Thrombin-induced increase of F-actin in human umbilical vein endothelial cells.
The actin cytoskeleton of the endothelium plays a key role in the maintenance of an endothelial permeability barrier. Inflammatory agonists, such as thrombin, cause an increase in vascular permeability associated with changes in the actin filament system. However, the full nature and extent of agonist-induced changes to endothelial actin have not been documented. We have studied the actin cytoskeleton in human umbilical vein endothelial cells (HUVEC) growing on tissue culture plastic coverslips or 0.4-micron pore-size polycarbonate membranes. We found: (1) Thrombin (0.3 U/ml) induced a rapid (within 5 min) increase in the number of microfilaments in HUVEC. (2) Using a quantitative assay for cellular filamentous actin (F-actin), thrombin induced a 1.7-fold increase in HUVEC F-actin within 1 min which persisted for at least 30 min. (3) Blockage of the thrombin-induced intracellular calcium ion ([Ca2+)i) signal did not block the thrombin-induced increase in F-actin, and calcium ionophores did not cause an increase in F-actin. (4) Protein kinase C inhibitors (calphostin C and staurosporine both at 100 nM) partially blocked the actin increase. Higher doses of staurosporine (500 nM) resulted in complete blockage of the thrombin-induced increase in F-actin. (5) Treatment with phorbol ester (100 nM PMA) or mezerein (100 nM) did not produce significant changes in F-actin content. These results suggest that an increase in [Ca2+]i is not necessary for the thrombin-induced increase in endothelial F-actin and, further, that the effect is mediated by protein kinases not yet identified.
Thurston
G
G
Department of Ophthalmology, University of British Columbia, Vancouver, Canada.
Turner
D
D
D000199:Actins / Q000096:biosynthesis*; D000470:Alkaloids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004224:Diterpenes; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D009281:Naphthalenes; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D019311:Staurosporine; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D013917:Thrombin / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology
D000199:Actins; D000470:Alkaloids; D004224:Diterpenes; D009281:Naphthalenes; D011083:Polycyclic Compounds; D013729:Terpenes; C011309:mezerein; D019284:Thapsigargin; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium