LOW-DOSE ALCOHOL REDIRECTS POSTPRANDIAL CHOLINERGIC-STIMULATED APICAL EXOCYTOSIS TO THE BASOLATERAL MEMBRANE IN PANCREATIC ACINAR CELLS, THE SUSCEPTIBILITY MECHANISM OF ALCOHOLIC PANCREATITIS

Abstract

Background: We previously demonstrated that alcohol (EtOH) pretreatment of pancreatic acini caused the basal plasma membrane (BPM) to become receptive to aberrant BPM exocytosis upon stimulation with submaximal CCK-8. These actions of alcohol on the acinar cell were due to induction of PM-bound Munc18c to dissociate into the cytosol, rendering the PM Syntaxin4 to interact with PM-SNAP-23 and granule-VAMP to form a complex capable of mediating basolateral exocytosis into the interstitial space, which we had postulated to be a mechanism of alcoholic pancreatitis. Rat pancreas however lacks CCK receptors and instead responds to cholinergic stimulation. Aim: We therefore examined whether 1- low EtOH concentration (20 mM) is able to alter submaximal cholinergic stimulation of rat pancreatic acinar cell secretion and exocytosis; 2- alcohol diet-fed rats are susceptible to develop pancreatitis upon low postprandial cholinergic stimulation. Methods: 1- Dispersed rat pancreatic acini were pretreated with non-stimulatory EtOH (20 mM-1 h) followed by stimulation with postprandial carbachol (3 uM-1 h), a dose stimulating only half maximal amylase secretion; 2- rats were fed with either a control or ethanol (36% of total calories intake replacement) liquid diet for 6 weeks and then challenged with low carbachol (5 ip injections of 0.5 uM/kg/h) stimulation.We then examined: a) PM levels of Munc18c and SNARE proteins, and Munc18c displacement into the cytosol by confocal microscopy and cell subfractionation; b) amylase release; c) real-time epifluorescent imaging of exocytosis using FM1-43; d) electron microscopy to visualize the distribution of granules within the cell; e) tissue hematoxylin and eosin (H&E) staining to examine morphologic changes of pancreatitis; f) serum amylase, lipase and ethanol concentrations as clinical parameters of pancreatitis. Results: 1- a) Whereas 20 mM EtOH has no effect on amylase secretion, pretreatment with 20 mM EtOH inhibited 3 uM carbachol stimulated secretion. b) Whereas 3 uM carbachol evokes apical exocytosis, pretreatment with 20 mM EtOH redirected exocytosis to the basal (by FM1-43 imaging) and lateral plasma membrane (by E.M.). c) While neither 20 mM EtOH nor 3 uM carbachol affected PM-Munc18c (confocal microscopy and subcellular fractionation), pretreatment of acini with 20 mM ETOH induced 3 uM carbachol to displace Munc18c from the BPM into the cytosol. 2-Alcohol diet-fed rats (serum ETOH levels of 20-30 mM) were normal and did not exhibit major morphologic defects in the pancreas. However, subsequent exposure to low-dose carbachol induced mild pancreatitis, observed as altered morphology (E.M and H&E) showing vacuolization in acinar cells, leukocyte infiltrate, hemorrhage and necrotic spots; along with clinical parameters of elevated serum levels of amylase and lipase. These morphologic changes correlated to Munc18c displacement from the BPM and its degradation in the cytosol. Conclusion: Postprandial EtOH renders acini PM-Munc18c conducive for postprandial cholinergic stimulation to displace into the cytosol, relieving Syntaxin4 to bind SNAP-23 and ZG VAMP proteins to promote basolateral exocytosis. We propose this to be a 'susceptibility' mechanism by which alcohol predisposes to human pancreatitis. (Funded by: Alcohol Beverage Medical Research Foundation and NIH -R21 AA015579-01).