Differential effects of P2Y1 and P2Y12 nucleotide receptors on ERK1/ERK2 and phosphatidylinositol 3-kinase signalling and cell proliferation in serum-deprived and nonstarved glioma C6 cells

1 We have previously shown that, in glioma C6 cells, two nucleotide ADP-sensitive receptors coexist: P2Y(1), coupled to PLC and responsible for Ca2+ 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. 2 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 Ca2+ 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. 3 ERK1/2 activity was positively correlated with cell proliferation of cultured glioma C6 cells. 4 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. 5 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.