Reactive blue 2 inhibition of cyclic AMP-dependent differentiation of rat C6 glioma cells by purinergic receptor-independent inactivation of phosphatidylinositol 3-kinase

Abstract

Cyclic AMP-dependent differentiation of rat C6 glioma cells into an astrocyte type II is characterized by inhibition of cell growth and induction of glial fibrillary acidic protein (GFAP) synthesis. Activation of the P2Y 12 receptor with 2-methylthioadenosine-5′-diphosphate inhibited β-adrenergic receptor-induced differentiation. The selective P2Y 12 receptor antagonist N 6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-β,γ-dichloromethylene ATP abolished the receptor-mediated effect on differentiation. In contrast non-selective antagonists of P2Y receptors did not revert the inhibiting effect of the P2Y 12 receptor on differentiation. Reactive blue 2 (RB2), a potent P2Y 12 receptor antagonist, completely inhibited the synthesis of GFAP, while the P2Y receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid were less efficient. However, although P2Y receptor antagonists inhibited GFAP synthesis to a different extent they were unable to relieve the growth inhibition that accompanied induction of differentiation, whereas stimulation of the P2Y 12 receptor with 2-methylthioadenosine-5′-diphosphate inhibited GFAP expression and restored cell proliferation. Assay of the activity of phosphatidylinositol 3-kinase (PI 3-K), an enzyme required for GFAP expression [J. Neurochem. 76 (2001) 610], showed that RB2 inhibited this enzyme after cellular uptake, while suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid inhibited PI 3-K to a lesser extent. The intracellular concentration of RB2 increased in time and attained the ic 50 for PI 3-K inhibition (4 μM) after 40-min incubation with 50 μM RB2. In conclusion, cAMP-induced differentiation in C6 cells is inhibited by activation of the P2Y 12 receptor. In addition, synthesis of GFAP is also inhibited by cellular uptake of non-selective nucleotide receptor antagonists that inhibit PI 3-K, a kinase required for the cAMP-dependent induction of differentiation.