P269	Long term inhibition of Rho Kinase opposes its endothelial barrier protective effect.
G.P.van Nieuw Amerongen, S.Zeeman, R.J.P.Musters, M.E.Engelse, C.Jungerius, I.J.M.Korstjens, M.H.van Wijhe, P.Sipkema, V.W.M.van Hinsbergh
Institute for Cardiovascular Research, Lab for Physiology, VU medical center, Amsterdam, NL.

Background Endothelial hyperpermeability is regulated by a myosin light chain (MLC) phosphorylation-dependent mechanism. Myosin phosphatase (MP) plays a critical role in the Ca2+-sensitivity of MLC phosphorylation. We have previously shown that thrombin is a potent inducer of hyperpermeability of cultured endothelial monolayers via Rho kinase-mediated MLC phosphorylation. It has been suggested that Rho kinase-mediated phosphorylation of MP at Thr695 of the myosin phosphatase targeting subunit (MYPT1) results in inhibition, and provides a surrogate marker for Rho kinase activity. Aim To study the role of Rho kinase-mediated MP activities in regulation of EC barrier function in vitro and ex vivo. Methods In vitro permeability was evaluated in a model of HUVECs grown on porous filters. Ex vivo, regions of paracellular flux in rat kidney arterioles were identified by perfusion with Concanavalin A. MLC/MYPT1 phosphorylation was visualized by digital 3D imaging microscopy. Results Thrombin induced a 3-fold increase in MYPT1-phosphorylation in HUVECs, indicative of inhibition of MP. Inhibition of MP with calyculin A or cantharidin induced a rapid decrease in endothelial barrier function in a dose-dependent manner, similar to the decrease induced by thrombin and was accompanied by disruption of cell-cell contacts and an increase in MLC phosphorylation. This was observed both in intact vessels and in endothelial monolayers in vitro. Phosphorylated MYPT1 was found to colocalize with thrombin-induced F-actin stress fibers. Interestingly, in non-stimulated endothelial cells, a Rho kinase-mediated peripheral band of phosphorylated MYPT1 colocalized with cortical F-actin, and disappeared upon thrombin stimulation. This suggested that basal inactivation of MYPT1 in junctional areas contributes to barrier integrity. In accordance, inhibition of basal Rho kinase activity for 24 hours disrupted endothelial barrier integrity, opposing previously observed protection from the thrombin response. Conclusions We show for the first time that Rho kinase has opposing activities in regulation of endothelial barrier function: 1- an intrinsic activity at the cell margins that is essential for proper barrier integrity, and 2- an induced activity at F-actin stress fibers, resulting in barrier disruption. Our findings warrant attention to the time window for treatment of patients suffering from vascular leakage with Rho kinase-interfering drugs. Supported by NHF grant 2003T032

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