Sakamoto T, Hinton D R, Sakamoto H, Gopalakrishna R, Ryan S J, McDonnell P J
Doheny Eye Institute, Los Angeles, CA 90033, USA.
Curr Eye Res. 1995 Jan;14(1):35-45. doi: 10.3109/02713689508999912.
We studied the participation of the protein kinase C pathway in thrombin-induced cytoskeletal alterations in confluent cultured bovine corneal endothelial (BCE) cells. Cultured BCE cells were exposed to alpha-thrombin (0.1-10 U/ml for 15-60 min) and the distribution of F-actin and vinculin plaques was examined using immunofluorescent staining and electron microscopy. Phorbol 12-myristate 13-acetate (PMA, 10 nM for 15 min), the broad spectrum protein kinase inhibitors staurosporine (10 nM) and H-7 (10 nM), and highly specific PKC inhibitor calphostin C (10 nM) were used to evaluate the role of PKC/phosphorylation in this phenomenon. HA-1004 (10 nM) was used as a negative control for these inhibitors. In a parallel experiment, PKC activity of cytosol and membrane of BCE cells was also evaluated. In control samples, F-actin was distributed mainly at the periphery of cells, where it formed dense peripheral bundles; vinculin plaques were also present at the cell boundary. Exposure of BCE cells to thrombin changed the distribution of F-actin and vinculin into a diffuse pattern; a similar alteration was also induced by incubation with PMA. These phenomena were blocked by incubation with H-7, staurosporine and calphostin C. Both cytosolic and membrane PKC activity was increased after 5 to 30 min exposure of alpha-thrombin and returned to the control level after 1 h. alpha-Thrombin induces alteration in the cytoskeleton of BCE cells, and this message is transduced at least in part by PKC dependent pathways. PKC/phosphorylation may thus play an important role in physiological processes that involve alterations of the cytoskeleton.
我们研究了蛋白激酶C途径在凝血酶诱导的汇合培养牛角膜内皮（BCE）细胞细胞骨架改变中的作用。将培养的BCE细胞暴露于α-凝血酶（0.1 - 10 U/ml，作用15 - 60分钟），并使用免疫荧光染色和电子显微镜检查F-肌动蛋白和纽蛋白斑的分布。使用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA，10 nM，作用15分钟）、广谱蛋白激酶抑制剂星形孢菌素（10 nM）和H-7（10 nM）以及高度特异性的蛋白激酶C抑制剂钙泊三醇C（10 nM）来评估蛋白激酶C/磷酸化在这一现象中的作用。HA-1004（10 nM）用作这些抑制剂的阴性对照。在平行实验中，还评估了BCE细胞胞质溶胶和膜的蛋白激酶C活性。在对照样品中，F-肌动蛋白主要分布在细胞周边，在那里形成密集的周边束；纽蛋白斑也存在于细胞边界。BCE细胞暴露于凝血酶会使F-肌动蛋白和纽蛋白的分布变为弥散模式；与PMA孵育也会诱导类似的改变。用H-7、星形孢菌素和钙泊三醇C孵育可阻断这些现象。暴露于α-凝血酶5至30分钟后，胞质溶胶和膜的蛋白激酶C活性均增加，并在1小时后恢复到对照水平。α-凝血酶诱导BCE细胞细胞骨架改变，并且这一信号至少部分通过蛋白激酶C依赖性途径转导。因此，蛋白激酶C/磷酸化可能在涉及细胞骨架改变的生理过程中起重要作用。
