1 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 þ 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 antiphosphop42/p44 MAPK antibody. The phosphorylation was reduced both by removal of the extracellular Ca 2 þ 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 serumdeprived cells the expression of P2Y 12 mRNA becomes more pronounced.

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 and P2Y 12 family members while among P2X receptors P2X 4 , P2X 5 and P2X 7 were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca 2+ ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X 5 play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y 2 -dependent Ca 2+ signaling, while the mRNA expression of P2Y 2 receptor reminds still on the high level. Moreover, P2X 2 receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.Abbreviations: 2MeSADP -2-methylthio-ADP; BzATP -3 0 -0(4-benzoyl)benzoyl ATP; [Ca 2+ ]i -intracellular Ca 2+ concentration; DMEM -Dulbecco's modified essential medium; ERK -Ras/extracellular signal-regulated kinase; FCS -fetal calf serum; GAPDH -glycerol 3-phosphate dehydrogenase; HS -horse serum; InsP 3 -inositol 1,4,5-trisphosphate; PLCphospholipase C; PPADS -pirydoxal-phosphate-6-azophenyl-2 0 , 4 0 -disulphonic acid