Role of Ca2+ in differentiation mediated by nerve growth factor and dibutyryl cyclic AMP in PC12 cells

Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca2+ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca2+ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca2+ influx, and Ca2+ mobilization from intracellular Ca2+ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca2+ accumulation by nonmitochondrial Ca2+ pools or the thapsigargin (TG)-sensitive Ca2+ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca2+ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca2+ . Therefore, NGF and dbcAMP induced different effects on Ca2+ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca2+ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca2+ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.