Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y1 and P2Y12 receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5′-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y1 receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y12 receptor behaved similarly. We demonstrated here that, unlike with the Gq-coupled P2Y1 receptor, activation of the Gi-coupled P2Y12 receptor does not induce apoptosis. Furthermore, activation of the P2Y12 receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor α (TNFα)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y12 receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y1 receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y12 receptor activation by 10 nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100 pM 2MeSADP, activation of the hP2Y12 receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y12 receptor activation. Apyrase did not affect the ability of TNFα to induce apoptosis in hP2Y12-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y1 and P2Y12 receptors and their opposing effects on cell death pathways.