Citi S
Department of Cell Biology and Anatomy, Cornell University Medical College, New York 10021.
J Cell Biol. 1992 Apr;117(1):169-78. doi: 10.1083/jcb.117.1.169.
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.
当上皮细胞培养物从具有正常细胞外钙浓度（1 - 2 mM）的培养基转移至具有低细胞外钙浓度（LC，游离Ca2+小于50 microM）的培养基时，细胞间接触被破坏，紧密连接依赖的跨上皮电阻下降。在本研究中，我使用MDCK上皮细胞来研究低钙浓度对紧密连接蛋白cingulin定位的影响，以及蛋白激酶在低钙浓度诱导的事件中的作用。免疫荧光分析表明，在低钙浓度下汇合单层培养物孵育15分钟内，cingulin标记从细胞周边移位，形成一系列颗粒，构成环状结构。在去除钙后的后期，cingulin标记大多出现在细胞质中，呈弥散和颗粒状分布，细胞呈现圆形且变小。这些事件不受血清缺乏的影响，也不受用10 mM叠氮化钠或6 mg/ml放线菌酮预孵育的影响。然而，当细胞用蛋白激酶抑制剂H - 7（大于或等于30 microM）预处理时，低钙浓度诱导的细胞间接触破坏、细胞形状变化以及cingulin和其他连接蛋白的重新分布受到抑制。抑制剂H - 8以及程度较轻的星形孢菌素也有效，而HA - 1004和ML - 7基本无活性，这表明不同抑制剂作用具有特异性。跨上皮电阻的测量表明，能够防止连接解体的激酶抑制剂也能减少低钙浓度诱导的跨上皮电阻下降。剂量反应曲线表明，H - 7是抑制剂中最有效的，去除钙后长达1小时，跨上皮电阻为对照的70%。这些结果表明，低细胞外钙通过涉及蛋白激酶的效应系统调节连接完整性和细胞骨架组织。
