Agonists of the P2Y(AC)-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-med iated P2Y(AC)-receptor activation in rat C6 glioma. In the present study, 2 MeSADP 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 MA P kinase (ERK1/2) activation that returned to the basal level 4 h after sti mulation and was correlated with a gradual desensitization of the P2Y(AC)-p urinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase a ctivation. An IP3-independent Ca2+ influx was observed after P2Y(AC)-recept or activation. Inhibition of this influx by Ca2+-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors a nd a specific MEK-inhibitor inhibited the 2MeSADP- and AP(3)A-induced MAP k inase activation. In addition, transfection with dominant negative RhoA(ASn 19) rendered C6 cells insensitive to P2Y(AC)-receptor-mediated MAP kinase a ctivation whereas dominant negative ras was without effect. Immunoprecipita tion experiments indicated a significant increase in the phosphorylation of raf-1 after P2Y(AC)-receptor activation. We may conclude that P2Y(AC)-puri noceptor agonists activate MAP kinase through a G(i)-RhoA-PKC-raf-MEK-depen dent, but ras- and Ca2+-independent cascade.