Activation of RhoA by thrombin in endothelial hyperpermeability - Role of Rho kinase and protein tyrosine kinases

Endothelial cells (ECs) actively regulate the extravasation of blood consti tuents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cel ls. These changes partly depend on a rise in [Ca2+](i) and activation of th e Ca2+/calmodulin-dependent myosin light chain kinase, In this study, mecha nisms that contribute to the thrombin-enhanced endothelial permeability wer e further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under t he same conditions, the activity of the related protein Rac was not affecte d, This was accompanied by an increase in myosin light chain phosphorylatio n, the generation of F-actin stress fibers, and a prolonged increase in end othelial permeability, Inhibition of the RhoA target Rho kinase with the sp ecific inhibitor Y-27632 reduced all of these effects markedly. In the pres ence of Y-27632, the thrombin-enhanced permeability was additionally reduce d by chelation of [Ca2+](i) by BAPTA. These data indicate that RhoA/Rho kin ase and Ca2+ represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin- induced endothelial permeability without affecting activation of RhoA by th rombin. Our data support a model of thrombin-induced endothelial permeabili ty that is regulated by 3 cellular signal transduction pathways.