Effects of ethanol metabolites on exocytosis of pancreatic acinar cells in rats.
OBJECTIVE
During development of alcoholic pancreatitis, oxidative (acetaldehyde) and nonoxidative metabolites (ethyl palmitate, ethyl oleate), rather than ethanol itself, mediate toxic injury. Exposure of pancreatic acini to ethanol blocks cholecystokinin (CCK)-8-stimulated apical exocytosis and redirects exocytosis to the basolateral plasma membrane, causing interstitial pancreatitis. We examined how each ethanol metabolite contributes to these changes in exocytosis.


METHODS
Rat pancreatic acini were incubated with concentrations of ethanol associated with alcoholic pancreatitis (20-50 mmol/L) or ethanol metabolites (1-3 mmol/L) and then stimulated with CCK-8. We performed single zymogen granule (ZG) exocytosis assays, Ca(2+) imaging studies, ultrastructural analyses (with electron microscopy), and confocal microscopy to assess the actin cytoskeleton and track the movement of vesicle-associated membrane protein (VAMP)-8-containing ZGs. Coimmunoprecipitation assays were used to identify complexes that contain the distinct combinations of Munc18 and the soluble N-ethylmaleimide sensitive factor attachment protein receptor proteins, which mediate apical (ZG-apical plasma membrane) and basolateral exocytosis and fusion between ZGs (ZG-ZG).


RESULTS
The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate reduced CCK-8-stimulated apical exocytosis and formation of apical exocytotic complexes (between Munc18b and Syntaxin-2, synaptosomal-associated protein of 23 kilodaltons [SNAP23], and VAMP2) in rat pancreatic acini. Acetaldehyde and ethyl oleate redirected CCK-8-stimulated exocytosis to the basal and lateral plasma membranes and translocation of VAMP8-containing ZGs toward the basolateral plasma membrane. This process was mediated primarily via formation of basolateral exocytotic complexes (between Munc18c and Syntaxin-4, SNAP23, and VAMP8). Exposure of the acini to acetaldehyde and ethyl oleate followed by CCK-8 stimulation mildly perturbed the actin cytoskeleton and Ca(2+) signaling; exposure to ethyl palmitate severely affected Ca(2+) signaling. Acetaldehyde, like ethanol, promoted fusion between ZGs by the formation of ZG-ZG exocytotic complexes (between Munc18b and Syntaxin-3, SNAP23, and VAMP8), whereas ethyl palmitate and ethyl oleate reduced ZG-ZG fusion and formation of these complexes.


CONCLUSIONS
The ethanol metabolites acetaldehyde, ethyl palmitate, and ethyl oleate perturb exocytosis processes in cultured rat pancreatic acini (apical blockade, basolateral exocytosis, and fusion between ZGs). Acetaldehyde and, to a lesser degree, ethyl oleate produce many of the same pathologic effects of ethanol on CCK-8-stimulated exocytosis in pancreatic acini.
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Subhankar
S
Dolai
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Tao
T
Liang
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Patrick P L
PPL
Lam
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
Nestor A
NA
Fernandez
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Subbulaksmi
S
Chidambaram
Department of Medicine, University of Toronto, Toronto, Ontario, Canada; Department of Physiology, University of Toronto, Toronto, Ontario, Canada. Electronic address: herbert.gaisano@utoronto.ca.
Herbert Y
HY
Gaisano
D000079:Acetaldehyde / Q000378:metabolism / Q000633:toxicity; D008841:Actin Cytoskeleton / Q000378:metabolism; D000681:Amylases / Q000378:metabolism*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000378:metabolism / Q000633:toxicity*; D005089:Exocytosis / Q000187:drug effects*; D047468:Immunoprecipitation; D008297:Male; D008561:Membrane Fusion / Q000187:drug effects; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D051938:Munc18 Proteins / Q000378:metabolism; D009829:Oleic Acids / Q000378:metabolism / Q000633:toxicity; D010169:Palmitic Acids / Q000378:metabolism / Q000633:toxicity; D046790:Pancreas, Exocrine / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D019512:Pancreatitis, Alcoholic / Q000201:enzymology / Q000209:etiology* / Q000473:pathology; D050765:Qa-SNARE Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D022142:Secretory Vesicles / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D012844:Sincalide / Q000494:pharmacology; D013997:Time Factors; D046509:Tissue Culture Techniques; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
D051938:Munc18 Proteins; D009829:Oleic Acids; D010169:Palmitic Acids; D050765:Qa-SNARE Proteins; C498858:Snap23 protein, rat; C494706:Vamp2 protein, rat; D050988:Vesicle-Associated Membrane Protein 2; D033921:Vesicular Transport Proteins; D000431:Ethanol; D000681:Amylases; D000079:Acetaldehyde; C007680:ethyl palmitate; D012844:Sincalide; C033180:ethyl oleate