title: Mechanism of extracellular calcium regulation of intestinal epithelial tight junction permeability: role of cytoskeletal involvement.
abstract: 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.
authors.affiliation: Division of Gastroenterology, Department of Medicine, DVA Medical Center, Long Beach, California 90822, USA. tyma@uci.edu
authors.forename: T Y
authors.initials: TY
authors.lastname: Ma
authors.forename: D
authors.initials: D
authors.lastname: Tran
authors.forename: N
authors.initials: N
authors.lastname: Hoa
authors.forename: D
authors.initials: D
authors.lastname: Nguyen
authors.forename: M
authors.initials: M
authors.lastname: Merryfield
authors.forename: A
authors.initials: A
authors.lastname: Tarnawski
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000378:metabolism; D001381:Azepines / Q000494:pharmacology; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors; D018938:Caco-2 Cells; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D003599:Cytoskeleton / Q000502:physiology*; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D019108:Tight Junctions / Q000187:drug effects* / Q000502:physiology; D062826:Zonula Occludens-1 Protein
chemical_list: D000199:Actins; D001381:Azepines; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009281:Naphthalenes; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C070571:ML 7; D009219:Myosin-Light-Chain Kinase; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins; D002118:Calcium