© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
ATP antagonism of thrombin-induced endothelial barrier permeability
aPhysiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany
bInstitut für Vegetative Physiologie, Universität zu Köln, Köln, Germany
* Corresponding author. Tel.: +49-641-994-7215; fax: +49-641-994-7219. dursun.guenduez{at}med.uni-giessen.de
Objectives: Thrombin induces endothelial barrier failure by activating the contractile machinery of endothelial cells. Contractile activation is due to an increase in myosin light chain (MLC) phosphorylation. Here, it was investigated whether stimulation of endothelial cells with ATP can interrupt this thrombin-induced pathomechanism. Methods: In cultured human umbilical vein endothelial cells, cytosolic calcium [Ca2+]i (Fura 2 method), phosphorylation of MLC, isometric tension and permeability for albumin were studied. Results: Thrombin (0.2 U/ml) increased [Ca2+]i from a basal level of 78±8 to 570±63 nM (mean±S.D., n = 5, P<0.05), MLC phosphorylation from 71±7 to 163±18%, isometric tension from 157±17 to 232±26 µN, and permeability from 2.8±0.4 to 11.6±1x10–6 cm/s. Co-presence of ATP (10 µM) and thrombin did not alter the [Ca2+]i rise, but reduced MLC phosphorylation to 59.8±10%, isometric tension to 174±14 µN, and permeability to 5.4±0.6x10–6 cm/s. The thrombin-induced rise in MLC phosphorylation was sensitive to reduction of [Ca2+]i It was accompanied by an increase in Rho activation, and was inhibited by Y-27632 (10 µM), a Rho-kinase blocker. The ATP-induced decrease in MLC phosphorylation was not sensitive to [Ca2+]i. It was not accompanied by changes in RhoA activation, and could not by suppressed by Y-27632. Conclusions: ATP antagonizes the Ca2+- and Rho-dependent effects of thrombin on MLC phosphorylation most likely by a Ca2+- and Rho-independent activation of MLC phosphatase. It thereby functionally antagonizes the thrombin-induced increase in monolayer tension and permeability.
KEYWORDS Endothelial function; Contractile apparatus; Signal transduction; Protein phosphorylation
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