Thrombin induced cytoskeletal change in cultured bovine corneal endothelial cells mediated via protein kinase C pathway

Abstract

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 α-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 α-thrombin and returned to the control level after 1 h. α-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.

Key Words:

• corneal endothelium
• protein kinase C
• phorbol ester
• calcium
• inositol phosphate
• thrombin-receptor
• phosphorylation
• cyclic AMP
• cattle (bovine)