Brône Bert, Moechars Diederik, Marrannes Roger, Mercken Marc, Meert Theo
Gobal Preclinical Development, Johnson and Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B2340 Beerse, Belgium.
Immunol Lett. 2007 Nov 15;113(2):83-9. doi: 10.1016/j.imlet.2007.07.015. Epub 2007 Aug 22.
In this study the ATP-induced (P2X) currents in isolated peritoneal macrophages of wild type (WT) and P2X(4) knockout (P2X(4)(-/-)) mice were studied by means of whole-cell patch clamp in order to (1) survey the P2X currents of native macrophages and (2) to investigate the expression of P2X(4)-like currents in the WT versus P2X(4)(-/-) mice. Three types of currents were observed in the isolated macrophages: (1) in approximately 10% of both WT and P2X(4)(-/-) macrophages a fast activating and inactivating P2X1-like current was recorded with low concentrations (0.1-1 microM) of ATP; (2) 85% of wild type and 100% of P2X(4)(-/-) macrophages exhibited a non-desensitizing P2X(7)-like current activated at high concentrations of ATP (10mM). The identity of the P2X(7) current was confirmed using the specific blocker A-740003; (3) 88.6% of the WT but none of the P2X(4)(-/-) macrophages showed a small P2X(4)-like current that desensitized slowly upon ATP application at intermediate concentrations (3-300 microM). Several observations indicated that the slowly desensitizing current in WT macrophages was P2X(4). The EC50 value of 5.3 microM ATP was as expected for P2X(4) and the current induced by 3-300 microM ATP was absent in P2X(4)(-/-) mice. Upon application of 3 microM ivermectin, a P2X(4)-selective modulator, the amplitude of this current was increased and the desensitization was inhibited in WT cells. In addition, this current was facilitated by 10 microM Zn(2+) but inhibited by Cu(2+) (in contrast to P2X(2)). We conclude that the P2X(4) and P2X(7) currents are functionally expressed in recruited peritoneal macrophages of WT mice and that the P2X(4)-like current is absent in P2X(4)(-/-) mice.
在本研究中，通过全细胞膜片钳技术研究了野生型（WT）和P2X(4)基因敲除（P2X(4)(-/-)）小鼠分离的腹腔巨噬细胞中ATP诱导的（P2X）电流，目的是：（1）检测天然巨噬细胞的P2X电流；（2）研究WT小鼠与P2X(4)(-/-)小鼠中P2X(4)样电流的表达情况。在分离的巨噬细胞中观察到三种类型的电流：（1）在WT和P2X(4)(-/-)巨噬细胞中约10%的细胞，用低浓度（0.1 - 1 microM）的ATP记录到一种快速激活和失活的P2X1样电流；（2）85%的野生型和100%的P2X(4)(-/-)巨噬细胞表现出一种在高浓度ATP（10 mM）时激活的非脱敏性P2X(7)样电流。使用特异性阻滞剂A - 740003证实了P2X(7)电流的特性；（3）88.6%的WT巨噬细胞但P2X(4)(-/-)巨噬细胞均未表现出一种小的P2X(4)样电流，该电流在中等浓度（3 - 300 microM）ATP作用下缓慢脱敏。多项观察表明，WT巨噬细胞中缓慢脱敏的电流是P2X(4)。5.3 microM ATP的EC50值符合P2X(4)的预期，且3 - 300 microM ATP诱导的电流在P2X(4)(-/-)小鼠中不存在。施加3 microM伊维菌素（一种P2X(4)选择性调节剂）后，WT细胞中该电流的幅度增加且脱敏受到抑制。此外，该电流受到10 microM Zn(2+)的促进但受到Cu(2+)的抑制（与P2X(2)相反）。我们得出结论，P2X(4)和P2X(7)电流在WT小鼠募集的腹腔巨噬细胞中功能性表达，且P2X(4)样电流在P2X(4)(-/-)小鼠中不存在。
