Add time:07/30/2019         Source:sciencedirect.com

We report here that human Ntera-2/D1 (NT-2) cells, an undifferentiated committed neuronal progenitor cell line, endogenously express a functional P2Y1 receptor, while other P2Y subtypes, except perhaps P2Y4, are not functionally expressed. Quantitative RT-PCR analysis showed that NT-2 cells abundantly express mRNA for P2Y1 and P2Y11 receptors, while P2Y2 and P2Y4 receptors were detected at considerably lower levels. Western blot analysis also demonstrated expression of P2Y1 receptors and Gαq/11 subunits. Various nucleotides induced intracellular Ca2+ mobilisation in NT-2 cells in a concentration-dependent manner with a rank order potency of 2-MeSADP > 2-MeSATP > ADP > ATP > UTP > ATPγS, a profile resembling that of human P2Y1 receptors. Furthermore, P2Y1 receptor-specific (A3P5P) and P2Y-selective (PPADS, suramin) antagonists inhibited adenine nucleotide-induced Ca2+ responses in a concentration-dependent manner, consistent with expression of a P2Y1 receptor. Moreover, of seven adenine nucleotides tested, only Bz-ATP and ATPγS elicited small increases in cAMP formation suggesting that few, if any, functional P2Y11 receptors were expressed. P2Y1 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 P2Y1 receptor.

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