PROTEIN-KINASE INHIBITORS PREVENT JUNCTION DISSOCIATION INDUCED BY LOW EXTRACELLULAR CALCIUM IN MDCK EPITHELIAL-CELLS

When epithelial cell cultures are transferred from a medium with a nor mal extracellular calcium concentration (1-2 mM) to a medium with a lo w extracellular calcium concentration (LC, < 50-mu-M free Ca2+) cell-c ell contacts are disrupted, and the tight junction-dependent transepit helial resistance drops. In this study, I used MDCK epithelial cells t o investigate the effects of LC on the localization of the tight junct ion protein cingulin, and the role of protein kinases in the events in duced by LC. Immunofluorescence analysis showed that within 15 min of incubation of confluent monolayers in LC, cingulin labeling was disloc ated from the cell periphery, as an array of granules forming a ring-l ike structure. At later times after calcium removal, cingulin labeling appeared mostly cytoplasmic, in a diffuse and granular pattern, and c ells 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 junct ional proteins induced by LC were inhibited when cells were pretreated with the protein kinase inhibitor H-7 (greater-than-or-equal-to 30-mu -M). The inhibitors H-8 and, to a lesser degree, staurosporine were al so effective, whereas HA-1004 and ML-7 showed essentially no activity, suggesting a specificity of action of different inhibitors. Measureme nt 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 demons trated that H-7 is the most effective among the inhibitors, and the tr ansepithelial resistance was 70% of control up to 1 h after calcium re moval. These results suggest that low extracellular calcium modulates junctional integrity and cytoskeletal organization through an effector system involving protein kinases.