School of Biological Sciences, University of Wollongong, Wollongong, NSW, Australia.
Purinergic Signal. 2013 Dec;9(4):609-19. doi: 10.1007/s11302-013-9371-6. Epub 2013 Jun 21.
The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the 'low affinity' IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium(+) uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.
P2X7 受体是一种三聚体 ATP 门控阳离子通道，在健康和疾病中都具有重要作用。我们观察到，特定的磷脂酶 D（PLD）1 拮抗剂 CAY10593 可损害 P2X7 诱导的“低亲和力”IgE 受体 CD23 的脱落。本研究探讨了该化合物对 P2X7 激活的作用方式。ATP 诱导的 ethidium(+)摄取测量表明，CAY10593 损害了人 RPMI 8226 B 细胞、转染 P2X7 的 HEK-293 细胞和外周血单核细胞中的 P2X7 诱导的孔形成。浓度反应曲线表明，CAY10593 对 RPMI 8226 细胞中 P2X7 诱导的孔形成的损害作用比 PLD2 拮抗剂 CAY10594 和非特异性 PLD 拮抗剂 halopemide 更强。电生理学测量表明，CAY10593 还抑制了 P2X7 诱导的内向电流。值得注意的是，RT-PCR 表明 RPMI 8226 细胞中缺乏 PLD1，而胆碱-Cl 培养基或 1-丁醇分别阻断 PLD 刺激和信号转导，不会损害这些细胞中的 P2X7 激活。这些数据表明，CAY10593 损害人 P2X7 独立于 PLD1 刺激，强调了在信号研究中使用的化合物必须确保不影响 P2X7 激活下游信号转导的受体本身的重要性。
