Acetaldehyde disrupts tight junctions and adherens junctions in human colonic mucosa: protection by EGF and L-glutamine.

Acetaldehyde, a toxic metabolite of ethanol oxidation, is suggested to play a role in the increased risk for gastrointestinal cancers in alcoholics. In the present study, the effect of acetaldehyde on tyrosine phosphorylation, immunofluorescence localization, and detergent-insoluble fractions of the tight junction and the adherens junction proteins was determined in the human colonic mucosa. The role of EGF and L-glutamine in prevention of acetaldehyde-induced effects was also evaluated. Acetaldehyde reduced the protein tyrosine phosphatase activity, thereby increasing the tyrosine phosphorylation of occludin, E-cadherin, and beta-catenin. The levels of occludin, zonula occludens-1, E-cadherin, and beta-catenin in detergent-insoluble fractions were reduced by acetaldehyde, while it increased their levels in detergent-soluble fractions. Pretreatment with EGF or L-glutamine prevented acetaldehyde-induced protein tyrosine phosphorylation, redistribution from intercellular junctions, and reduction in the levels of detergent-insoluble fractions of occludin, zonula occludens-1, E-cadherin, and beta-catenin. These results demonstrate that acetaldehyde induces tyrosine phosphorylation and disrupts tight junction and adherens junction in human colonic mucosa, which can be prevented by EGF and glutamine.

L-Glutamine ameliorates acetaldehyde-induced increase in paracellular permeability in Caco-2 cell monolayer.

Role of L-glutamine in the protection of intestinal epithelium from acetaldehyde-induced disruption of barrier function was evaluated in Caco-2 cell monolayer. L-Glutamine reduced the acetaldehyde-induced decrease in transepithelilal electrical resistance and increase in permeability to inulin and lipopolysaccharide in a time- and dose-dependent manner; d-glutamine, L-aspargine, L-arginine, L-lysine, or L-alanine produced no significant protection. The glutaminase inhibitor 6-diazo-5-oxo-L-norleucine failed to affect the L-glutamine-mediated protection of barrier function. L-Glutamine reduced the acetaldehyde-induced redistribution of occludin, zonula occludens-1 (ZO-1), E-cadherin, and beta-catenin from the intercellular junctions. Acetaldehyde dissociates occludin, ZO-1, E-cadherin, and beta-catenin from the actin cytoskeleton, and this effect was reduced by L-glutamine. L-Glutamine induced a rapid increase in the tyrosine phosphorylation of EGF receptor, and the protective effect of L-glutamine was prevented by AG1478, the EGF-receptor tyrosine kinase inhibitor. These results indicate that L-glutamine prevents acetaldehyde-induced disruption of the tight junction and increase in the paracellular permeability in Caco-2 cell monolayer by an EGF receptor-dependent mechanism.

Epidermal growth factor prevents acetaldehyde-induced paracellular permeability in Caco-2 cell monolayer.

BACKGROUND: Intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Previous studies showed that acetaldehyde disrupts intestinal epithelial barrier function and increases paracellular permeability by a tyrosine kinase-dependent mechanism. In the present study, the role of epidermal growth factor (EGF) in protection of epithelial barrier function from acetaldehyde was evaluated in Caco-2 intestinal epithelial cell monolayer. METHODS: Caco-2 cells on Transwell inserts were exposed to acetaldehyde in the absence or presence of EGF, and the paracellular permeability was evaluated by measuring transepithelial electrical resistance and unidirectional flux of inulin. Integrity of epithelial tight junctions and adherens junctions was analyzed by confocal immunofluorescence microscopy and immunoblot analysis of occludin, zonula occludens (ZO)-1, E-cadherin, and beta-catenin in the actin cytoskeleton. Reorganization of actin cytoskeletal architecture was examined by confocal microscopy. RESULTS: Acetaldehyde increased paracellular permeability to inulin and lipopolysaccharide, and EGF significantly reduced these effects of acetaldehyde in a time- and dose-dependent manner. EGF prevented acetaldehyde-induced reorganization of occludin, ZO-1, E-cadherin, and beta-catenin from the cellular junctions to the intracellular compartments. Acetaldehyde treatment induced a reorganization of actin cytoskeletal network and reduced the levels of occludin, ZO-1, E-cadherin, and beta-catenin associated with the actin cytoskeleton. EGF effectively prevented acetaldehyde-induced reorganization of actin cytoskeleton and the interaction of occludin, ZO-1, E-cadherin, and beta-catenin with the actin cytoskeleton. CONCLUSION: These results indicate that EGF attenuates acetaldehyde-induced disruption of tight junctions and adherens junctions and prevents acetaldehyde-induced reorganization of actin cytoskeleton and its interaction with occludin, ZO-1, E-cadherin, and beta-catenin.