title: Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells.
abstract: BACKGROUND
  Apoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5'-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC.
  
  
  RESULTS
  To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po(2)<4mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/-0.4; P<0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/-0.5; P<0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αβ-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP.
  
  
  CONCLUSIONS
  The data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.
authors: Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany. Danurban@gmx.de
authors: D
authors: D
authors: Urban
authors: F V
authors: FV
authors: Härtel
authors: K
authors: K
authors: Gadiraju
authors: D
authors: D
authors: Gündüz
authors: M
authors: M
authors: Aslam
authors: H M
authors: HM
authors: Piper
authors: T
authors: T
authors: Noll
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002070:Butadienes / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D019610:Cytoprotection; D004791:Enzyme Inhibitors / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D007511:Ischemia / Q000201:enzymology* / Q000473:pathology; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D058912:Purinergic P2Y Receptor Agonists / Q000494:pharmacology*; D058470:Receptors, Purinergic P2Y / Q000378:metabolism*
chemical_list: D000255:Adenosine Triphosphate; D002070:Butadienes; D053148:Caspase 3; D002867:Chromones; D004791:Enzyme Inhibitors; D020930:MAP Kinase Kinase Kinases; D009025:Morpholines; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D051057:Proto-Oncogene Proteins c-akt; D058912:Purinergic P2Y Receptor Agonists; D058470:Receptors, Purinergic P2Y; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one