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. 1992 Apr 1;117(1):169–178. doi: 10.1083/jcb.117.1.169

Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells

PMCID: PMC2289405  PMID: 1556151

Abstract

When epithelial cell cultures are transferred from a medium with a normal extracellular calcium concentration (1-2 mM) to a medium with a low extracellular calcium concentration (LC, less than 50 microM free Ca2+) cell-cell contacts are disrupted, and the tight junction- dependent transepithelial resistance drops. In this study, I used MDCK epithelial cells to investigate the effects of LC on the localization of the tight junction protein cingulin, and the role of protein kinases in the events induced by LC. Immunofluorescence analysis showed that within 15 min of incubation of confluent monolayers in LC, cingulin labeling was dislocated from the cell periphery, as an array of granules forming a ring-like structure. At later times after calcium removal, cingulin labeling appeared mostly cytoplasmic, in a diffuse and granular pattern, and cells appeared rounded and smaller. These events were not influenced by lack of serum, or by preincubation with 10 mM sodium azide or 6 mg/ml of cycloheximide. However, the disruption of cell-cell contacts, the cell shape changes, and the redistribution of cingulin and other junctional proteins induced by LC were inhibited when cells were pretreated with the protein kinase inhibitor H-7 (greater than or equal to 30 microM). The inhibitors H-8 and, to a lesser degree, staurosporine were also effective, whereas HA-1004 and ML-7 showed essentially no activity, suggesting a specificity of action of different inhibitors. Measurement of the transepithelial resistance showed that the kinase inhibitors that could prevent junction disassembly could also reduce the drop in transepithelial resistance induced by LC. Dose-response curves demonstrated that H-7 is the most effective among the inhibitors, and the transepithelial resistance was 70% of control up to 1 h after calcium removal. These results suggest that low extracellular calcium modulates junctional integrity and cytoskeletal organization through an effector system involving protein kinases.

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Selected References

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