ABSTRACT
BACKGROUND & AIMS: Acute or chronic alcohol treatment does little to the exocrine pancreas but predisposes the pancreas to postprandial cholinergic stimulation that triggers cellular events leading to pancreatitis. This alcohol-induced susceptibility mechanism of pancreatitis is unknown. METHODS: We employed alcohol-treated dispersed rat pancreatic acini and alcohol diet-fed rats to examine the effects of submaximal carbachol-induced changes in exocytosis (FM1-43 epifluorescence imaging and electron microscopy), Munc18c cellular translocation (confocal microscopy and subcellular fractionation), and protein kinase C (PKC) alpha-induced phosphorylation in relation to pancreatitis. RESULTS: Acute low-dose alcohol (20 mmol/L) in vitro exposure or chronic alcohol diet reduces postprandial cholinergic-stimulated amylase secretion from rat pancreatic acinar cells by blocking apical exocytosis and redirecting exocytosis to less efficient basolateral plasma membrane sites. This ectopic exocytosis is mediated by PKCalpha-induced phosphorylation of Munc18c, causing Munc18c displacement from the basolateral plasma membrane into the cytosol in which it undergoes proteolytic degradation; these processes can be blocked by PKCalpha inhibition. CONCLUSIONS: We conclude that sequential low-dose alcohol and postprandial cholinergic stimulation can induce PKCalpha-mediated Munc18c plasma membrane displacement. This relieves cognate SNARE proteins on zymogen granules and basolateral membrane to complex and consummate pathologic ectopic exocytosis at the basolateral surface. This change in vesicle trafficking may be related to the pathogenesis of pancreatitis.
Subject(s)
Carbachol/pharmacology , DNA/genetics , Exocytosis/genetics , Gene Expression , Munc18 Proteins/genetics , Pancreatitis, Alcoholic/genetics , Protein Kinase C-alpha/genetics , Amylases/metabolism , Animals , Cholinergic Agonists/pharmacology , Ethanol/toxicity , Fluorescent Dyes , Immunoblotting , Immunoprecipitation , Male , Microscopy, Confocal , Microscopy, Electron , Munc18 Proteins/drug effects , Pancreas/drug effects , Pancreas/enzymology , Pancreas/ultrastructure , Pancreatitis, Alcoholic/metabolism , Pancreatitis, Alcoholic/pathology , Peroxidase/metabolism , Phosphorylation/drug effects , Protein Kinase C-alpha/drug effects , Protein Kinase C-alpha/metabolism , Pyridinium Compounds , Quaternary Ammonium Compounds , Rats , Rats, Wistar , Solvents/toxicity , Translocation, GeneticABSTRACT
Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.
