Kaiho H, Matsuoka I, Kimura J, Nakanishi H
Department of Pharmacology, Fukushima Medical College, Japan.
J Neurochem. 1998 Mar;70(3):951-7. doi: 10.1046/j.1471-4159.1998.70030951.x.
ATP activates a nonselective cation current by stimulating the P2Z receptor in NG108-15 cells--a hybrid cell line of the mouse neuroblastoma N18TG-2 cells and the rat glioma C6Bu-1 cells. Recently, the P2X7 receptor was cloned from the rat brain and was found to have electrophysiological properties similar to those of the P2Z receptor. We examined the expression of P2X7 receptor mRNA in NG108-15 cells as well as in their parent cell lines, N18TG-2 and C6Bu-1 cells, by reverse transcription-polymerase chain reaction (RT-PCR). The cDNA templates from these cell lines were amplified with primers specific to the P2X7 receptor sequence. Positive signals were detected in the RT-PCR products from NG108-15 and N18TG-2 cells but not from C6Bu-1 cells. We next examined the effect of ATP on the membrane current in N18TG-2 cells and C6Bu-1 cells by whole-cell voltage clamp. In N18TG-2 cells, ATP induced a sustained current with a reversal potential of 9.3 +/- 1.2 mV (n = 22) in a concentration-dependent manner with an EC50 of 1.76 +/- 0.18 mM (n = 36). In contrast, ATP (1 mM) did not induce any current in C6Bu-1 cells. The ATP-induced current in N18TG-2 cells resembled that in NG108-15 cells in the following points: (a) The currents did not desensitize significantly. (b) EC50 values of ATP are of millimolar order. (c) Benzoylbenzoyl-ATP was a more potent agonist than ATP. (d) The current was larger in methanesulfonate than in Cl- external solution. (e) The current was larger at lower external Mg2+ concentrations. These results suggest that the hybrid NG108-15 cells possess a P2X7 receptor like the P2Z receptor and that the ability of expressing this channel originates from N18TG-2 cells but not from C6Bu-1 cells.
ATP通过刺激NG108 - 15细胞中的P2Z受体来激活一种非选择性阳离子电流，NG108 - 15细胞是小鼠神经母细胞瘤N18TG - 2细胞和大鼠胶质瘤C6Bu - 1细胞的杂交细胞系。最近，P2X7受体从大鼠脑中克隆出来，发现其具有与P2Z受体相似的电生理特性。我们通过逆转录 - 聚合酶链反应（RT - PCR）检测了P2X7受体mRNA在NG108 - 15细胞及其亲本细胞系N18TG - 2和C6Bu - 1细胞中的表达。用针对P2X7受体序列的特异性引物扩增这些细胞系的cDNA模板。在NG108 - 15和N18TG - 2细胞的RT - PCR产物中检测到阳性信号，但在C6Bu - 1细胞中未检测到。接下来，我们通过全细胞膜片钳检测了ATP对N18TG - 2细胞和C6Bu - 1细胞膜电流的影响。在N18TG - 2细胞中，ATP以浓度依赖性方式诱导出持续电流，其反转电位为9.3±1.2 mV（n = 22），EC50为1.76±0.18 mM（n = 36）。相比之下，1 mM的ATP在C6Bu - 1细胞中未诱导出任何电流。N18TG - 2细胞中ATP诱导的电流在以下方面与NG108 - 15细胞中的电流相似：（a）电流没有明显脱敏。（b）ATP的EC50值为毫摩尔级。（c）苯甲酰苯甲酰 - ATP是比ATP更有效的激动剂。（d）甲磺酸盐中的电流比Cl - 外液中的电流大。（e）在较低的细胞外Mg2 +浓度下电流更大。这些结果表明，杂交的NG108 - 15细胞拥有类似于P2Z受体的P2X7受体，并且表达该通道的能力源自N18TG - 2细胞而非C6Bu - 1细胞。
