Human Ntera-2/D1 neuronal progenitor cells endogenously express a functional P2Y1 receptor

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.