Department of Neurobiology, Neuroscience Program, Institute of Biology, Fluminense Federal University, Cx. Postal 100180, Niterói, RJ, 24001-970, Brazil.
Purinergic Signal. 2013 Mar;9(1):15-29. doi: 10.1007/s11302-012-9324-5. Epub 2012 Jun 26.
Previous data suggest that nucleotides are important mitogens in the developing retina. Here, the effect of ATP on the death of cultured chick embryo retina cells was investigated. In cultures obtained from retinas of 7-day-old chick embryos (E7) that were cultivated for 2 days (E7C2), both ATP and BzATP induced a ∼30 % decrease in cell viability that was time- and dose-dependent and that could be blocked by 0.2 mM oxidized ATP or 0.3 μM KN-62. An increase in cleaved caspase-3 levels and in the number of TUNEL-positive cells was observed when cultures were incubated with 3 mM ATP and immunolabeling for cleaved-caspase 3 was observed over neurons but not over glial cells. ATP-dependent cell death was developmentally regulated, the maximal levels being detected by E7C2-3. Nucleotides were able to increase neuronal ethidium bromide and sulforhodamine B uptake in mixed and purified neuronal cultures, an effect that was blocked by the antagonists Brilliant Blue G and oxidized ATP. In contrast, nucleotide-induced cell death was observed only in mixed cultures, but not in purified cultures of neurons or glia. ATP-induced neuronal death was blocked by the glutamatergic antagonists MK801 and DNQX and activation of P2X7 receptors by ATP decreased the uptake of [(3)H]-D-aspartate by cultured glial cells with a concomitant accumulation of it in the extracellular medium. These results suggest that ATP induces apoptosis of chick embryo retinal neurons in culture through activation of P2X7 and glutamate ionotropic receptors. Involvement of a P2X7 receptor-mediated inhibition of the glial uptake of glutamate is suggested.
先前的数据表明核苷酸在发育中的视网膜中是重要的有丝分裂原。在这里，研究了 ATP 对培养的鸡胚视网膜细胞死亡的影响。从 7 日龄鸡胚（E7）的视网膜获得的培养物（E7C2）培养 2 天后，ATP 和 BzATP 均诱导细胞活力下降约 30%，这种下降具有时间和剂量依赖性，并且可以被 0.2mM 氧化型 ATP 或 0.3μM KN-62 阻断。当培养物用 3mM ATP 孵育时，观察到 cleaved caspase-3 水平升高和 TUNEL 阳性细胞增多，并且在神经元上观察到 cleaved-caspase 3 的免疫标记，但在神经胶质细胞上没有观察到。ATP 依赖性细胞死亡受到发育调控，最大水平在 E7C2-3 时检测到。核苷酸能够增加混合和纯化神经元培养物中 ethidium bromide 和 sulforhodamine B 的摄取，该效应被 Brilliant Blue G 和氧化型 ATP 拮抗剂阻断。相反，仅在混合培养物中观察到核苷酸诱导的细胞死亡，而在纯化的神经元或神经胶质细胞培养物中未观察到。ATP 诱导的神经元死亡被谷氨酸能拮抗剂 MK801 和 DNQX 阻断，并且 ATP 对 P2X7 受体的激活减少了培养的神经胶质细胞对 [(3)H]-D-天冬氨酸的摄取，同时使其在细胞外介质中积累。这些结果表明，ATP 通过激活 P2X7 和谷氨酸离子型受体诱导鸡胚视网膜神经元在培养物中发生细胞凋亡。提示涉及 P2X7 受体介导的对谷氨酸摄取的抑制。
