1. Volume 132, Issue 4, Pages 1527-1545 (April 2007)
Alcohol-Induced Protein Kinase Cα Phosphorylation of Munc18c in Carbachol- Stimulated Acini Causes Basolateral Exocytosis 
Laura I. Cosen–Binker, Patrick P.L. Lam, Marcelo G. Binker, Herbert Y. Gaisano 
Gastroenterology 
Volume 132, Issue 4, Pages (April 2007)
DOI: /j.gastro
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2. Figure 1
Alcohol reduces Cch-stimulated amylase secretion from dispersed rat pancreatic acini. (A) Optimal duration for 3 μmol/L Cch stimulation to attain maximal amylase secretion from dispersed rat pancreatic acini, which is 1 hour. (B) Optimal time of preincubation with 20 mmol/L EtOH required to inhibit 3 μmol/L Cch-stimulated amylase release from dispersed rat pancreatic acini, which is 1 hour. Using the optimal conditions from A and B, we performed the experiment in C. (C) Effect of 20 mmol/L EtOH (1-hour preincubation) on submaximal (3 μmol/L, 1 hour), maximal (80 μmol/L, 1 hour), and supramaximal (2 mmol/L, 1 hour) Cch-stimulated amylase release. (D) Effect of 6-week alcohol diet on 3 μmol/L Cch-stimulated amylase release. Here, dispersed acini were obtained from CD or ED-fed rats and then stimulated with 3 μmol/L Cch (1 hour), 1 μmol/L Atp (1 hour) followed by 3 μmol/L Cch (1 hour), or KRH (control). The amylase secreted into the media was determined and expressed as a percentage of the total cellular amylase of the respective sample. Each value is the mean ± SD of triplicate samples per experiment from 5 independent experiments (n = 15). *P < .05, **P < .01.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Alcohol blocks Cch-evoked apical exocytosis and redirects exocytosis to the basal plasma membrane surface of the pancreatic acinar cell. A, C, and F are representative sequences of static FM1-43 epifluorescence images, and B, E, and G are their respective real-time fluorescent tracings of each region (apical lumen, apical ZG pole, BPM, or BPM hot spots) of the acinus analyzed. (A and B) For 20 mmol/L EtOH stimulation. No exocytosis was observed in. (C and E) Twenty-millimolar EtOH (preincubation) + 3 μmol/L Cch stimulation. The 3 indicated hot spots (HS) on the BPM (indicated by arrowheads) of 3 adjacent acinar cells in C are analyzed in E, where HS1 and HS2 are de novo hot spots occurring during Cch stimulation, and HS3 is exocytosis occurring at a previously docked ZG. (D) Shows 2 of the images in C at 1 minute and 31 minutes in pseudocolor to visualize better the fluorescence intensity changes of these 3 hot spots on the BPM indicating basolateral exocytosis (indicated by arrows at 31 minutes; arrowheads at 1 minute). No exocytosis occurred in the apical lumen or apical ZG pole (in C, D, E). *Adhering debris. (F and G) Twenty-millimolar EtOH + 1 μmol/L Atp + 3 μmol/L Cch stimulation, where Atp completely blocked exocytosis, in particular, BPM exocytosis that would have occurred (as in C, D, E). In these studies, rat pancreatic acini were preincubated at 37°C with 2 μmol/L FM1-43 until stable fluorescence was attained on the PM, and the indicated stimulation protocols were carried out. Images were taken at 1 frame per second for 30 minutes. In E, the intensity for each HS was normalized to the intensity of the PM adjacent to it, where the relative intensity of BPM/PM where no HS appears is 1. All experiments shown are representative of 3 experiments per condition performed on 4 acini isolations (n = 12 experiments per condition).
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Alcohol causes apical blockade of Cch-evoked exocytosis and redirection of exocytosis to the lateral plasma membrane surface. Electron microscopy was performed on (A) dispersed pancreatic acini from rats on regular chow diet preincubated with KRH or 20 mmol/L ethanol for 1 hour and then stimulated with 3 μmol/L Cch (KRH/Cch and ethanol/Cch) for 1 hour or further incubated with KRH (KRH/KRH) or 20 mmol/L ethanol (ethanol/ethanol) for 1 hour and (C) pancreatic tissue from rats either on control diet or ethanol diet that were subjected to 5 IP injections of saline, Atp (1 μmol/L/kg/h) treatment followed by Cch (0.5 μmol/L/kg/h) (Atp + Cch), or Cch (0.5 μmol/L/kg/h) alone (Cch). Asterisks indicate the location of the apical ductal lumens, and open arrows indicate the junctional complexes (more clearly shown on B, middle panel) that separate the apical lumen from the lateral PM. B and D show high-power magnification of ethanol/Cch stimulation in A and ethanol diet + Cch treatment in C, respectively. Open arrows indicate the junctional complexes, and arrows indicate the locations of lateral PM; omega (Ω) indicates dilated interstitial spaces between apposing lateral PM of 2 cells; open arrowheads in lower panels of B and D indicate ZGs docked onto the lateral PM, whereas open arrowheads in upper panel of D indicate ZG-ZG fusions with a ZG that has partially emptied its contents; and small solid arrowheads in B, upper panel, indicate fusion pore formation. We find minimal lateral exocytosis in the other groups (data not shown, as described in EtOH blocks Cch-evoked apical exocytosis and redirects exocytosis to the lateral plasma surface to cause pancreatitis). (E) Further characterization of pancreatic tissues of ED-fed rats injected with Cch (0.5 μmol/L/kg/h), where we find necrotic spots (pyknotic nuclei, disrupted cytoskeleton, organelle disintegration) (i, ii) adjacent to healthy appearing cells (i). iv Is a healthy acinus within this pancreatitis tissue. In both acini undergoing necrosis (ii) and in apparent good health (iv), we find abundant ZGs undergoing various stages of exocytosis with the lateral PM (indicated by arrows) shown better on higher magnification (iii and vi), including compound exocytosis (indicated by series of open arrowheads) of ZGs, which are either still filled (iii, upper panel) or almost completely emptied (iii, lower panel) of dense core contents; fusion pore formation (small solid arrowheads); dilatation of interstitial spaces (Ω); and disruption of lateral PM. vi Also shows a ZG (open arrowhead) forming a fusion pore (small solid arrowhead) into a partially emptying ZG (solid arrowhead). Bars, 2 μm. ZG quantification of these EM data in A–D was performed in a blinded manner by 2 independent observers, and this analysis is shown in Supplementary Figure 1 (see Supplementary Figures 1 and 2 online at (dispersed acinar cells in A and B) and Supplementary Figure 2 (pancreatic tissues in C and D).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
EtOH diet (6 weeks) + low-dose Cch induced mild rat pancreatitis. Rats were pair fed with either a control diet (CD) or ethanol diet (ED) for 6 weeks and then treated with 5 IP injections of either saline or Cch (0.5 μmol/L/kg/h) or Atp (1 μmol/L/kg/h) followed by Cch (0.5 μmol/L/kg/h). (A) Serum amylase, lipase, and TNF-α levels were determined at death, whereas serum ethanol levels were measured at 3 and 6 weeks, n = 4 rats per group. (B) Histology of pancreatic tissues is normal in all the indicated groups except the ED/Cch group, which showed cytoplasmic vacuoles, leukocyte infiltration, hemorrhage, and necrosis. (C) Determinations of indicated parameters of pancreatitis in pancreatic tissues in B, n = 4 rats per group. (D) Histopathologic score of pancreatitis in pancreatic tissues in B assessed in a blinded manner by 2 independent observers. Results are expressed as the mean ± SD.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Alcohol enables submaximal Cch to displace Munc18c from the pancreatic acinar cell basal membrane into the cytosol. Confocal microscopy (A and C) and corresponding subcellular fractionations (B and D) were performed on dispersed acini from rats on (A and B) regular chow diet, where acini were preincubated in KRH (control) or 20 mmol/L EtOH for 1 hour and then stimulated with 3 μmol/L Cch for 1 hour, or further incubated with KRH or 20 mmol/L EtOH (1 hour), or on (C and D) liquid control diet (CD) or ethanol diet (ED), where acini were treated for 1 hour with KRH or 3 μmol/L Cch or 1 μmol/L Atp 1 hour followed by 3 μmol/L Cch for 1 hour. In A and C, double labeling for Munc18c and actin was performed to show their locations. Images for CD acini treated with 3 μmol/L Cch (1 hour) or 1 μmol/L Atp (1 hour) followed by 3 μmol/L Cch (1 hour) are similar to those treated with KRH (data not shown). This is representative of 4 independent experiments performed in triplicate per experiment, n = 12. Bars, 10 μm. In B and D, subcellular fractions of PM and cytosol and total cell lysates were probed with the indicated antibodies for Munc18c and SNARE proteins (Syn-4, SNAP-23), and irrelevant resident proteins (Na+/K+ATPase, tubulin) in these compartments. Blots are representative of 3 independent experiments, n = 3. Analyses are shown in Supplementary Data Figure 3 (see Supplementary Data Figure 3 online at
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Alcohol followed by submaximal Cch stimulation induces PKCα-mediated threonine-phosphorylation of Munc18c, causing Munc18c displacement from the BPM. PKCα inhibition blocks EtOH + Cch-induced Munc18c displacement from the BPM demonstrated by subcellular fractionation (in A) and confocal microscopy (in B). Dispersed acini were incubated for 5 hours in KRH or DMSO (1% in KRH, vehicle control) as controls or 3 hours with 10 μmol/L translocation inhibitor (inh) of PKCδ (δV1-1) or PKCε (εV1-2), or PKCζ pseudosubstrate (psS); or 30 minutes with 5 μmol/L PKCα inhibitor or 30–40 minutes with 200 nmol/L Gö6976 or 500 nmol/L Calphostin C. The acini were then treated with 20 mmol/L EtOH for 1 hour followed by 3 μmol/L Cch for 1 hour. (A) The treated acini were then fractionated into plasma membrane and cytosol fractions and total cell lysates. Ten micrograms protein of each fraction was separated on SDS-PAGE and immunoblotted with antibodies against the indicated proteins. Blots are representative of 3 independent experiments, n = 3. (B) The treated acini were subjected to confocal microscopy to show the localization of Munc18c and actin. Only DMSO, EtoH + Cch, PKCα inhibitor + EtOH + Cch, and Gö EtOH + Cch are shown. Bars, 10 μm. This is representative of 4 independent experiments performed in triplicate per experiment, n = 12. (C) PKCα inhibition blocks threonine-phosphorylation of Munc18c. Dispersed acini were treated with 1 μmol/L TPA for 10 minutes, 20 mmol/L EtOH (70 minutes), 20 mmol/L EtOH (1 hour) + 3 μmol/L Cch (10 minutes), KRH (70 minutes), or KRH (1 hour) + 3 μmol/L Cch (10 minutes) or preincubated with 5 μmol/L PKCα inhibitor (30 minutes) or 200 nmol/L Gö6976 (30 minutes) or 500 nmol/L Calphostin C (40 minutes) prior to stimulation with 20 mmol/L EtOH (1 hour) + 3 μmol/L Cch (10 minutes). Whole cell lysates were immunoprecipitated with anti-Munc18c antibody, and precipitated proteins were separated on SDS-PAGE and blotted with antiphosphothreonine, Munc18c, and PKCα antibodies. Blot is representative of 3 independent experiments, n = 3. Analyses are shown in Supplementary Data Figure 4 (see Supplementary Data Figure 4 online at
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Role of PKCα activation on low ethanol + submaximal Cch-stimulated exocytosis. (A) EtOH + Cch stimulation causes PKCα translocation to the PM in dispersed acini (in i) and pancreatic tissues of ethanol diet-fed rats (in ii). (i) Twenty-millimolar EtOH (1-hour preincubation) +3 μmol/L Cch stimulation (10 minutes) of dispersed rat acini vs KRH or 3 μmol/L Cch alone. (ii) Pancreatic tissues from the indicated ethanol diet- and control diet-treated groups of animals shown in Figure 5D. In i and ii, the acini or pancreatic tissues were fractionated into plasma membrane and cytosol fractions and total cell lysates. Ten micrograms protein of each fraction was separated on SDS-PAGE and immunoblotted with antibodies to PKCα and the other indicated proteins, which show that PKCα is translocated from the cytosol to the PM. Blots are representative of 3 independent experiments, n = 3. Analyses are shown in Supplementary Data Figure 5 (see Supplementary Data Figure 5 online at (B) PKCα activity resulting from EtOH + Cch stimulation of (i) dispersed acini and (ii) pancreatic tissues of ED (vs CD) fed rats. Ten micrograms protein of lystates from dispersed acini (in i) or pancreatic tissues (in ii) subjected to the indicated treatments was loaded into each well and PKCα activity determined by EIA, with each sample performed in triplicate. Data shown in i are the mean ± SD from 3 separate portions of acini per experiment (from 1 rat) of 3 different acini preparations (n = 9), and values shown were normalized to basal activity in unstimulated (KRH only) control acini. Data shown in ii are the mean ± SD from n = 4 rats per group; values shown were from the indicated pancreatitic tissues expressed as the fold increase of CD + saline control. Results were analyzed by Student t test, with significance determined to be P < .05. (C) Amylase secretion stimulated by EtOH + Cch is blocked by PKCα inhibition, which did not affect Cch-stimulated secretion. Dispersed acini were incubated as indicated, and, for PKCα inhibition, acini were preincubated with PKCα inhibitor (30 minutes) or 200 nmol/L Gö6976 (30 minutes) prior to further incubation with 3 μmol/L Cch or 20 mmol/L EtOH + 3 μmol/L Cch The amylase secreted into the media was determined and expressed as a percentage of the total cellular amylase of the respective sample. Each value is the mean ± SD of triplicate samples per experiment from 5 independent experiments (n = 15). *P < .05. (D and E) Exocytosis. PKCα inhibition by 200 nmol/L Gö6976 for 30 minutes (in D) or 5 μmol/L PKCα inhibitor peptide for 30 minutes (in E) blocks 20 mmol/L EtOH + 3 μmol/L Cch stimulated BPM exocytosis but does not rescue apical exocytosis. Acini were preincubated at 37°C with 2 μmol/L FM1-43 until stable fluorescence was attained on the PM, and images were taken 1 per second for 30 minutes. Images in i show representative sequence of static images and ii shows the corresponding real-time recording of the kinetics of relative FM1-43 fluorescence of the different regions of a cell within the acinus. Each shown is representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Susceptibility mechanism for alcohol-mediated pancreatitis. Chronic alcohol diet followed by postprandial submaximal Cch stimulation can cause PKCα-mediated threonine-phosphorylation of Munc18c, which would induce Munc18c displacement from the basolateral PM into the cytosol. This would relieve and activate BPM-bound Syn-4 to bind SNAP-23 and VAMP from ZGs to form an exocytic SNARE complex. The presence of Ca2+ would then trigger basolateral exocytosis of digestive enzymes into the interstitial space, which we postulate to contribute to the pathogenesis of interstitial pancreatitis.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions