Human embryonic kidney 293 (HEK293) cells stably transfected with the rat P2X2 receptor subunit were preincubated with 200 nM progesterone (HEK293-P2X2-PROG), a potent positive allosteric modulator of homomeric P2X2 receptors, and used to detect low nanomolar concentrations of extracellular ATP. Fura-2-loaded HEK293-P2X2-PROG cells were acutely plated on top of cultured DH glial cells to quantify ATP release from single DH glial cells. Application of the α1 adrenoceptor agonist phenylephrine (PHE, 20 μM) or of a low K⁺ (0.2 mM) solution evoked reversible increases in the intracellular calcium concentration ([Ca²⁺]_i) in the biosensor cells. A reversible increase in [Ca²⁺]_i was also detected in half of the biosensor cells following the interruption of general extracellular perfusion. All increases in [Ca²⁺]_i were blocked in the presence of the P2X2 antagonist PPADS or after preloading the glial cells with the calcium chelator BAPTA, indicating that they were due to calcium-dependent ATP release from the glial cells. ATP release induced by PHE was blocked by -l-phenylalanine 2-naphtylamide (GPN) that permeabilizes secretory lysosomes and bafilomycin A1 (Baf A1), an inhibitor of the H⁺-pump of acidic secretory vesicles. By contrast, ATP release induced by application of a low-K⁺ solution was abolished by Baf A1 but not by GPN. Finally, spontaneous ATP release observed after interrupting general perfusion was insensitive to both GPN and Baf A1 pretreatment. Our results indicate that ATP is released in a calcium-dependent manner from two distinct vesicular pools and one non-vesicular pool coexisting in DH glial cells and that noradrenaline and PHE selectively target the secretory lysosome pool.

中文翻译：

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高分辨率检测单培养小鼠背角脊髓神经胶质细胞释放的ATP及其受去甲肾上腺素的调节作用。

将稳定转染了大鼠P2X2受体亚基的人胚胎肾293（HEK293）细胞与200 nM孕酮（HEK293-P2X2-PROG）进行预孵育，这是一种有效的同型P2X2受体的正构构调节剂，用于检测低纳摩尔浓度的细胞外ATP 。将装有Fura-2的HEK293-P2X2-PROG细胞急性接种在培养的DH胶质细胞顶部，以定量从单个DH胶质细胞释放的ATP。α1肾上腺素受体激动剂去氧肾上腺素（PHE，20μM）或低K ⁺（0.2 mM）溶液的应用引起生物传感器细胞内细胞内钙浓度（[Ca ²⁺ ] _i）的可逆增加。[Ca ²⁺ ] _i的可逆增加在一般的细胞外灌注中断后，一半的生物传感器细胞中也检测到了这种蛋白。在存在P2X2拮抗剂PPADS的情况下或在向神经胶质细胞预加载钙螯合剂BAPTA之后，[Ca ²⁺ ] _i的所有增加均被阻止，这表明它们是由于钙依赖性ATP从神经胶质细胞中释放所致。由PHE诱导的ATP释放被能使分泌性溶酶体透化的-1-苯丙氨酸2-萘酰胺（GPN）和酸性分泌囊泡H ⁺泵的抑制剂bafilomycin A1（Baf A1）阻断。相比之下，低钾⁺引起的ATP释放Baf A1取消了该解决方案，但GPN则没有。最后，中断一般灌注后观察到的自发ATP释放对GPN和Baf A1预处理均不敏感。我们的结果表明，ATP以钙依赖的方式从DH胶质细胞中共存的两个不同的囊泡池和一个非囊泡池中释放，并且去甲肾上腺素和PHE选择性靶向分泌性溶酶体池。