EXTRACELLULAR ATP TRIGGERS 2 FUNCTIONALLY DISTINCT CALCIUM SIGNALING PATHWAYS IN PC12 CELLS

We have investigated the effects of extracellular ATP on Ca2+ signalli ng, and its relationship to secretion in rat pheochromocytoma (PC12) c ells. In single cells, extracellular ATP evoked two very distinct subc ellular distributions of intracellular calcium concentration ([Ca2+](i )), only one of which could be mimicked by the pyrimidine nucleotide U TP, 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 th e 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 intracellu lar Ca2+ through a 'nucleotide' receptor sensitive to ATP and UTP, the results indicate that ATP also activates divalent cation entry throug h an independent receptor-operated channel. Firstly, ATP-induced entry of Ca2+ or Mn2+ was independent of Ca2+ mobilisation, as prior treatm ent of cell populations with UTP abolished the ATP-evoked release of i ntracellular Ca2+ stores, but left the Ca2+- and Mn2+-entry components uneffected. Secondly, although UTP and ATP were equally effective in generating inositol phosphates, only ATP stimulated divalent cation en try, indicating that ATP-activated influx was independent of phosphoin ositide turnover. Thirdly, single cell experiments revealed a subpopul ation of cells that responded to ATP with divalent cation entry withou t mobilising Ca2+ from intracellular stores. Lastly, the dihydropyridi ne antagonist, nifedipine, reduced the ATP-induced rise in [Ca2+](i) b y only 24%, suggesting that Ca2+ entry was largely independent of L-ty pe voltage-operated Ca2+ channels. The Ca2+ signals could also be dist inguished at a functional level, Activation of ATP-induced divalent ca tion influx was absolutely required to evoke transmitter release, beca use ATP triggered secretion of [H-3]dopamine only in the presence of e xternal Ca2+, and UTP was unable to promote secretion, irrespective of the extracellular [Ca2+]. The results suggest that the same extracell ular stimulus can deliver different Ca2+ signals into the same cell by activating different Ca2+ signalling pathways, and that these Ca2+ si gnals can be functionally distinct.