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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.
Thomas et al. (Sat,) studied this question.