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 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 [Ca(2+)](i) and activation of the Ca(2+)/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 [Ca(2+)](i) by BAPTA. These data indicate that RhoA/Rho kinase and Ca(2+) 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.
van Nieuw Amerongen
G P
GP
Gaubius Laboratory TNO Health and Prevention, Leiden.
van Delft
S
S
Vermeer
M A
MA
Collard
J G
JG
van Hinsbergh
V W
VW
D000577:Amides / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011725:Pyridines / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000577:Amides; D004791:Enzyme Inhibitors; D006490:Hemostatics; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011725:Pyridines; C108830:Y 27632; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D054460:rho-Associated Kinases; D013917:Thrombin; D020742:rhoA GTP-Binding Protein; D002118:Calcium