ATP antagonism of thrombin-induced endothelial barrier permeability.
OBJECTIVE
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 [Ca(2+)](i) (Fura 2 method), phosphorylation of MLC, isometric tension and permeability for albumin were studied.


RESULTS
Thrombin (0.2 U/ml) increased [Ca(2+)](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 microN, and permeability from 2.8+/-0.4 to 11.6+/-1 x 10(-6) cm/s. Co-presence of ATP (10 microM) and thrombin did not alter the [Ca(2+)](i) rise, but reduced MLC phosphorylation to 59.8+/-10%, isometric tension to 174+/-14 microN, and permeability to 5.4+/-0.6 x 10(-6) cm/s. The thrombin-induced rise in MLC phosphorylation was sensitive to reduction of [Ca(2+)](i) It was accompanied by an increase in Rho activation, and was inhibited by Y-27632 (10 microM), a Rho-kinase blocker. The ATP-induced decrease in MLC phosphorylation was not sensitive to [Ca(2+)](i). It was not accompanied by changes in RhoA activation, and could not by suppressed by Y-27632.


CONCLUSIONS
ATP antagonizes the Ca(2+)- and Rho-dependent effects of thrombin on MLC phosphorylation most likely by a Ca(2+)- and Rho-independent activation of MLC phosphatase. It thereby functionally antagonizes the thrombin-induced increase in monolayer tension and permeability.
Gündüz
Dursun
D
Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany. dursun.guenduez@med.uni-giessen.de
Hirche
Frank
F
Härtel
Frauke Viola
FV
Rodewald
Christoph Walter
CW
Schäfer
Matthias
M
Pfitzer
Gabriele
G
Piper
Hans Michael
HM
Noll
Thomas
T
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000704:Analysis of Variance; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects*; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism; D010766:Phosphorylation; D013917:Thrombin / Q000494:pharmacology*
D018994:Myosin Light Chains; D000255:Adenosine Triphosphate; D013917:Thrombin; D002118:Calcium