School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
Int J Mol Sci. 2017 Oct 27;18(11):2261. doi: 10.3390/ijms18112261.
Monocytes and macrophages express a repertoire of cell surface P2 receptors for adenosine 5'-triphosphate (ATP) a damage-associated molecular pattern molecule (DAMP), which are capable of raising cytoplasmic calcium when activated. This is achieved either through direct permeation (ionotropic P2X receptors) or by mobilizing intracellular calcium stores (metabotropic P2Y receptors). Here, a side-by-side comparison to investigate the contribution of P2X4 receptor activation in ATP-evoked calcium responses in model human monocytes and macrophages was performed. The expression of P2X1, P2X4, P2X5 and P2X7 was confirmed by qRT-PCR and immunocytochemistry in both model monocyte and macrophage. ATP evoked a concentration-dependent increase in intracellular calcium in both THP-1 monocyte and macrophages. The sarco/endoplasmic reticulum Ca-ATPase inhibitor thasigargin (Tg) responses to the maximal ATP concentration (100 μM) in THP-1 monocytes, and responses in macrophage were significantly attenuated. Tg-resistant ATP-evoked calcium responses in the model macrophage were dependent on extracellular calcium, suggesting a requirement for calcium influx. Ivermectin (IVM) potentiated the magnitude of Tg-resistant component and slowed the decay of response in the model macrophage. The Tg-resistant component was attenuated by P2X4 antagonists 5-BDBD and PSB-12062 but not by the P2X1 antagonist Ro0437626 or the P2X7 antagonist A438079. shRNA-mediated P2X4 knockdown resulted in a significant reduction in Tg-resistant ATP-evoked calcium response as well as reduced sensitivities towards P2X4-specific pharmacological tools, IVM and PSB-12062. Inhibition of endocytosis with dynasore significantly reduced the magnitude of Tg-resistant component but substantially slowed decay response. Inhibition of calcium-dependent exocytosis with vacuolin-1 had no effect on the Tg-resistant component. These pharmacological data suggest that P2X4 receptor activation contributed significantly towards the ionotropic calcium response evoked by ATP of the model human macrophage.
单核细胞和巨噬细胞表达一系列细胞表面 P2 受体，用于结合腺苷 5'-三磷酸 (ATP) 这种损伤相关分子模式分子 (DAMP)，受体激活时能够提高细胞质内钙离子浓度。这可以通过直接渗透 (离子型 P2X 受体) 或动员细胞内钙储存库 (代谢型 P2Y 受体) 来实现。在这里，我们并排比较了 P2X4 受体激活在模型人单核细胞和巨噬细胞中 ATP 诱导的钙反应中的作用。通过 qRT-PCR 和免疫细胞化学，在两种模型单核细胞和巨噬细胞中均证实了 P2X1、P2X4、P2X5 和 P2X7 的表达。ATP 在 THP-1 单核细胞和巨噬细胞中均引起浓度依赖性的细胞内钙离子增加。在 THP-1 单核细胞中，肌浆/内质网 Ca-ATP 酶抑制剂硫代甘肽 (Tg) 对最大 ATP 浓度 (100 μM) 的反应以及巨噬细胞中的反应均显著减弱。模型巨噬细胞中 Tg 抵抗的 ATP 诱导的钙反应依赖于细胞外钙，表明需要钙内流。伊维菌素 (IVM) 增强了 Tg 抵抗成分的幅度，并减缓了模型巨噬细胞中反应的衰减。Tg 抵抗成分被 P2X4 拮抗剂 5-BDBD 和 PSB-12062 减弱，但不被 P2X1 拮抗剂 Ro0437626 或 P2X7 拮抗剂 A438079 减弱。shRNA 介导的 P2X4 敲低导致 Tg 抵抗的 ATP 诱导钙反应显著减少，以及对 P2X4 特异性药理学工具、IVM 和 PSB-12062 的敏感性降低。用 dynasore 抑制内吞作用显著降低了 Tg 抵抗成分的幅度，但大大减缓了反应的衰减。用 vacuolin-1 抑制钙依赖性胞吐作用对 Tg 抵抗成分没有影响。这些药理学数据表明，P2X4 受体激活对模型人巨噬细胞中由 ATP 诱导的离子型钙反应有重要贡献。
