Activation of Src/kinase/phospholipase C/mitogen-activated protein kinase and induction of neurite expression by ATP, independent of nerve growth factor

Extracellular ATP has been reported to potentiate the neurite outgrowth induced by nerve growth factor. In the present study the neurotrophic effect of ATP and other nucleotides was examined in mouse neuroblastoma neuro2a cells which lack nerve growth factor receptor. Exposure of neuro2a cells to ATP resulted in a dramatic increase in neurite bearing cells as compared with untreated control cells. Experiments performed with purinergic receptor agonists and antagonists suggest that the ATP stimulates neurite outgrowth via P2 receptors. Neurite outgrowth was completely blocked by P2 receptor antagonist suramin whereas the P1 receptor antagonist CGS15943 was ineffective. P1 receptor agonist 5'-(N-ethylcarboxamido)adenosine failed to induce neurite outgrowth. The potency order of different P2 receptor agonists was ATP=ATPgammaS>ADP>2Me-S-ATP. It was insensitive to UTP and antagonist pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) suggesting the involvement of P2Y11 receptor in the observed neuritogenic effect. The signaling pathway leading to ATP-induced neuritogenesis was investigated. The neuritogenic effect of ATP is independent of rise in intracellular Ca(2+) as pharmacological profile of neuritogenic P2Y receptor does not match with that of P2Y2 receptor associated with [Ca(2+)](i) signaling cascade. Exposure of cells to ATP caused activation of Src kinase, phospholipase Cgamma and extracellular signal-regulated kinases ERK1/2. Mitogen-activated protein kinase (MAPK) inhibitor U0126 drastically reduced the number of neurite bearing cells in ATP-treated cultures implying that the neurotrophic effect of ATP is mediated by MAPK. Our results demonstrate that ATP can stimulate neurite outgrowth independent of other neurotrophic factors and can be an effective trophic agent.