Adenosine triphosphate induces proliferation of human neural stem cells: Role of calcium and p70 ribosomal protein S6 kinase

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Abstract
Human neural stem cells (NSCs) grown in culture responded to extracellularly applied adenosine triphosphate (ATP), and the rate of proliferation increased as shown by immunocytochemical and RT‐PCR analysis. Activation of P2 purinoceptors by ATP is coupled to the release of intracellular calcium ([Ca2+]i) from thapsigargin‐sensitive intracellular stores. ATP‐induced proliferation was blocked by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+‐ATPase. Neither EGTA, a calcium chelator, nor caffeine had any effect on ATP‐induced [Ca2+]i increases. Multiblot kinase analysis, by which activation of 24 different kinases could be determined, showed that application of ATP to NSCs predominantly activated p70 ribosomal protein S6 kinase (p70 S6 kinase). As well, rapamycin, a p70 S6 kinase inhibitor, blocked the ATP‐mediated proliferative response in NSCs. After outlining a role for p70 S6 kinase in ATP‐mediated NSC proliferation, we examined the possibility that phosphatidylinositol 3‐kinase (PI3‐kinase) acts upstream of p70 S6 kinase. The application of wortmannin, a PI3‐kinase inhibitor, decreased both ATP‐mediated p70 S6 kinase activation and NSC proliferation. From these results, we conclude that ATP application to NSCs induces release of Ca2+ from intracellular Ca2+ stores and that this increase in intracellular Ca2+ in turn promotes NSC proliferation. The increase in NSC proliferation observed following ATP application can also be mediated by PI3‐kinase‐dependent p70 S6 kinase activation.