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

Taiji Sakamoto, David R. Hinton, Hitomi Sakamoto, Rayudu Gopalakrishna, Stephen J. Ryan, Peter J. Mcdonnell

Research output: Contribution to journalArticlepeer-review

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 αthrombin (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 αthrombin and returned to the control level after 1 h. αThrombin 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.

Original languageEnglish (US)
Pages (from-to)35-45
Number of pages11
JournalCurrent Eye Research
Volume14
Issue number1
DOIs
StatePublished - 1995
Externally publishedYes

Keywords

  • Calcium
  • Cattle (bovine)
  • Corneal endothelium
  • Cyclic AMP
  • Inositol phosphate
  • Phorbol ester
  • Phosphorylation
  • Protein kinase C
  • Thrombin-receptor

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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