In rodents and humans, alcohol exposure has been shown to predispose the pancreas to cholinergic or viral induction of pancreatitis. We previously developed a rodent model in which exposure to an ethanol (EtOH) diet, followed by carbachol (Cch) stimulation, redirects exocytosis from the apical to the basolateral plasma membrane of acinar cells, resulting in ectopic zymogen enzyme activation and pancreatitis. This redirection of exocytosis involves a soluble NSF attachment receptor (SNARE) complex consisting of syntaxin-4 and synapseassociated protein of 23 kDa (SNAP-23). Here, we investigated the role of the zymogen granule (ZG) SNARE vesicle-associated membrane protein 8 (VAMP8) in mediating basolateral exocytosis. In WT mice, in vitro EtOH exposure or EtOH diet reduced Cch-stimulated amylase release by redirecting apical exocytosis to the basolateral membrane, leading to alcoholic pancreatitis. Further reduction of zymogen secretion, caused by blockade of both apical and basolateral exocytosis and resulting in a more mild induction of alcoholic pancreatitis, was observed in Vamp8 -/-mice in response to these treatments. In addition, although ZGs accumulated in Vamp8 -/-acinar cells, ZG-ZG fusions were reduced compared with those in WT acinar cells, as visualized by electron microscopy. This reduction in ZG fusion may account for reduced efficiency of apical exocytosis in Vamp8 -/-acini. These findings indicate that VAMP8 is the ZG-SNARE that mediates basolateral exocytosis in alcoholic pancreatitis and that VAMP8 is critical for ZG-ZG homotypic fusion.

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The pancreatic acinus is the functional unit of the exocrine pancreas whose role is to secrete zymogens into the gut lumen for food digestion via apical exocytosis. We previously reported that supramaximal CCK induced apical blockade and redirected exocytosis to ectopic sites on the basolateral plasma membrane (BPM) of this polarized cell, leading to pancreatitis. Basolateral exocytosis was mediated by protein kinase C phosphorylation of BPM Munc18c, causing its displacement into the cytosol and activation of BPMbound Syntaxin-4 to form a SNARE complex. To mimic the conditions of alcoholic pancreatitis, we now examined whether 20 mM alcohol followed by submaximal CCK might mimic supramaximal CCK in inducing these pathologic exocytotic events. We show that a non-secretory but clinically relevant alcohol concentration (20 mM) inhibited submaximal CCK (50 pM)-stimulated amylase secretion by blocking apical exocytosis and redirecting exocytosis to less efficient BPM, indeed mimicking supramaximal CCK (10 nM) stimulation. We further demonstrate that basolateral exocytosis caused by both stimulation protocols is mediated by PKC␣-induced phosphorylation of Munc18c: 1) PKC␣ is activated, which binds and induces phosphorylation of PM-Munc18c at a Thr site, and these events can be inhibited by PKC␣ blockade; 2) PKC␣ inhibition blocks Munc18c displacement from the BPM; 3) PKC␣ inhibition prevents basolateral exocytosis but does not rescue apical exocytosis. We conclude that 20 mM alcohol/submaximal CCK as well supramaximal CCK stimulation can trigger pathologic basolateral exocytosis in pancreatic acinar cells via PKC␣-mediated activation of Munc18c, which enables Syntaxin-4 to become receptive in forming a SNARE complex in the BPM; and we further postulate this to be an underlying mechanism contributing to alcoholic pancreatitis.The pancreatic acinar cell is a highly polarized epithelial cell designed for zymogen granules (ZG) 3 to undergo regulated exocytosis at the apical pole, thereby emptying the digestive zymogens into the gut lumen for food digestion. This well orchestrated exocytic pathway can be altered by supramaximal stimulation with cholecystokinin (CCK), which causes apical blockade and redirection of exocytosis to the basolateral plasma membrane (BPM) surface (1). Specifically, using epifluorescence imaging of the FM1-43 dye in pancreatic acinar cells, we showed real-time visualization of apical exocytosis induced by maximal CCK, and aberrant exocytosis at the BPM caused by supramaximal CCK stimulation (1). We further found that this basolateral exocytosis was consistent with the paradigm of the SNARE Hypothesis (2-5), involving a distinct set of cognate SNARE partners, Syntaxin-4 (Syn-4) and SNAP 23 on the BPM, and VAMP proteins on the ZG (1, 6, 7), and whose interactions were regulated by the SM protein Munc18c (1). Here, Munc18c on the acinar BPM binds Syn-4, and upon supramaximal CCK stimulation, becomes displaced into the cytosol via a PKC-mediated mechanism; which activates Syn-4 to become capable of ...

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