Antagonists of P2 receptor prevent NGF-dependent neuritogenesis in PC12 cells

The pheochromocytoma PC12 cell line that develops neuronal characteristics of sympathetic cells after treatment with nerve growth factor (NGF) represe nts a well-established cellular model system for studying NGF signalling. I nteresting information on the different mechanistic pathways of NGF can be obtained by adopting the pharmacological approach of inhibiting P2 receptor s, expressed in naive PC12 cells and recognised as important biological med iators of neurotransmitters and growth factors. We show here that Basilen B lue, an antagonist of P2 receptor, reversibly prevents NGF-dependent neurit e outgrowth with an IC50 in the 5-10 mu M range. Suramin, oxidised-ATP and diisothiocyanatostilbene-disulfonic acid, differently from other purinocept or ligands, are also effective in this regard. NGF-dependent regeneration a nd stability of neurites, selected NGF-dependent extracellular and intracel lular protein phosphorylations, binding of [H-3] ATP to PC12 cell membranes are also modulated by Basilen Blue. On the contrary, cell adhesion, cellul ar duplication, Sr-nucleotidase activity, NGF-induced tyrosine autophosphor ylation of TrkA receptors are not affected. NGF furthermore directly modula tes the extracellular release of ATP and especially the levels of P2X(2) re ceptor protein in PC12 cells. In addition, extracellular ATP improves the n euritogenic effect of sub-optimal concentrations of NGF. Our study identifies P2 receptor ligands, particularly Basilen Blue, as use ful tools to dissect different NGF-evoked functions, suggesting a mechanist ic role for P2 receptors in the signalling pathways of NGF. (C) 2000 Elsevi er Science Ltd. All rights reserved.