ROCK mediates thrombin's endothelial barrier dysfunction

Thrombin-induced endothelial monolayer hyperpermeability is thought to resu lt from increased F-actin stress fiber-related contractile tension, a proce ss regulated by the small GTP-binding protein Rho. We tested whether this p rocess was dependent on the Rho-associated protein kinase, ROCK, using a sp ecific ROCK inhibitor, Y-27632. The effects of Y-27632 on thrombin-induced myosin light chain phosphorylation (MLCP) and tyrosine phosphorylation of p 125 focal adhesion kinase (p125(FAK)) and paxillin were measured by Western blotting. F-actin organization and content were analyzed by digital imagin g, 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, reduc ed F-actin content, and reorganization of F-actin to beta-catenin-containin g 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-ind uced formation of large paracellular holes. These data indicate that thromb in-induced stress fiber formation is ROCK dependent. In contrast, thrombin- induced paracellular hole formation occurs in a ROCK-independent manner, wh ereas thrombin-induced monolayer hyperpermeability appears to be partially ROCK dependent.