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.
Moore
D J
DJ
Neurobiology Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK. darren.moore@bbsrc.ac.uk
Chambers
J K
JK
Murdock
P R
PR
Emson
P C
PC
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000242:Cyclic AMP; D005453:Fluorescence; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000145:classification / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D058473:Receptors, Purinergic P2Y2; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013234:Stem Cells / Q000378:metabolism*; D013498:Suramin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D000970:Antineoplastic Agents; D008565:Membrane Proteins; C546382:P2RY1 protein, human; C109734:P2RY11 protein, human; C548576:P2RY12 protein, human; C440036:P2RY13 protein, human; C546385:P2RY2 protein, human; D010975:Platelet Aggregation Inhibitors; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058925:Receptors, Purinergic P2Y12; D058473:Receptors, Purinergic P2Y2; D013873:Thionucleotides; C104749:purinoceptor P2Y4; C099845:purinoceptor P2Y6; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; C002480:methylthio-ADP; C022571:adenosine 5'-O-(3-thiotriphosphate); D011732:Pyridoxal Phosphate; D013498:Suramin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D020928:Mitogen-Activated Protein Kinases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins; D002118:Calcium; D014544:Uridine Triphosphate; C008501:2-methylthio-ATP