title: Thrombin-induced ATP release from human umbilical vein endothelial cells.
abstract: ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.
authors.affiliation: Department of Cardiovascular Physiology, Heinrich-Heine-University, Düsseldorf, Germany. stefanie.goedecke@uni-duesseldorf.de
authors.forename: Stefanie
authors.initials: S
authors.lastname: Gödecke
authors.forename: Claudia
authors.initials: C
authors.lastname: Roderigo
authors.forename: Christine R
authors.initials: CR
authors.lastname: Rose
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Rauch
authors.forename: Axel
authors.initials: A
authors.lastname: Gödecke
authors.forename: Jürgen
authors.initials: J
authors.lastname: Schrader
mesh_terms: D000255:Adenosine Triphosphate / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002229:Carbenoxolone / Q000494:pharmacology; D002478:Cells, Cultured; D018031:Connexin 43 / Q000037:antagonists & inhibitors; D017630:Connexins / Q000037:antagonists & inhibitors; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005682:Gadolinium / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors; D009842:Oligopeptides / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000819:agonists / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D018031:Connexin 43; D017630:Connexins; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; C492764:PANX1 protein, human; D034741:RNA, Small Interfering; D044463:Receptor, PAR-1; C508781:threonyl-phenylalanyl-leucyl-leucyl-arginyl-asparagine; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D000001:Calcimycin; D004533:Egtazic Acid; D006632:Histamine; D000255:Adenosine Triphosphate; D005682:Gadolinium; D013917:Thrombin; D002229:Carbenoxolone; C038958:gadolinium chloride; D002118:Calcium