title: Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation.
abstract: Evidence indicates that ethanol-induced intestinal barrier dysfunction and subsequent endotoxemia plays a key role in the pathogenesis of alcoholic liver disease. Recently, it has been demonstrated that ethanol induces RhoA kinase activation in intestinal epithelium, thereby disrupting barrier integrity. In this study, the role of a rise in intracellular calcium concentration ([Ca(2+)]i) in ethanol-induced Rho-associated coiled coil-forming kinase (Rho/ROCK) activation and barrier disruption was investigated in Caco-2 cell monolayers. Treatment of Caco-2 monolayers with 40 mmol/l ethanol induced [Ca(2+)]i release as indicated by increased relative fluorescent units of Fluo-3 from 0.06 ± 0.02 to 2.27 ± 1.96 (P < 0.0001). Pretreatment with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) completely inhibited the release, whereas the inositol 1,4,5-triphosphate receptor (IP3R)-antagonist, Xestospongin C, partially inhibited the ethanol-induced [Ca(2+)]i release (from 2.27 ± 1.96 to 0.03 ± 0.01; P < 0.0001 and from 2.27 ± 1.96 to 1.19 ± 1.80; P < 0.001, respectively). The rise in [Ca(2+)]i was paralleled with increased intestinal permeability, which could be attenuated by either BAPTA-AM or Xestospongin C. Furthermore, ethanol induced Rho/ROCK activation, as indicated by increased phosphorylation of myosin-binding subunit, which could be prevented either by BAPTA, Xestospongin C, or the specific Rho/ROCK inhibitor Y27632. Finally, inhibition of Rho/ROCK kinase by Y27632 ameliorated the ethanol-induced redistribution of zonula occluden-1, adherens junction proteins including E-cadherin and β-catenin, and also disorganization of F-actin. These findings suggest that ethanol-induced [Ca(2+)]i release, mediated by stimulating IP3R-gated Ca(2+) channel, activates Rho/ROCK in Caco-2 cells, thereby contributing to ethanol-induced intestinal barrier dysfunction.
authors.affiliation: Top Institute Food and Nutrition (TIFN), Wageningen;
authors.forename: Elhaseen
authors.initials: E
authors.lastname: Elamin
authors.forename: Ad
authors.initials: A
authors.lastname: Masclee
authors.forename: Jan
authors.initials: J
authors.lastname: Dekker
authors.forename: Daisy
authors.initials: D
authors.lastname: Jonkers
mesh_terms: D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism / Q000503:physiopathology; D008108:Liver Diseases, Alcoholic / Q000378:metabolism / Q000503:physiopathology; D010539:Permeability / Q000187:drug effects; D010766:Phosphorylation; D012997:Solvents / Q000494:pharmacology; D019108:Tight Junctions / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D002614:Chelating Agents; D012997:Solvents; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000431:Ethanol; D004533:Egtazic Acid; D054460:rho-Associated Kinases; D002118:Calcium
keywords: Caco-2; Rho kinase; ethanol; inositol 1,4,5-triphosphate receptor; intestinal epithelial barrier; intracellular calcium; tight junction