Extracellular ATP triggers two functionally distinct calcium signalling pathways in PC12 cells.
We have investigated the effects of extracellular ATP on Ca2+ signalling, and its relationship to secretion in rat pheochromocytoma (PC12) cells. In single cells, extracellular ATP evoked two very distinct subcellular distributions of intracellular calcium concentration ([Ca2+]i), only one of which could be mimicked by the pyrimidine nucleotide UTP, suggesting the involvement of more than one cell surface receptor in mediating the ATP-induced responses. ATP and UTP were equipotent in activating a receptor leading to inositol phosphate production and the mobilisation of intracellular Ca2+. In some cells (19%) this rise in [Ca2+]i initiated at a discrete site and then propagated across the cell in the form of a Ca2+ wave. In addition to mobilising intracellular Ca2+ through a 'nucleotide' receptor sensitive to ATP and UTP, the results indicate that ATP also activates divalent cation entry through an independent receptor-operated channel. Firstly, ATP-induced entry of Ca2+ or Mn2+ was independent of Ca2+ mobilisation, as prior treatment of cell populations with UTP abolished the ATP-evoked release of intracellular Ca2+ stores, but left the Ca(2+)- and Mn(2+)-entry components uneffected. Secondly, although UTP and ATP were equally effective in generating inositol phosphates, only ATP stimulated divalent cation entry, indicating that ATP-activated influx was independent of phosphoinositide turnover. Thirdly, single cell experiments revealed a subpopulation of cells that responded to ATP with divalent cation entry without mobilising Ca2+ from intracellular stores. Lastly, the dihydropyridine antagonist, nifedipine, reduced the ATP-induced rise in [Ca2+]i by only 24%, suggesting that Ca2+ entry was largely independent of L-type voltage-operated Ca2+ channels. The Ca2+ signals could also be distinguished at a functional level. Activation of ATP-induced divalent cation influx was absolutely required to evoke transmitter release, because ATP triggered secretion of [3H]dopamine only in the presence of external Ca2+, and UTP was unable to promote secretion, irrespective of the extracellular [Ca2+]. The results suggest that the same extracellular stimulus can deliver different Ca2+ signals into the same cell by activating different Ca2+ signalling pathways, and that these Ca2+ signals can be functionally distinct.
Barry
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VA
AFRC Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, UK.
Cheek
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TR
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006868:Hydrolysis; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects; D008345:Manganese / Q000378:metabolism; D016716:PC12 Cells; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D014544:Uridine Triphosphate / Q000494:pharmacology
D007473:Ion Channels; D010716:Phosphatidylinositols; D008345:Manganese; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate; D004298:Dopamine