Ethanol disrupts intestinal epithelial tight junction integrity through intracellular calcium-mediated Rho/ROCK activation.

E. Elamin, A.A. Masclee, J. Dekker, D.M. Jonkers

Research output: Contribution to journalArticleAcademicpeer-review

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, role of a rise in intracellular calcium concentration ([Ca2+]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 [Ca2+]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, whereas the inositol 1, 4, 5-triphosphate receptor (IP3R)-antagonist, Xestospongin C, partially inhibited the ethanol-induced [Ca2+]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 [Ca2+]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 ZO-1, adherens junction proteins including E-cadherin, and beta-catenin, and also disorganization of F-actin. These findings suggest that ethanol-induced [Ca2+]i release, mediated by stimulating IP3R-gated Ca2+ channel, activates Rho/ROCK in Caco-2 cells, and thereby contributing to ethanol-induced intestinal barrier dysfunction.
Original languageEnglish
Pages (from-to)G677-G685
Number of pages9
JournalAmerican Journal of Physiology-Gastrointestinal and Liver Physiology
Volume306
Issue number8
DOIs
Publication statusPublished - Apr 2014

Keywords

  • intestinal epithelial barrier
  • Caco-2
  • ethanol
  • inositol 1,4,5-triphosphate receptor
  • intracellular calcium
  • Rho kinase
  • tight junction
  • LIGHT-CHAIN KINASE
  • PROTEIN-KINASE
  • PARACELLULAR PERMEABILITY
  • BARRIER DYSFUNCTION
  • ADHERENS JUNCTIONS
  • PLASMA ENDOTOXIN
  • LIVER-DISEASE
  • STRESS FIBERS
  • RHO GTPASES
  • PHOSPHORYLATION

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