Volume 143, Issue 3, Pages 832-843.e7 (September 2012) Effects of Ethanol Metabolites on Exocytosis of Pancreatic Acinar Cells in Rats  Subhankar Dolai, Tao Liang, Patrick P.L. Lam, Nestor A. Fernandez, Subbulaksmi Chidambaram, Herbert Y. Gaisano  Gastroenterology  Volume 143, Issue 3, Pages 832-843.e7 (September 2012) DOI: 10.1053/j.gastro.2012.06.011 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Effect of EtOH and EtOH metabolites on CCK-8–stimulated amylase secretion and Ca2+ signaling in rat pancreatic acini. (A) Rat pancreatic acini (∼106) were incubated with KRBH buffer, 20 mmol/L EtOH, 1 mmol/L acetaldehyde, 3 mmol/L ethyl oleate, or 3 mmol/L ethyl palmitate for 2 hours; a similar set was incubated with these reagents (1 hour) followed by 100 pmol/L CCK-8 stimulation (1 hour). Supplementary Figure 1 shows that these are the minimal effective concentrations of the EtOH metabolites. Amylase secreted was expressed as percentage of total cellular amylase of the respective sample. Each value is mean ± SEM of triplicate samples per experiment from 5 independent experiments (n = 15). *P < .05 compared with 100 pmol/L CCK-8. (B) Ca2+ i/Fluo 4 fluorescence is shown as fluorescence ratio (F/F0), in which F is fluorescence changes with time and F0 is prestimulation basal fluorescence. Pancreatic acini were preincubated with EtOH metabolites (20 minutes, 37°C), recording was started in the last 5 minutes, and 200 pmol/L CCK-8 was then added. Pretreatment with ethyl palmitate (Biv, n = 6) generated sustained Ca2+ elevation, in contrast to CCK-8–evoked oscillations **P < .01. ***P < .001 (Bi, n = 8). (C) Although acetaldehyde (Bii, n = 7) and ethyl oleate (Biii, n = 8) pretreatment did not alter Ca2+ spike frequency (Ciii), they altered Ca2+ spike amplitudes (Ci) and broadened the spikes (full-width at half maximum [FWHM]) (Cii). Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Alcohol metabolite redirects CCK-8–evoked exocytosis from apical pole to basolateral PM surface. (A) CCK-8 (100 pmol/L) stimulation caused prominent exocytosis within the apical pole, with no exocytosis found on basolateral PM (flat fluorescent intensity graph in Aii; n = 5). Acini preincubated with (B) 1 mmol/L acetaldehyde (Bii, n = 5) or (C) 3 mmol/L ethyl oleate (Cii, n = 5) followed by 100 pmol/L CCK-8 stimulation showed complete to near-complete blockade of apical exocytosis and abundant exocytosis at the basolateral PM. (D) Pretreatment with 3 mmol/L ethyl palmitate (Dii, n = 6) showed milder reduction of apical exocytosis, with little basolateral exocytosis. Di shows representative sequences of static FM1-43 epifluorescence images, and Dii shows respective averaged real-time fluorescent tracings of apical pole versus basal PM regions of acini analyzed from 3 independent experiments. Hotspots in apical ZG poles are indicated by arrowheads, and hotspots in basolateral PM are indicated by arrows. Scale bar = 10 μm. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Acetaldehyde and ethyl oleate induce CCK-8–evoked ZG fusion events at basal and lateral PM. (A) After Ad-syncollin-pHluorin infection, acini treated with (i) KRBH control followed by 200 pmol/L CCK-8 caused apical exocytosis, and (ii) 1 mmol/L acetaldehyde or (iii) 3 mmol/L ethyl oleate followed by 200 pmol/L CCK-8 caused basolateral exocytosis and apical blockade. The left panels are DIC images with superimposed syncollin-pHluorin green images (scale bar = 10 μm). The right panels are timed sequences of enlarged views of indicated boxed regions shown in DIC images on the left (scale bar = 1 μm). Red arrows point to apical lumen. In Ai, syncollin-pHluorin hotspots were confined to the apical region (inner dashed circle), with box 1 indicating ZG exocytosis at the apical lumen and box 2 showing sequential images indicating rapid sampling (within seconds), with appearance and disappearance of a single fluorescent ZG indicating full fusion; the lower panel shows a corresponding fluorescence intensity trace. Box 3 is an example of exocytosis at the basal PM, and box 4 is an example of exocytosis at the lateral PM. (B) Summary of acetaldehyde (n = 8 cells) and ethyl oleate (n = 12 cells) effects on CCK-8–evoked single ZG fusion events; CCK-8 only (n = 10 cells). We counted all single ZG exocytosis events, categorized into basolateral and apical exocytosis, summated into total fusion events, and then normalized to cell area and recording time. As controls, incubation with KRBH or EtOH metabolites alone (KRBH, n = 3; acetaldehyde, n = 4; ethyl oleate, n = 5; ethyl palmitate, n = 4) evoked very few fusion events. **P < .01, ***P < .001, NS, not significant. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Alcohol metabolites cause apical blockade of CCK-8-evoked exocytosis and redirection of exocytosis to the lateral PM surface. Electron microscopy was performed on rat pancreatic acini subjected to the following conditions: (A) 100 pmol/L CCK-8 (45 minutes) and pretreatment (1 hour) with (B) 20 mmol/L EtOH, (C) 1 mmol/L acetaldehyde, or (D) 3 mmol/L ethyl oleate, followed by 100 pmol/L CCK-8 stimulation (45 minutes). Low-magnification images are on the left, and boxed sections are enlarged on the right. Enlarged (high-magnification) images highlight exocytosis occurring at the lateral PM, causing expansion of interstitial spaces that are normally collapsed. Arrowheads indicate ZGs undergoing exocytosis at the lateral PM. Arrows in B and D indicate junctional complexes separating lateral PM and interstitial spaces from the apical lumen. E shows the apical luminal area as an indicator of apical exocytosis and apical blockade (n = 7 acini from each treatment group, 3 independent experiments). **P < .01 compared with CCK-8 or ethyl palmitate as indicated; NS, not significant. Scale bar = 100 μm unless otherwise mentioned. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Alcohol metabolites cause CCK-8 to relocate VAMP8-containing ZGs from the apical to basolateral region of pancreatic acinar cells. Acini are double labeled with VAMP8 antibody (green) and rhodamine-conjugated phalloidin (red), the latter to indicate the location of apical ductal lumen. Merge shows relative positions of VAMP8 and actin. Acini were pretreated with (A) KRBH buffer control, (B) 20 mmol/L EtOH, (C) 1 mmol/L acetaldehyde, or (D) 3 mmol/L ethyl oleate and then stimulated with 100 pmol/L CCK-8. In the merge images, arrows indicate apical lumen and arrowheads indicate VAMP8 at basal and lateral PM. Micrographs shown are each representative of >40 images from 2 independent experiments performed in a blinded manner. Scale bar = 10 μm. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Effects of EtOH metabolites on actin cytoskeleton and junctional complexes in rat pancreatic acini stimulated with CCK-8. Rat acini were incubated for 20 minutes with (Ai, ii, F) KRBH or (20 mmol/L, Bi, ii) EtOH or EtOH metabolites, (Ci, ii) 1 mmol/L acetaldehyde, (Di, ii) 3 mmol/L ethyl oleate, or (Ei, ii) 3 mmol/L ethyl palmitate, followed by incubation for 20 minutes of the same (i; no CCK-8 stimulation) or stimulated with 100 pmol/L CCK-8 (ii, A–E). F is stimulation with supramaximal CCK-8 (100 nmol/L). Acini were double labeled with fluorescein isothiocyanate–conjugated phalloidin and Alexa 594–conjugated ZO-1 antibody to assess the actin cytoskeleton and junctional complex, respectively. These images are superimposed on merge images; merge images are superimposed on DIC images. Each micrograph is representative of >10 images from 5 independent experiments performed in a blinded manner. Scale bar = 10 μm. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Alcohol metabolites alter CCK-8–stimulated Munc18/SNARE complex formation in rat pancreatic acini. Rat acini were treated as in Figure 1A. A total of 500 μg protein of acini lysates was immunoprecipitated with (A) anti–Syntaxin-2, (B) anti–Syntaxin-3, or (C) anti–Syntaxin-4 antibodies; preimmune immunoglobulin G is control. Precipitated proteins were identified with the indicated antibodies. (D) To examine whether the Syntaxin-4 coprecipitated complex of proteins might be affected by intracellular Ca2+, acini treated as previously described were incubated in 3 mmol/L Ca2+ (control) versus low Ca2+ (0.1 mmol/L Ca2 plus 50 μmol/L BAPTA-AM). (ii) For each condition in i, total lysates (25 μg protein) served as input controls. These blots are representative of 3 independent experiments. Statistical analysis is shown in Supplementary Figure 6. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Determination of the minimal effective concentration of EtOH metabolites on CCK-8–stimulated amylase secretion. Approximately 106 dispersed rat pancreatic acini were preincubated with 0.1 to 5 mmol/L (0.1, 0.5, 1, 2, 3, 4, and 5 mmol/L) of each of the metabolites (acetaldehyde, ethyl oleate, and ethyl palmitate) in KRBH for 1 hour at 37°C. Cells were subsequently stimulated with 100 pmol/L CCK-8 for 1 hour, and secreted amylase was expressed as a percentage of total cellular amylase. All experiments were performed in triplicate for 3 independent experiments (n = 9). The minimal effective inhibitory concentrations appeared to be 1 mmol/L acetaldehyde, 3 mmol/L ethyl oleate, and 3 mmol/L ethyl palmitate, which we used for all subsequent experiments. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 EtOH metabolites alone do not induce exocytosis in rat pancreatic acini. Dispersed rat acini were preincubated for 20 minutes with (A) KRBH alone (Aii, n = 3) or KRBH with (B) 1 mmol/L acetaldehyde (Bii, n = 3), (C) 3 mmol/L ethyl oleate (Cii, n = 3), and (D) 3 mmol/L ethyl palmitate (Dii, n = 3). Exocytosis events were monitored by FM1-43 epifluorescence microscopy. i shows representative sequences of static FM1-43 epifluorescence images, and ii shows the respective averaged real-time fluorescent tracings of apical poles and basolateral PM of acini analyzed from 3 independent experiments. No detectable exocytosis was found, as indicated by the flat fluorescence intensity graph in Aii, Bii, Cii, and Dii. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 FM1-43 imaging. Low extracellular Ca2+ plus chelation of intracellular Ca2+ with BAPTA prevent CCK-8–stimulated exocytosis in rat pancreatic acini. Dispersed rat pancreatic acini were incubated in KRBH buffer (0.1 mmol/L CaCl2 + 50 μmol/L BAPTA-AM) for 20 minutes with (A) 1 mmol/L acetaldehyde (Aii, n = 3), (B) 3 mmol/L ethyl oleate (Bii, n = 3), or (C) 3 mmol/L ethyl palmitate (Cii, n = 3). Exocytosis events were then recorded by real-time FM1-43 epifluorescence microscopy as in Figure 2. A total of 100 pmol/L CCK-8 was added after 10 minutes of recording, and images were captured up to 25 minutes. No exocytosis (apical/basolateral) was observed throughout the recording time, as indicated by the flat fluorescence intensity graph in Aii, Bii, and Cii. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 Syncollin-pHluorin imaging. Low extracellular Ca2+ plus chelation of intracellular Ca2+ with BAPTA prevent stimulated exocytosis in rat pancreatic acini. (A) Rat pancreatic acini infected with Ad-syncollin-pHluorin as in Figure 4 were incubated in KRBH buffer (0.1 mmol/L CaCl2 + 50 μmol/L BAPTA-AM) for 20 minutes with 1 mmol/L acetaldehyde (n = 5), 3 mmol/L ethyl oleate (n = 4), or 3 mmol/L ethyl palmitate (n = 4), and exocytosis events were recorded by spinning disk microscopy as in Figure 3. A total of 200 pmol/L CCK-8 was added, and images were captured up to 15 minutes. (B) Summary of BAPTA chelation of intracellular Ca2+ effects on CCK-8–evoked single ZG fusion events; CCK-8 only (n = 10 cells). We counted all single ZG exocytosis events, categorized into basolateral and apical exocytosis, summated into total fusion events, and then normalized by the cell area and recording time. Very few exocytosis (apical/basolateral) was observed throughout the recording time in the presence of BAPTA. A total of 200 pmol/L CCK-8 in extracellular Ca2+ buffer (3 mmol/L) in the absence of BAPTA was used as positive control, which showed apical exocytosis. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Alcohol metabolite treatment followed by CCK-8 stimulation causes Munc18c displacement from PM into the cytosol and cytosolic degradation. Rat acini were pretreated (1 hour) with KRBH buffer, 20 mmol/L EtOH, 1 mmol/L acetaldehyde, 3 mmol/L ethyl oleate, or 3 mmol/L ethyl palmitate, followed by 100 pmol/L CCK-8 (1 hour) or no CCK-8. (Ai) Whole acini cell lysates and (Bi) PM fractionation were then prepared and subjected to immunoblotting to identify the indicated proteins. Lower levels in cell lysates indicate degradation had occurred; lower PM levels indicate displacement into cytosol. This is representative of 5 independent experiments. Densitometry analysis (Aii and Bii) from 5 independent experiments of the whole cell lysate and plasma membrane fractionation levels of Munc18c. *P < .05; **P < .01. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 6 Summary of the densitometry analysis of the coimmunoprecipitation data from Figure 7. Shown is analysis of 3 independent experiments of the immunoprecipitated proteins by (A) Syntaxin-2, (B) Syntaxin-3, or (C and D) Syntaxin-4 antibodies in i and their corresponding input controls in ii. Input results (mean ± SEM) are expressed as a percentage of the maximal value determined with each antibody. The intensities were analyzed, taking the most intense band in the blot as 100. Quantification of the immunoprecipitated syntaxins and coimmunoprecipitated proteins in i was performed by normalizing the IPed protein band intensity to the corresponding input protein band intensity in ii and then comparing that value with total protein (500 μg) used for each immunoprecipitation assay. This explains why the relative amounts of Munc18c coimmunoprecipitated were calculated to be similar in Ci and Di with the other treatments, because the reduced Munc18c signals in Figure 7Ci for EtOH + CCK-8, acetaldehyde + CCK-8, and ethyl oleate + CCK-8 were calculated against the corresponding reduced input levels as shown in Figure 7Cii and Figure 7Dii, respectively. *P < .05. Gastroenterology 2012 143, 832-843.e7DOI: (10.1053/j.gastro.2012.06.011) Copyright © 2012 AGA Institute Terms and Conditions