Agonists of the P2YAC-receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma

We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap(3)A induced no detectable PI turnover and were identified as specific agonists of the P2Y(AC)-receptor with EC50 values of 250 +/- 37 pm and 1 +/- 0.5 mum, respectively. P2Y(AC)-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2Y(AC)-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP3-independent Ca2+ influx was observed after P2Y(AC)-receptor activation. Inhibition of this influx by Ca2+-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and AP(3)A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA(ASn19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-purinoceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-dependent, but ras- and Ca2+-independent cascade.