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Abstract：
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.
摘要：
将用大鼠P2X2受体亚基稳定转染的人胚肾293（HEK293）细胞与200nM孕酮（HEK293-P2X2-PROG）预孵育，同型P2X2受体的有效正变构调节剂，并用于检测低纳摩尔浓度的细胞外ATP。将呋喃-2负载的HEK293-P2X2-PROG细胞急性铺板在培养的DH神经胶质细胞的顶部以定量从单个DH神经胶质细胞释放的ATP。α1肾上腺素受体激动剂苯肾上腺素（PHE，20μM）或低K（0.2mM）溶液引起生物传感器细胞中细胞内钙浓度（[Ca2]i）的可逆增加。在一般细胞外灌注中断后，在一半的生物传感器细胞中也检测到[Ca2]i的可逆增加。在P2X2拮抗剂PPADS存在下或在用钙螯合剂BAPTA预加载神经胶质细胞后，[Ca2]i的所有增加都被阻断，表明它们是由于神经胶质细胞释放钙依赖性ATP所致。PHE诱导的ATP释放被-L-苯丙氨酸2-萘胺（GPN）阻断，该-L-苯丙氨酸2-萘胺能渗透分泌性溶酶体和巴弗洛霉素A1（BafA1），酸性分泌囊泡H+泵的抑制剂。相比之下,BafA1消除了由低K溶液引起的ATP释放，但GPN并未消除。最后,中断一般灌注后观察到的自发ATP释放对GPN和BafA1预处理均不敏感。我们的结果表明，ATP以钙依赖性方式从DH神经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，去甲肾上腺素和PHE选择性地靶向分泌性溶酶体池。
