q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 1325±1338 1325 Journal of Neurochemistry, 2001, 78, 1325±1338 Agonists of the P2Y AC -receptor activate MAP kinase by a ras-independent pathway in rat C6 glioma Bert Grobben,* ,1 Patrik Claes,* ,1 Kristof Van Kolen,* Dirk Roymans,* Paul Fransen,² Stanislas U. Sys² and Herman Slegers* *Department of Biochemistry, Cellular Biochemistry, Universiteit Antwerpen, Universitaire Instelling Antwerpen, Wilrijk, Belgium ²Department of Medicine, Human Physiology and Pathophysiology, Universiteit Antwerpen, RUCA, Antwerpen, Belgium Abstract 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 identi®ed as speci®c agonists of the P2Y AC -receptor with EC 50 values of 250 ^ 37 pM and 1 ^ 0.5 mM, 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 IP 3 -independent Ca 21 - in¯ux was observed after P2Y AC -receptor activation. Inhibition of this in¯ux by Ca 21 -chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a speci®c MEK-inhibitor inhibited the 2MeSADP- and Ap 3 A-induced MAP kinase activation. In addition, transfection with dominant negative RhoA Asn19 rendered C6 cells insensi- tive to P2Y AC -receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immuno- precipitation experiments indicated a signi®cant 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 Ca 21 -independent cascade. Keywords: adrenergic receptor signalling, MAP kinase, P2Y AC -purinoceptor, purinoceptor agonists, rat C6 glioma cells, RhoA. J. Neurochem. (2001) 78, 1325±1338. The original proposition for the existence of P2-purinocep- tors was made on the basis of smooth muscle contractile responses to adenine nucleotides (Burnstock 1978). Subse- quent pharmacological research, predominantly based on the rank order of agonist potency of ATP analogues, subdivided these receptors into ionotrophic P2X- and metabotrophic P2Y-purinoceptors. To date, seven P2X-receptors (P2X 127 ) and eight P2Y-receptors (P2Y 124, 6, 8, 11212 ) have been cloned and characterized (Webb et al. 1996b; Communi et al. 1997; Boarder and Hourani 1998; Ralevic and Burnstock 1998; Hollopeter et al. 2001). Inositol lipid hydrolysis and intracellular Ca 21 mobilization were initially proposed to be the predominant responses to P2Y-receptor activation (Irving and Exton 1987; Nicholas et al. 1996). However, some purinoceptor subtypes are also linked to phospholipase A 2 (Gupta et al. 1990; Bruner and Murphy 1993) and phospholipase D (Martin and Michaelis 1989). Whereas the P2Y 11 -receptor is positively linked to adenylate cyclase and phospholipase C (PLC) (Communi et al. 1997), the blood platelet P2Y 12 -receptor (P2T AC -), the rat brain capillary endothelial B10 cell P2Y T -receptor and P2Y 1 -like- receptor inhibit adenylate cyclase (Le Âon et al. 1999; Simon et al. 2001). A P2Y-receptor, negatively linked to adenylate cyclase, was also identi®ed on rat C6 glioma cells (Pianet et al. 1989; Received March 2, 2001; revised manuscript received June 22, 2001; accepted June 28, 2001. Address correspondence and reprint requests to H. Slegers, Department of Biochemistry, Cellular Biochemistry, Universiteit Antwerpen, Universitaire Instelling Antwerpen, Universiteitplein 1, B-2610 Antwerpen-Wilrijk, Belgium. E-mail: slegers@uia.ua.ac.be 1 B. Grobben and P. Claes contributed equally to the work. Abbreviations used: DIDS, 4,4 0 -diisothiocyanatostilbene-2,2 0 -disul- phonic acid; ecto-ATPDase, ecto-ATP diphosphohydrolase; ecto- NPPase, ecto-nucleotide pyrophosphatase; MBP, myelin-basic protein; MEM, minimal essential medium; PA, phosphatidic acid; PAP, adenosine-3 0 ,5 0 -biphosphate; PPADS, pyridoxalphosphate-6-azophe- nyl-2 0 ,4 0 -disulphonic acid; PSS, physiological salt solution; PTX, pertussis toxin; RB2, reactive blue 2; MAP kinase, mitogen-activated protein kinase; PI 3-K, phosphatidylinositol-3-phosphate kinase; SDS± PAGE, sodium dodecyl sulphate±polyacrylamide gel electrophoresis.