Ethanol modulation of intestinal epithelial tight junction barrier.
Previous studies have shown that high concentrations of ethanol (>/=40%) cause functional damage of the gastrointestinal epithelial barrier by direct cytotoxic effect on the epithelial cells. The effects of lower noncytotoxic doses of ethanol on epithelial barrier function are unknown. A major function of gastrointestinal epithelial cells is to provide a barrier against the hostile substances in the gastrointestinal lumen. The apicolaterally located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent cells and act as a paracellular barrier. In this study, we investigated the effects of lower doses of ethanol on intestinal epithelial TJ barrier function using filter-grown Caco-2 intestinal epithelial monolayers. The Caco-2 TJ barrier function was assessed by measuring epithelial resistance or paracellular permeability of the filter-grown monolayers. Ethanol (0, 1, 2.5, 5, 7.5, and 10%) produced a dose-related drop in Caco-2 epithelial resistance and increase in paracellular permeability. Ethanol also produced a progressive disruption of TJ protein (ZO-1) with separation of ZO-1 proteins from the cellular junctions and formation of large gaps between the adjacent cells. Ethanol, at the doses used (</=10%), did not cause cytotoxicity (lactate dehydrogenase release) to the Caco-2 cells. Ethanol produced a disassembly and displacement of perijunctional actin and myosin filaments from the perijunctional areas. On ethanol removal, actin and myosin filaments rapidly reassembled at the cellular borders. Ethanol stimulated the Caco-2 myosin light chain kinase (MLCK) activity but did not affect the MLCK protein levels. Specific MLCK inhibitor ML-7 inhibited both ethanol increases in MLCK activity and TJ permeability without affecting the MLCK protein levels. Consistent with these findings, metabolic inhibitors sodium azide and 2,4-dinitrophenol significantly prevented ethanol-induced increase in Caco-2 TJ permeability, whereas cycloheximide or actinomycin D had no effect. The results of this study indicate that ethanol at low noncytotoxic doses causes a functional and structural opening of the Caco-2 intestinal epithelial TJ barrier by activating MLCK.
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Division of Gastroenterology, Department of Medicine, Department of Veterans Affairs Medical Center, Long Beach 90822, USA. ma.thomas_y@long-beach.va.gov
Nguyen
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Bui
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Nguyen
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Hoa
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D001381:Azepines / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000235:genetics; D009281:Naphthalenes / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured
D001381:Azepines; D004791:Enzyme Inhibitors; D009281:Naphthalenes; C070571:ML 7; D000431:Ethanol; D003513:Cycloheximide; D009219:Myosin-Light-Chain Kinase