Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement

Microsc Res Tech. 2000 Oct 15;51(2):156-68. doi: 10.1002/1097-0029(20001015)51:2<156::AID-JEMT7>3.0.CO;2-J.


Recent studies suggest that an abnormal increase in intestinal tight junction (TJ) permeability may be an important etiologic factor in number of diseases including Crohn's disease, NSAID-associated enteritis, and various infectious diarrheal syndromes. The intracellular processes involved in regulation of intestinal epithelial TJ permeability, however, remain poorly understood. In this study, we used cultured Caco-2 intestinal epithelial cells to examine the intracellular processes involved in extracellular Ca(++) modulation of intestinal epithelial monolayer TJ barrier. Incubation of the filter-grown Caco-2 intestinal monolayers in Ca(++)-free solution (CFS), consisting of modified Krebs-buffer solution containing 0 mM Ca(++) and 1 mM EGTA, resulted in a rapid drop in Caco-2 epithelial resistance and increase in epithelial permeability to paracellular markers mannitol and inulin, indicating an increase in TJ permeability. The increase in Caco-2 TJ permeability was rapidly reversed by the re-introduction of Ca(++) (1.8 mM) into the incubation medium. The CFS-induced increase in Caco-2 TJ permeability was associated with separation of the cytoplasmic and transmembrane TJ proteins, ZO-1 and occludin, and formation of large intercellular openings between the adjoining cells. The CFS-induced modulation of TJ barrier was associated with activation of myosin light chain kinase (MLCK) activity and centripetal retraction of peri-junctional actin and myosin filaments. The inhibition of CFS-induced activation of Caco-2 MLCK with MLCK inhibitor (ML-7) prevented the CFS-induced retraction of actin and myosin filaments and the subsequent alteration of TJ barrier function and structure. Our results suggested that the CFS-induced alteration of TJ proteins and functional increase in TJ permeability was mediated by Caco-2 MLCK activation and the resultant contraction of the peri-junctionally located actin-myosin filaments. Consistent with the role of MLCK in this process, selected inhibitors of Mg(++)-myosin ATPase and metabolic energy, but not protein synthesis inhibitors, also prevented the CFS-induced retraction of actin and myosin filaments and the subsequent increase in TJ permeability. In conclusion, our results indicate that extracellular Ca(++) is crucial for the maintenance of intestinal epithelial TJ barrier function. The removal of extracellular Ca(++) from the incubation medium causes activation of Caco-2 MLCK, which in turn leads to an increase in intestinal monolayer TJ permeability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / antagonists & inhibitors
  • Actins / metabolism
  • Azepines / pharmacology
  • Ca(2+) Mg(2+)-ATPase / antagonists & inhibitors
  • Caco-2 Cells
  • Calcium / pharmacology*
  • Cell Membrane Permeability / drug effects
  • Cytoskeleton / physiology*
  • Drug Resistance
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Intestinal Mucosa / drug effects*
  • Intestinal Mucosa / physiology
  • Membrane Proteins / metabolism
  • Myosin-Light-Chain Kinase / antagonists & inhibitors
  • Myosin-Light-Chain Kinase / metabolism
  • Myosins / antagonists & inhibitors
  • Myosins / metabolism
  • Naphthalenes / pharmacology
  • Occludin
  • Phosphoproteins / metabolism
  • Tight Junctions / drug effects*
  • Tight Junctions / physiology
  • Zonula Occludens-1 Protein


  • Actins
  • Azepines
  • Enzyme Inhibitors
  • Membrane Proteins
  • Naphthalenes
  • OCLN protein, human
  • Occludin
  • Phosphoproteins
  • TJP1 protein, human
  • Zonula Occludens-1 Protein
  • ML 7
  • Myosin-Light-Chain Kinase
  • Ca(2+) Mg(2+)-ATPase
  • Myosins
  • Calcium