Volume 139, Issue 5, Pages 1711-1720.e5 (November 2010) Protons Released During Pancreatic Acinar Cell Secretion Acidify the Lumen and Contribute to Pancreatitis in Mice  Natasha Behrendorff, Matthias Floetenmeyer, Christof Schwiening, Peter Thorn  Gastroenterology  Volume 139, Issue 5, Pages 1711-1720.e5 (November 2010) DOI: 10.1053/j.gastro.2010.07.051 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Zymogen granules are an acidic compartment. (A) Mouse pancreatic acinar fragments loaded with Acridine Orange show zymogen granules as red, indicating dye accumulation in an acidic compartment. Cells also were bathed in an aqueous extracellular dye SRB (pseudocolored green) as a locator of the lumen (seen lying between the cells). (B) Graphs show that Acridine Orange red intensity increases away from the lumen and the size of the red objects increases. (C) Cells loaded with Lysosensor Yellow/Blue dye, with staining in the granular region and bright puncta (presumably lysosomes) throughout the cell. Raw emission images (blue–green) are shown of a cluster of cells with the enlarged region (bottom right) as a calibrated pseudocolor ratio. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Stimulated secretion promotes large extracellular acidification. (A) Pancreatic fragments bathed in the dyes SRB (red, left) and HPTS (pseudocolor, right) show the dye diffuses into the branching lumen between the cells. In the enlarged time sequence CCK-stimulated secretory activity induces an exocytic event (arrowhead) recorded as SRB entry into the fused granule. The simultaneously recorded HPTS signal, converted to a pseudocolor representation of the pH change (using a ratio method according to the calibration curve in Supplementary Figure 1), shows large acidification in the lumen (note SRB also enters the fused granule but cannot be displayed as a ratio in these images and is shown as a white circle). (B) The fluorescence changes in regions of interest (circles shown in A) within the fused granule (upper) shows a peak as SRB enters the granule and accumulates on granule content, then decays to a plateau as content is lost. In contrast, the HPTS signal shows a slow increase, suggesting acidity is quenching the fluorescence increase. In the lumen (lower panel), SRB signal shows a small increase as the content moves out of the granules with its associated accumulated dye. The lumenal HPTS signal is dramatically different; HPTS fluorescence decreases seconds before SRB enters the vesicle to a nadir of around pH 7.0. Scale, 5 μm. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Extracellular acidification diffuses away from the site of granule fusion. (A) HPTS ratiometric pseudocolor image of a pancreatic fragment showing dye in the lumen and, in the enlarged time sequence of images (i, ii, iii at time points indicated in panel B), shows that a single fusion event induces luminal acidification that starts at the point of fusion and then spreads along the lumen. (B) Regions of interest placed at the points indicated along the lumen (1, 2, 3, and 4) show the time course and magnitude of the pH changes are faster and larger in regions close to the point of fusion, both for this example and in (C) the population of granules analyzed. The granule SRB signal in the granule is shown in panel B as a point of reference. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Changing pH buffering or imposing an acid shift do not affect fusion pore kinetics. (A) Example images taken in cells that were stimulated with CCK in the presence of the lysine fixable dye fluorescein isothiocyanate (fitc; green) with the dye tetramethylrhodamine (TMRE; red) added 5 minutes after stimulation and the cells then were paraformaldehyde-fixed 5 minutes later. The distribution of FITC and TMRE are similar in the lumens and fused granules are seen under all conditions. (B) Analysis of the TMRE:FITC ratio is used as an indication of fusion pore closure. Conditions that promote either opening or closure of the fusion pore are expected to shift the ratio distribution to the right or left, respectively.24 The distribution of dye ratios for the population of granules shows no change with or without HEPES, or at an imposed extracellular pH of 7.0. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Extracellular acid decreases the amount of exocytosis with no effect on endocytosis. (A and B) Cells bathed in SRB (red), were stimulated with acetylcholine for 2 minutes, bathed in atropine for 30 minutes, then washed and resuspended in MPTS (blue). The obtained images show red-only vesicles (enlarged in B, 2 structures circled in ii) as well as blue vesicles (indicating these are still in continuity with the lumen and therefore exocytic). (C) Counts of the numbers of structures show that an acid pH of 7 significantly decreases the numbers of exocytic vesicles per cell but has no effect on endocytic vesicles (numbers of tissue clusters shown at the bottom of the bars). There was no change in the relative ratio of exocytic to endocytic events. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Ca2+ spike duration is shortened by extracellular acidification. (A) Trains of Ca2+ spikes, induced by Ins(2,4,5)P3 and recorded using whole-cell patch clamp because inward (downward) Cl- currents have relatively similar kinetics over long periods of time. Changing extracellular pH from 7.4 to 7.0 reversibly shortened spike duration seen in the expanded sections (i, ii) and (B) quantified in the overlay of the means of all the spikes (aligned to peak) as a faster recovery time. (C–E) In imaging experiments, Fluo-4 loaded into acinar cells records oscillatory Ca2+ signals induced by CCK. Those spikes recorded in high pH buffer (high HEPES) were significantly slower to recover at each spike without any change on spike increase time. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Supramaximal cerulein induces massive, long-lasting extracellular acidifications that damage cell junctions. (A) A time sequence of images of a pancreatic fragment stimulated with supramaximal cerulein and bathed in SRB (red) and HPTS (blue), here not pseudocolored because of the large unstable structural changes seen as vacuolation within the cells. The graph shows the lumen become extremely acidic; changes that last for many minutes. (B) In fixed cells, supramaximal cerulein induces a significant loss in the immunolocalization of occludin from the cell junctions (Caer -) that is prevented by high pH buffering (Caer +) and not seen with normal CCK stimulation (CCK+ and CCK-). Scale, 50 μm. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 (A) pH calibration curves for the fluorescent dyes used in this work. SRB and MPTS are pH-insensitive, and HPTS is pH-sensitive with a decrease in fluorescence on acidification. The HPTS line was fitted with the following equation: pH = 7.4 + log (f/f0) − log (1 − (f/f0 − 1))× 10(7.4-Kd) with a dissociation constant of pH 6.79. (B) MPTS, the pH-insensitive analogue of HPTS, behaves similarly to SRB. In experiments identical to those of Figure 2, only substituting MPTS for HPTS, the fluorescence changes in the vesicle and in the lumen show that the time course and extent of the MPTS signal are very similar to SRB. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 High pH buffering attenuates extracellular acidification. Experiments identical to those of Figure 2, only increasing the pH buffering to 10 mmol/L HEPES, show that the HPTS signal in the vesicle are similar to the SRB signal, indicating that HEPES is buffering intravesicular protons. In the lumen the HPTS changes are much smaller than in low pH buffering. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 3 Blocking the V-ATPase does not abolish the extracellular acidification. In experiments identical to those of Figure 2, now compare control (left-hand curves) with responses after pretreatment for 30 minutes with bafilomycin (right-hand curves) to block the V-ATPase pumps. Although the kinetics of the intravesicular HPTS changes are different after bafilomycin treatment there is still a suppression of the HPTS signal indicative of an acid luminal environment and a luminal acidification also is seen. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 4 The Ca2+-dependent Cl- current is not affected by changes to an extracellular pH of 7.0. Whole-cell patch clamp with low intracellular Ca2+ (left) or high intracellular Ca2+ (right) with voltage-clamp step changes to the voltage shown build up a family of current curves and show the Ca2+-dependent Cl- current activated in the right-hand column. Changing the extracellular pH had no affect on either the resting currents or those activated by Ca2+. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 5 Supramaximal cerulein induces massive exocytosis and subsequent vacuolization that retain continuity with the lumen for prolonged periods of time. (A) Three images taken from a time series of the response to 100 nmol/L cerulein measured with SRB (upper panel). The lumen between cells is labeled with SRB (i), 15 minutes after stimulation dye has entered the granules and vacuoles along the lumen (ii), and 30 minutes after stimulation, large vacuoles are seen in the cell (iii). The simultaneously recorded images in the blue emission channel show HPTS fluorescence (note here we use an excitation wavelength of 850 nm at which HPTS is essentially pH-independent). HPTS is added to the extracellular media at 15 minutes and, over time, (A) fills the vacuolar structures, (B) enlarged, and (C) from a region of interest placed over the vacuolar/luminal region. We conclude that over these time periods the vacuoles are in continuity with the outside. Scale, 10 μm. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 6 (A) F-actin (phalloidin staining) and Zona occludens-1 (ZO-1) colocalize in the apical region and define the lumens in a tissue fragment. Treatment with physiological concentrations of CCK does not alter their distribution but supramaximal concentrations of cerulein dramatically disrupt the F-actin cytoskeleton and action partially ameliorated by high extracellular pH buffer. Supramaximal cerulein also affects the ZO-1 localization; it is still present along the lumen but now appears as puncta within the cell and again this change is partly ameliorated by high extracellular pH buffer. (B) E-cadherin immunostaining was conducted with methanol fixation. E-cadherin localizes to the apical and lateral plasma membrane; its distribution is not affected by CCK, but is disrupted with supramaximal cerulein treatment where it becomes diffuse at the membrane and appears in the subapical region; both effects are essentially prevented in high pH buffered solutions. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 7 Experiments stimulating with either 10 μm bombesin or 100 nm cerulein in 2 mmol/L HEPES buffer (low buffering) show similar proton releases in the lumen as measured by HPTS fluorescence. In both cases the lumen is acidified for more than 10 minutes and shows similar total decreases in luminal pH. However, E-cadherin distribution does not change as dramatically after bombesin stimulation even in low buffering, unlike with cerulein (see Supplementary Figure 6B). Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 8 (A) Our data show that under normal stimulation the exocytosis of zymogen granules releases substantial amounts of protons into the luminal space causing rapid and significant acidification. We show that this acidification is sufficient to accelerate recovery of cytosolic calcium after cell stimulation. (B) With pathophysiological stimulation, massive exocytic activity leads to a large and sustained luminal acidification. Our data show that this acidification disrupts the tight and adherens junctions and we speculate that this loss of junctional integrity is part of the pathology associated with the early phase of disease. Gastroenterology 2010 139, 1711-1720.e5DOI: (10.1053/j.gastro.2010.07.051) Copyright © 2010 AGA Institute Terms and Conditions