title: ATP antagonism of thrombin-induced endothelial barrier permeability.
abstract: 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.
authors.affiliation: Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany. dursun.guenduez@med.uni-giessen.de
authors.forename: Dursun
authors.initials: D
authors.lastname: Gündüz
authors.forename: Frank
authors.initials: F
authors.lastname: Hirche
authors.forename: Frauke Viola
authors.initials: FV
authors.lastname: Härtel
authors.forename: Christoph Walter
authors.initials: CW
authors.lastname: Rodewald
authors.forename: Matthias
authors.initials: M
authors.lastname: Schäfer
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pfitzer
authors.forename: Hans Michael
authors.initials: HM
authors.lastname: Piper
authors.forename: Thomas
authors.initials: T
authors.lastname: Noll
mesh_terms: 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*
chemical_list: D018994:Myosin Light Chains; D000255:Adenosine Triphosphate; D013917:Thrombin; D002118:Calcium