Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells

作者：Urban D^*; Haertel F V; Gadiraju K; Guenduez D; Aslam M; Piper H M; Noll T

来源：Biochemical and Biophysical Research Communications, 2012, 425(2): 230-236.

DOI：10.1016/j.bbrc.2012.07.073

• Endothelial cells
• Ischemic injury
• Apoptosis
• ATP
• P2-receptors

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%26apos;-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC. %26lt;br%26gt;Methods and results: To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po-2 %26lt; 4 mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/-0.4; P %26lt; 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 %26lt; 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 alpha beta-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. %26lt;br%26gt;Conclusion: 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.

• 出版日期2012-8-24