ATP and its metabolites are important extracellular signal transmitters acting on purinergic P2 and P1 receptors. Most cells can actively secrete ATP in response to a variety of external stimuli such as gating of the P2X7 receptor. We used Yac-1 murine lymphoma cells to study P2X7-mediated ATP release. These cells co-express P2X7 and ADP-ribosyltransferase ARTC2, permitting gating of P2X7 by NAD⁺-dependent ADP-ribosylation without the need to add exogenous ATP. Yac-1 cells released ATP into the extracellular space within minutes after stimulation with NAD⁺. This was blocked by pre-incubation with the inhibitory P2X7-specific nanobody 13A7. Gating of P2X7 for 3 h significantly decreased intracellular ATP levels in living cells, but these had returned to normal by 20 h. P2X7-mediated ATP release was dependent on a rise in cytosolic calcium and the depletion of intracellular potassium, but was not blocked by inhibitors of pannexins or connexins. We used genetically encoded FRET-based ATP sensors targeted to the cytosol to image P2X7-mediated changes in the distribution of ATP in 3T3 fibroblasts co-expressing P2X7 and ARTC2 and in Yac-1 cells. In response to NAD⁺, we observed a marked depletion of ATP in the cytosol. This study demonstrates the potential of ATP sensors as tools to study regulated ATP release by other cell types under other conditions.

中文翻译：

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P2X7介导的ATP分泌伴随着胞质ATP的消耗。

ATP及其代谢物是作用于嘌呤能P2和P1受体的重要细​​胞外信号传递者。大多数细胞可以响应各种外部刺激（例如P2X7受体的门控）而主动分泌ATP。我们使用Yac-1鼠淋巴瘤细胞研究P2X7介导的ATP释放。这些细胞共表达P2X7和ADP-核糖基转移酶ARTC2，从而允许通过NAD ⁺依赖性ADP-核糖基化对P2X7进行门控，而无需添加外源ATP。在用NAD ⁺刺激后几分钟内，Yac-1细胞将ATP释放到细胞外空间。通过与抑制性P2X7特异性纳米抗体13A7预先孵育来封闭。门控P2X7 3小时显着降低了活细胞的细胞内ATP水平，但到20小时它们已恢复正常。P2X7介导的ATP释放取决于胞质钙的增加和细胞内钾的消耗，但不受pannexins或connexins抑制剂的阻止。我们使用针对细胞溶质的基因编码的基于FRET的ATP传感器对P2X7介导的在共同表达P2X7和ARTC2的3T3成纤维细胞以及Yac-1细胞中ATP分布的变化进行成像。响应NAD ⁺，我们观察到胞浆中ATP的明显消耗。这项研究证明了ATP传感器作为研究其他条件下其他细胞类型调节的ATP释放的工具的潜力。