) accelerated the pHi decrease (0.069±0.010 pH unit min-1). In slc26a6-/-ducts, the rate of pHi decrease was 0.058±0.009 pH unit min-1, which was significantly (p<0.05) faster than that in wild-type ducts and this time significantly (p<0.05) inhibited by luminal application of CFTRinh-172.②We repeated the same protocol of experiments in ducts luminally-perfused with the high-HCO3- solution containing 125 mM HCO3-and 24 mM Cl-. In this condition, luminal application of CFTRinh-172 significantly (p<0.05) inhibited apical HCO3- secretion both in wild-type and slc26a6-/-ducts. The data confirm that HCO3- conductance of CFTR provides a major route for apical HCO3- secretion in this condition. The rate of pHi decrease in slc26a6-/-ducts (0.063±0.008 pH unit min-1) was significantly (p <0.05) faster than that in wild-type ducts (0.052±0.009 pH unit min-1).③When the lumen was perfused with the high-Cl" solution containing 25 mM HCO3- and 124 mM Cl-, there was no significant difference in the rate of pH; decrease after alkaline-pulse between wild-type andΔF ducts. In contrast, when the lumen was perfused with the high-HCO3- solution containing 125 mM HCO3- and 24 mM Cl-, the rate of pH; decrease inΔF ducts (0.031±0.005 pH unit min-1) was significantly (p<0.05) slower than that in wild-type ducts (0.044±0.008 pH unit min-1).④There are no significant differences (p<0.05) on the relative quantification of CFTR expression between wild-type and slc26a6 knock-out mice.⑤The lumen was microperfused with the Cl--rich (25 mM HCO3--124 mM Cl-) solution so that apical Cl--HCO3- exchange was active. Luminal application of H2DIDS (200μM) resulted in a reversible hyperpolarization in wild-type ducts (10.5±1.7 mV) but not in slc26a6-/-ducts. In wild-type ducts removal of luminal Cl- resulted in a transient depolarization followed by sustained hyperpolarization (9.8±1.9 mV). Conclusion:SLC26A6 and CFTR compensated with each other when luminal Cl- concentration is high and CFTR provides a major route for apical HCO3- secretion when luminal HCO3- concentration is high. The data are compatible with the reported 1:2 stoichiometry of SLC26A6 1Cl--2HCO3-exchange.