HCO3- Transport through Anoctamin/Transmembrane Protein ANO1/TMEM16A in Pancreatic Acinar Cells Regulates Luminal pH

Abstract

The identification of ANO1/TMEM16A as the likely calcium-dependent chloride channel of exocrine glands has led to a more detailed understanding of its biophysical properties. This includes a calcium-dependent change in channel selectivity and evidence that HCO3 (-) permeability can be significant. Here we use freshly isolated pancreatic acini that preserve the luminal structure to measure intraluminal pH and test the idea that ANO1/TMEM16A contributes to luminal pH balance. Our data show that, under physiologically relevant stimulation with 10 pm cholesystokinin, the luminal acid load that results from the exocytic fusion of zymogen granules is significantly blunted by HCO3 (-) buffer in comparison with HEPES, and that this is blocked by the specific TMEM16A inhibitor T16inh-A01. Furthermore, in a model of acute pancreatitis, we observed substantive luminal acidification and provide evidence that ANO1/TMEM16A acts to attenuate this pH shift. We conclude that ANO1/TMEM16A is a significant pathway in pancreatic acinar cells for HCO3 (-) secretion into the lumen.

Keywords: bicarbonate; epithelial cell; exocytosis; ion channel; pancreas; secretion; transporter.

FIGURE 1.

Luminal acidification after zymogen granule exocytosis is decreased in an HCO₃⁻ buffer. A, two-photon live-cell imaging of pancreatic fragments, bathed in HEPES, SRB, and HTPS, showing the dyes outline the cells and enter the lumen between the cells. Zymogen granule exocytosis, triggered by stimulation with CCK (10–20 pm), is observed as the sudden appearance of fluorescent spots (two events are shown, arrows) as the dyes enter the granule shown here at time points i, ii, and iii (see also B and supplemental Movie 1). The pseudocolored pH changes were obtained from calibrated ratios of the HPTS fluorescence and show spatial pH changes in the lumen synchronous with the granule fusion events (see also supplemental Movie 2. B, plots of fluorescence over time (baseline subtracted, n ≥ 33 events, n = 3+ animals) within regions of interest (A, arrows) over the region of the fusing granules showing that both SRB and HPTS enter the granules. In the lumen (A, circles), SRB shows a small fluorescence increase, but HPTS shows a decrease, consistent with luminal acidification and quenching of HPTS fluorescence. C, pancreatic fragments bathed in HCO₃⁻ buffer show similar fluorescent changes in the granule and the lumen as those in HEPES buffer (see also supplemental Movies 3 and 4). D and E, comparison of the responses in HEPES and HCO₃⁻ showing that the changes of fluorescence within the granule are similar but that, in the lumen, the pH nadir and recovery were significantly (Student's t test; *, p < 0.05; **, p < 0.01) less acidic in HCO₃⁻. Scale bars = 5 μm.

FIGURE 2.

TMEM16A is a significant transporter of HCO₃⁻ in to the acinar lumen. A and B, two-photon live-cell imaging of pancreatic fragments bathed in HCO₃⁻ stimulated with CCK (10–20 pm) in the absence (DMSO control, A; see also supplemental Movies 5 and 6) or presence of 10 μm T16Ainh-AO1 (B, see also supplemental Movies 7 and 8). The SRB fluorescence shows an example granule fusion event (arrow). Also shown are pseudocolored pH changes as assessed with the HPTS fluorescence changes. C, D, and E, the inhibitor leads to a significant (Student's t test; ****, p < 0.001; **, p < 0.01) increase in stimulus-induced luminal acidification (n ≥ 10 events, n > 3 mice). Scale bars = 5 μm.

FIGURE 3.

T16Ainh-AO1 blocks the agonist-evoked calcium-dependent chloride current in pancreatic acinar cells but has no effect on zymogen granule acidification. A and B, whole-cell patch clamp with voltage clamp steps showing activation of a large current after stimulation with 10 pm CCK that is blocked when cells were stimulated in the presence of 10 μm T16AinhAO1, as shown in the current-voltage relationships (B). C and D, cells incubated in Lysosensor were two-photon-imaged with excitation at 750 nm and emission collected at wavelength >520 nm and <490 nm. Images were ratioed (C and D) and calibrated across a range of pH values (D). E, the cellular pH was not different (Student's t test) after treatment with 10 μm T16Ainh-AO1. Scale bar = 5 μm. pF, picofarad; ns, not significant.

FIGURE 4.

T16Ainh-AO1 does not affect the agonist-evoked calcium response. A and B, application of 200 nm acetylcholine (Ach) induced a rapid rise in intracellular calcium in control (DMSO-treated, n = 10 acini, n = 3 mice) and T16Ainh-A01-treated (10 μm, n = 6 acini, n = 3 mice) pancreatic acini as measured with calibrated Fura-2-loaded cells. A, example images taken at time points i, ii, iii, and iv (indicated in B). B, time traces from the examples shown in A. C and D, the average calcium responses were not different (Student's t test) in the peak (C) or temporal changes (D) between control and T16Ainh-A01 treatment. ns, not significant.

FIGURE 5.

Supramaximal CCK stimulation (10 nm) induces a large exocytic response; the consequent acid load in the lumen with HEPES buffer is significantly smaller with HCO₃⁻ buffer. A, two-photon live-cell imaging in HEPES buffer before (C, i) and after (C, ii) supramaximal CCK stimulation showing a large exocytic response (SRB) and significant luminal acidification (A, C, and D; see also supplemental Movies 9 and 10). In the presence of HCO₃⁻ buffer (B), the exocytic response is similar, but the luminal acidification is much less (B–D, see also supplemental Movies 11 and 12). Scale bars = 5 μm. n ≥ 9 lumens, n ≥ 3 mice. Student's t test; **, p < 0.01.

FIGURE 6.

TMEM16A transports HCO₃⁻ into the lumen during supramaximal agonist stimulation. Addition of 10 μm T16Ainh-AO1 to HCO₃⁻-buffered solution significantly increased luminal acidification (B–D, see also supplemental Movies 15 and 16) compared with the HCO₃⁻-buffered control responses (A, C, and D, DMSO; see also supplemental Movies 13 and 14). Counts of the numbers of exocytic events per luminal length show that this is not altered in the presence of T16Ainh-AO1 (E). Scale bars = 5 μm. ns, not significant. Student's t test; *, p < 0.05.

FIGURE 7.

Niflumic acid also enhances luminal acidification in response to supramaximal agonist stimulation. Addition of 100 μm niflumic acid (NFA) to HCO₃⁻-buffered solution significantly increased luminal acidification (B–D, see also supplemental Movies 19 and 20) compared with the HCO₃⁻-buffered control responses (A, C, and D, DMSO; see also supplemental Movies 17 and 18). Counts of the numbers of exocytic events per luminal length show that this was not altered in the presence of niflumic acid (E). Scale bars = 5 μm. ns, not significant. Student's t test; *, p < 0.05.