ROCK mediates thrombin's endothelial barrier dysfunction.
Thrombin-induced endothelial monolayer hyperpermeability is thought to result from increased F-actin stress fiber-related contractile tension, a process regulated by the small GTP-binding protein Rho. We tested whether this process was dependent on the Rho-associated protein kinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imaging, and endothelial monolayer permeability was measured in bovine pulmonary artery endothelial cell (EC) monolayers using a size-selective permeability assay. Y-27632 enhanced EC monolayer barrier function due to a decline in small-pore number that was associated with increased EC surface area, reduced F-actin content, and reorganization of F-actin to beta-catenin-containing cell-cell adherens junctions. Although Y-27632 prevented thrombin-induced MLCP, stress fiber formation, and the increased phosphotyrosine content of paxillin and p125(FAK), it attenuated but did not prevent the thrombin-induced formation of large paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.
Carbajal
J M
JM
Department of Physiology, The University of Arizona, and The Benjamin W. Zweifach Microcirculation Laboratories, Department of Veteran Affairs Medical Center, Tucson, Arizona 85723, USA.
Gratrix
M L
ML
Yu
C H
CH
Schaeffer
R C
RC
D000199:Actins / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D013917:Thrombin / Q000502:physiology*; D000267:Tissue Adhesions / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control; D014443:Tyrosine / Q000037:antagonists & inhibitors / Q000378:metabolism; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D051419:Paxillin; D010750:Phosphoproteins; D011725:Pyridines; C108830:Y 27632; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D013917:Thrombin; D009218:Myosins