Physiologisches Institut, Justus-Liebig-Universität, Giessen, Germany.
Biochem Biophys Res Commun. 2012 Aug 24;425(2):230-6. doi: 10.1016/j.bbrc.2012.07.073. Epub 2012 Jul 22.
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.
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.
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.
内皮细胞（EC）的凋亡死亡在缺血性损伤的发展中起着至关重要的作用。在本研究中，我们研究了细胞外腺苷-5'-三磷酸（ATP）的影响，无论是从细胞中释放还是外源性添加，对人 EC 缺血诱导凋亡的影响。
为了模拟缺血条件，培养的人脐静脉内皮细胞（HUVEC）在无血清培养基中暴露于 2 小时的缺氧（Po（2）<4mmHg）。与正常氧对照相比，缺血导致 EC 凋亡增加 1.7 倍（+/-0.4；P<0.05），通过免疫印迹和半胱天冬酶-3 的免疫细胞化学检测到。缺血诱导的凋亡伴随着细胞外 ATP 的 2.3 倍（+/-0.5；P<0.05）增加，通过使用荧光素/荧光素酶测定法检测到。添加可溶性外核苷酸酶 apyrase，增强 ATP 降解，增加了半胱天冬酶-3 的切割。相应地，通过添加选择性外核苷酸酶抑制剂 ARL67156 抑制 ATP 分解显著减少了缺血诱导的凋亡。细胞外 ATP 作用于膜结合的 P2Y-和 P2X-受体，诱导细胞内信号转导。ATP 和 P2Y-受体激动剂 UTP 以等效的方式显著降低缺血诱导的凋亡，而 P2X-受体激动剂 αβ-me-ATP 则不改变半胱天冬酶-3 的切割。当用苏拉明或 PPADS 阻断 P2 受体时，ARL67156 和 UTP 的抗凋亡作用被阻断。此外，细胞外 ATP 导致 MEK/ERK-和 PI3K/Akt-信号通路的激活。相应地，UO126 抑制 MEK/ERK 信号或 LY294002 抑制 PI3K/Akt 信号阻断了 ATP 的抗凋亡作用。
本研究的数据表明，细胞外 ATP 通过激活 P2Y-受体介导的信号转导，减少半胱天冬酶-3 的切割，拮抗人 EC 缺血诱导的凋亡。
