Effects of ethanol metabolites on exocytosis of pancreatic acinar cells in rats.

Abstract

BACKGROUND & AIMS:

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.

• Authors: Subhankar Dolai, Tao Liang, Patrick P L Lam, Nestor A Fernandez, Subbulaksmi Chidambaram, Herbert Y Gaisano
• Journal: Gastroenterology (Gastroenterology) Vol. 143 Issue 3 Pg. 832-843.e7 (Sep 2012) ISSN: 1528-0012 [Electronic] United States
• PMID: 22710192 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)
• Copyright: Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

Chemical References

• Munc18 Proteins
• Oleic Acids
• Palmitic Acids
• Qa-SNARE Proteins
• Snap23 protein, rat
• Vamp2 protein, rat
• Vesicle-Associated Membrane Protein 2
• Vesicular Transport Proteins
• Ethanol
• Amylases
• Acetaldehyde
• ethyl palmitate
• Sincalide
• ethyl oleate

Topics

• Acetaldehyde (metabolism, toxicity)
• Actin Cytoskeleton (metabolism)
• Amylases (metabolism)
• Animals
• Calcium Signaling (drug effects)
• Dose-Response Relationship, Drug
• Ethanol (metabolism, toxicity)
• Exocytosis (drug effects)
• Immunoprecipitation
• Male
• Membrane Fusion (drug effects)
• Microscopy, Confocal
• Microscopy, Electron, Transmission
• Munc18 Proteins (metabolism)
• Oleic Acids (metabolism, toxicity)
• Palmitic Acids (metabolism, toxicity)
• Pancreas, Exocrine (drug effects, enzymology, metabolism, ultrastructure)
• Pancreatitis, Alcoholic (enzymology, etiology, pathology)
• Qa-SNARE Proteins (metabolism)
• Rats
• Rats, Sprague-Dawley
• Secretory Vesicles (drug effects, enzymology, metabolism)
• Sincalide (pharmacology)
• Time Factors
• Tissue Culture Techniques
• Vesicle-Associated Membrane Protein 2 (metabolism)
• Vesicular Transport Proteins (metabolism)