Differential effects of P2Y 1 and P2Y 12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3‐kinase signalling and cell proliferation in serum‐deprived and nonstarved glioma C6 cells

We have previously shown that, in glioma C6 cells, two nucleotide ADP‐sensitive receptors coexist: P2Y 1 , coupled to PLC and responsible for Ca 2+ release, and P2Y 12 , negatively coupled to adenylate cyclase. In the present study, we examined the effects of the stimulation of these two receptors on ERK1/2 and PI3‐K activation, and cell proliferation in either serum‐deprived or nonstarved C6 cells. In response to ADP and its analogues, in serum‐starved cells, both p44 ERK1 and p42 ERK2 were activated in a time‐dependent manner, as monitored by Western blot analysis using an antiphospho‐p42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca 2+ and partially or almost completely by MRS2179 or AR‐C69931MX, specific antagonists of the P2Y 1 and P2Y 12 receptors, respectively. The inhibitory effect of antagonists was additive. These data indicate the involvement of both receptors, P2Y 1 and P2Y 12 , in the ERK1/2 activation, but the P2Y 12 receptor contribution predominates. ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. In nonstarved cells, ADP markedly decreased the PI3‐K activity. In contrast, in serum‐starved cells, ADP evoked an increase in the PI3‐K activity. Blocking of the P2Y 1 receptor by MRS2179 additionally increased this ADP response. These results suggest that the P2Y 1 receptor has an inhibitory and the P2Y 12 receptor a stimulatory effect on PI3‐K signalling pathway. RT–PCR analysis revealed different mRNA expression of both receptors in starved and nonstarved cells. In nonstarved cells, the P2Y 1 receptor mRNA predominates, whereas in serum‐deprived cells the expression of P2Y 12 mRNA becomes more pronounced.British Journal of Pharmacology (2004) 141 , 497–507. doi: 10.1038/sj.bjp.0705639