Thrombin induced cytoskeletal change in cultured bovine corneal endothelial cells mediated via protein kinase C pathway.
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.
Sakamoto
T
T
Doheny Eye Institute, Los Angeles, CA 90033, USA.
Hinton
D R
DR
Sakamoto
H
H
Gopalakrishna
R
R
Ryan
S J
SJ
McDonnell
P J
PJ
D000199:Actins / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D016596:Vinculin / Q000378:metabolism*
D000199:Actins; D000911:Antibodies, Monoclonal; D016596:Vinculin; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate