Moore D J, Chambers J K, Murdock P R, Emson P C
Neurobiology Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK.
Neuropharmacology. 2002 Nov;43(6):966-78. doi: 10.1016/s0028-3908(02)00177-6.
We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y(1) receptor, while other P2Y subtypes, except perhaps P2Y(4), are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y(1) and P2Y(11) receptors, while P2Y(2) and P2Y(4) receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y(1) receptors and Galpha(q/11) subunits. Various nucleotides induced intracellular Ca(2+) mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPgammaS, a profile resembling that of human P2Y(1) receptors. Furthermore, P2Y(1) receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca(2+) responses in a concentration-dependent manner, consistent with expression of a P2Y(1) receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPgammaS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y(11) receptors were expressed. P2Y(1) receptor-selective adenine nucleotides, including 2-MeSADP and ADP, also induced concentration-dependent phosphorylation and hence, activation of the extracellular-signal regulated protein kinases (ERK1/2). NT-2 cells, therefore, provide a useful neuronal-like cellular model for studying the precise signalling pathways and physiological responses mediated by a native P2Y(1) receptor.
我们在此报告，人Ntera-2/D1（NT-2）细胞，一种未分化的定向神经元祖细胞系，内源性表达功能性P2Y(1)受体，而其他P2Y亚型，可能除了P2Y(4)之外，均未功能性表达。定量逆转录聚合酶链反应（RT-PCR）分析表明，NT-2细胞大量表达P2Y(1)和P2Y(11)受体的信使核糖核酸（mRNA），而P2Y(2)和P2Y(4)受体的检测水平则低得多。蛋白质免疫印迹分析也证实了P2Y(1)受体和Gα(q/11)亚基的表达。各种核苷酸以浓度依赖性方式诱导NT-2细胞内钙离子动员，其效力顺序为2-甲基硫代二磷酸腺苷（2-MeSADP）> 2-甲基硫代三磷酸腺苷（2-MeSATP）> 二磷酸腺苷（ADP）> 三磷酸腺苷（ATP）> 三磷酸尿苷（UTP）> 三磷酸腺苷γ-硫酯（ATPγS），这一特征与人P2Y(1)受体相似。此外，P2Y(1)受体特异性拮抗剂（A3P5P）和P2Y选择性拮抗剂（吡哆醛-5'-磷酸硫酸酯（PPADS）、苏拉明）以浓度依赖性方式抑制腺嘌呤核苷酸诱导的钙离子反应，这与P2Y(1)受体的表达一致。此外，在所测试的七种腺嘌呤核苷酸中，只有苯甲酰三磷酸腺苷（Bz-ATP）和ATPγS引起环磷酸腺苷（cAMP）生成的小幅增加，这表明几乎没有功能性P2Y(11)受体表达。包括2-MeSADP和ADP在内的P2Y(1)受体选择性腺嘌呤核苷酸也诱导浓度依赖性磷酸化，从而激活细胞外信号调节蛋白激酶（ERK1/2）。因此，NT-2细胞为研究天然P2Y(1)受体介导的精确信号通路和生理反应提供了一个有用的类神经元细胞模型。
