Ethanol modulation of intestinal epithelial tight junction barrier

Abstract

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.

Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: Summary of a symposium

“…However, ethanol up to 5% concentration failed to increase paracellular permeability, suggesting that ethanol metabolism to acetaldehyde is required for the action. This was confirmed by another study where ethanol up to 3% showed no significant effect on paracellular permeability in Caco-2 cell monolayers (Ma et al, 1999). This study showed that ethanol increased paracellular permeability at 7.5% concentration.…”

Intestinal permeability, gut-bacterial dysbiosis, and behavioral markers of alcohol-dependence severity

“…The possibility of a toxic effect of ethanol on the small-bowel epithelium has been described in healthy subjects (34,39) and in in vitro studies (40)(41)(42)(43). However, patients from both groups consumed the same amount of alcohol, which lessens the implication that ethanol itself is involved in permeability disturbance.…”

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

“…These include epithelial apoptosis, mucosal ulceration, and altered tight junction protein expression. 1,12,[22][23][24][25][26][27] While apoptosis and ulceration can certainly disrupt the mucosal barrier, abundant experimental evidence suggests that these alone cannot explain the barrier dysfunction observed. 23 One pair of recent reports has shown that increased expression of the tight junction protein claudin-2 can contribute to the reduced barrier function seen in response to IL-13, both in vitro and in vivo.…”

“…This is consistent with the documented role of MLCK in regulating epithelial tight junction permeability in response to physiological and pathophysiological stimuli in both in vitro and in vivo models. 13,15,19,27 The mechanism by which TNF induces epithelial MLCK activation is not well understood, 12 but studies in cultured epithelial monolayers suggest a role for TNF-dependent transcriptional activation of MLCK. 16,17 As a result, both MLCK expression and activity are increased, as demonstrated by increased MLC phosphorylation.…”