Department of Environmental Biochemistry, Kyoto Pharmaceutical University, Kyoto, Japan.
J Neurochem. 2012 Sep;122(6):1118-28. doi: 10.1111/j.1471-4159.2012.07868.x. Epub 2012 Aug 3.
P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.
P2X7 受体（P2X7R）在免疫细胞中被称为“死亡受体”，但其在神经元等非免疫细胞中的功能性表达存在争议。在这里，我们研究了 P2X7R 激活和线粒体功能障碍在培养的皮质神经元中 ATP 诱导的神经元死亡中的作用。在 P2X7R 和连接蛋白-1 表达的神经元培养物中，5mM 或更多的 ATP 或 0.1mM 或更多的 BzATP 诱导包括凋亡在内的神经元死亡，而 oxATP、P2X7R 选择性拮抗剂则可预防细胞死亡。用 ATP 处理的神经元表现出 Ca(2+)内流和 YO-PRO-1 摄取，前者被 oxATP 和 A438079 抑制，后者被 oxATP 和 carbenoxolone 抑制，而 oxATP 抑制 P2X7R，但 carbenoxolone 阻断连接蛋白-1 则不能阻止 ATP 诱导的神经元死亡。ATP 处理通过激活 NADPH 氧化酶诱导活性氧的产生，并激活聚（ADP-核糖）聚合酶，但两者对神经元死亡没有或几乎没有贡献。Rhodamine123 从神经元线粒体中的流出增加是由 ATP 处理引起的，被 oxATP 抑制，而线粒体通透性转换孔抑制剂环孢菌素 A 显著降低了 ATP 诱导的神经元死亡。在用 ATP 处理的神经元中，前胱天蛋白酶-3 的切割增加，而胱天蛋白酶抑制剂 Q-VD-OPh 和 Z-DEVD-FMK 抑制了神经元的死亡。凋亡诱导因子的切割增加，钙蛋白酶抑制剂 MDL28170 和 PD151746 抑制了神经元的死亡。这些发现表明，P2X7R 在皮质神经元培养物中功能性表达，其激活触发的 Ca(2+)内流和线粒体功能障碍在 ATP 诱导的神经元死亡中发挥了重要作用。
