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Activation of rhoa by thrombin in endothelial hyperpermeability: Role of rho kinase and protein tyrosine kinases

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Author: Nieuw Amerongen, G.P. van · Delft, S.V. · Vermeer, M.A. · Collard, J.G. · Hinsbergh, V.W.M. van
Institution: Gaubius Instituut TNO
Source:Circulation Research, 4, 87, 335-340
Identifier: 235643
Keywords: Amides · Calcium · Cell Membrane Permeability · Cells, Cultured · Cytoskeleton · Endothelium, Vascular · Enzyme Activation · Enzyme Inhibitors · Hemostatics · Humans · Intracellular Signaling Peptides and Proteins · Myosin Light Chains · Phosphorylation · Protein Binding · Protein-Serine-Threonine Kinases · Protein-Tyrosine Kinases · Proto-Oncogene Proteins c-akt · Pyridines · rhoA GTP-Binding Protein · Thrombin · Umbilical Veins


Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca2+](i) and activation of the Ca2+/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca2+](i) by BAPTA. These data indicate that RhoA/Rho kinase 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 thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways. Chemicals/CAS: Amides; Calcium, 7440-70-2; Enzyme Inhibitors; Hemostatics; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Protein-Serine-Threonine Kinases, EC; Protein-Tyrosine Kinases, EC; Proto-Oncogene Proteins c-akt, EC; Pyridines; Rho-associated kinase, EC 2.7.1.-; rhoA GTP-Binding Protein, EC; Thrombin, EC; Y 27632, 138381-45-0