Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction.

In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro. We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction. To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator. LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase. Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites. LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes. In endothelial cells, [Ca(2+)](i) was not elevated by LPA. Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP. Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage. Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage. Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability. These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in [Ca(2+)](i) and requires activation of RhoA and Rho kinase. Chemicals/CAS: Amides; Calcium, 7440-70-2; Cyclic AMP, 60-92-4; Enzyme Inhibitors; Horseradish Peroxidase, EC 1.11.1.-; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Phosphatidic Acids; Protein-Serine-Threonine Kinases, EC 2.7.1.37; Pyridines; Rho-associated kinase, EC 2.7.1.-; rhoA GTP-Binding Protein, EC 3.6.5.2; Thrombin, EC 3.4.21.5; Y 27632, 138381-45-