P295	Regulatory effects of NOS inhibitors on the activity of Rho GTPases and endothelial function in pulmonary macro and microvascular endothelial cells in vitro.
L.Yen Fen Tsang, P.Vallance, J.Leiper, B.Wojciak-Stothard
BHF Laboratories Department of Medicine UCL, London, GB.

Lack of nitric oxide and activation of RhoGTPase RhoA have been associated with a loss of endothelial function in chronic hypoxia-induced pulmonary hypertension (PH) but the links between those two regulatory mechanisms are not known. Decreased NO availability in PH has been attributed to the increased levels of NO inhibitors, methylarginines. Here we studied the in vitro effects of acute and chronic exposure of porcine pulmonary artery endothelial cells to nitric oxide synthase (NOS) inhibitors, ADMA and L-NAME, on changes in endothelial actin cytoskeleton, adherens junctions, endothelial barrier function, cell polarity and movement. These changes were correlated with the activities of Rho GTPases RhoA, Rac1 and Cdc42. Selected studies were also carried out on mouse pulmonary microvascular cells from normal mice and PH mice with increaed endogenous ADMA levels (DDAH I +/- mice). We show that naturally occurring substrate analogue asymmetric dimethylarginine (ADMA) and synthetic NO inhibitor L-NAME, but not inactive on NOS symmetric dimethylarginine (SDMA) induced a dose- and time-dependent activation of RhoA and inhibition of Rac1 and Cdc42, changes accompanied by the formation of stress fibres and a loss of cortical F-actin in endothelial cells. This resulted in an increase in endothelial permeability and reduction in a wound healing rate in confluent cell cultures. ADMA and L-NAME also reduced total cell translocation, speed of movement and inhibited cell polarity in sparse cells. The results suggest that PH-associated endothelial dysfunction manifested by increased permeability and abnormal remodelling may result from reduction in NO availability by asymmetric methylarginines and subsequent changes in the activity of the regulators of actin dynamics RhoA, Rac1 and Cdc42.

Copyright © 2005 S. Karger AG, Basel. Any further use of this abstract requires written permission from the publisher.