Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3- secretion: relevance to cystic fibrosis

Abstract

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.

Figure 1

Deletion of slc26a6 in mice, knock down of CFTR and images of sealed duct. (A) Shows the vector used to delete the slc26a6 gene in mice, Southern blot of ES cells used to generate the mice and PCR with DNA prepared from WT and slc26a6^−/− mice with the primers marked p1, p2 and p3. The two bands in the Southern blot of the ES cells are the WT (7.7 kb) and mutant (4.3 kb) alleles. In (B), mRNA was extracted from sealed WT and slc26a6^−/− ducts that were treated with scrambled (control) and three CFTR-specific dicer siRNA and was used to evaluate the level of CFTR and actin mRNA by RT–PCR. Actin mRNA was used to verify that the same amount of mRNA was used in each sample. (C) Shows the results of Q-PCR of WT ducts treated with scrabbled or CFTR-specific dicer siRNA2. The results show the mean±s.e. from three duct preparations. (D) Shows the images of sealed WT and slc26a6^−/− ducts incubated in HEPES and then HCO₃⁻-buffered media and stimulated with 30 nM secretin.

Figure 2

Fluid secretion by sealed intralobular pancreatic duct. In (A, C), WT (circles) and slc26a6^−/− ducts (squares) were treated with scrambled (filled symbols) and siRNA2 (open symbols) and cultured for 36–48 h. The ducts were incubated in HEPES-buffered media for 10 min and then in HCO₃⁻-buffered media for an additional 10 min before stimulation with 30 nM secretin (A) or 10 μM forskolin (C). Images were captured at a resolution of 2 (A) or 5 min/image (C). The means±s.e. of 9–12 ducts in (A) and 4–6 ducts in (C) from four (A) and three (C) mice of each line are shown. The average diameter and length of the ducts in (A) were used to calculate the secretory rates in terms of pl/min/mm² (B). Secretory rates were calculated for the 10 min intervals in which the ducts were incubated in HEPES-, then HCO₃⁻-buffered media and the first 10 min of stimulation with secretin for WT (green columns) and slc26a6^−/− ducts (blue columns). (For colour figure see online version.)

Figure 3

Expression of CFTR and slc26a3 mRNA in WT and slc26a6^−/− ducts. CFTR expression was evaluated by immunolocalization (A). Images captured randomly from slides prepared from the pancreas of two WT and two slc26a6^−/− mice were used to determine the intensity of duct staining. Images were captured under identical image capture setting and thresholded before measurement of intensity of individual ducts (at least 10 in each image). The intensities were averaged and the ratio of staining intensity in slc26a6^−/−/WT ducts is recorded in the first column in (B). mRNA was extracted from the pancreatic ducts of three WT and three slc26a6^−/− mice and the SMG of two of these WT and slc26a6^−/− mice. The mRNA was used to determine slc26a6 mRNA level by Q-PCR and the means±s.e. or the slc26a6^−/−/WT ratio is shown in (B).

Figure 4

Properties of Cl⁻/HCO₃⁻ exchange activity in WT and slc26a6^−/− ducts. (A) Sealed ducts prepared from WT (black and red traces and columns) and slc26a6^−/− ducts (blue and green traces and columns) were incubated for 15 min in HCO₃⁻-buffered media containing 5 μM CFTR_inh-172 (red and green) or vehicles (black and blue-controls) before perfusion with Cl⁻-free and Cl⁻-containing medium for the indicated period of time. The mean±s.e. of the rates of pH_i change obtained with 5–7 ducts prepared from two WT and two slc26a6^−/− mice are shown in the columns. ^#P<0.01 relative to WT, ^*P<0.05 relative to untreated slc26a6^−/− ducts. WT (B) and slc26a6^−/− ducts (C) were treated with scrambled (black and blue traces and columns) or sense siRNA2 (red and green traces and columns) and cultured for 36–48 h. The ducts were equilibrated in HCO₃⁻-buffered media and Cl⁻/HCO₃⁻ exchange activity was measured by alternately exposing the ducts to Cl⁻-free and Cl⁻-containing medium before and after stimulation with 30 nM secretin. The columns are the mean±s.e. of at least six experiments from three WT and three slc26a6^−/− mice. ^*P<0.05 of the respective control, ^#P<0.01 relative to unstimulated WT, ^‡better than P<0.001 relative to control slc26a6^−/− ducts. (For colour figure see online version.)

Figure 5

Luminal HCO₃⁻ permeability of WT and slc26a6^−/− ducts. WT (blue, red, gold, torques traces and/or columns and definition next to traces and columns) and slc26a6^−/− ducts (purple, violet, green, gray traces and/or columns) were treated with scrambled or sense dicer siRNA2, incubated in HCO₃⁻-buffered media and, as indicated, stimulated with 30 nM secretin. The ducts were then treated with 1 μM HOE694 and 0.2 mM H₂DIDS and the reduction in pH_i was measured. The rate of changes in pH_i (ΔpH/min) was calculated and expressed as % of that of WT. The results are the mean±s.e. of at least six ducts from three WT and three slc26a6^−/− mice. ^*P<0.01 relative to unstimulated WT, ^#P<0.01 relative to stimulated WT, ^‡P<0.01 relative to siRNA-treated WT. (For colour figure see online version.)

Figure 6

Regulation of CFTR by slc26a6. (A) Oocytes expressing CFTR (dark traces), CFTR and slc26a6 (green traces) or CFTR and slc26a3 (red trace) were stimulated with 1 or 10 μM forskolin, as indicated. The summary in (B) shows the mean±s.e of six experiments. (C) WT (blue traces and columns) and slc26a6^−/− (green traces and columns) parotid duct cells were used to measure CFTR current stimulated with 3 (n=7) or 10 μM (n=9) forskolin. The columns show the mean±s.e of the time to peak current. (For colour figure see online version.)