P2YAC−-receptor agonists enhance the proliferation of rat C6 glioma cells through activation of the p42/44 mitogen-activated protein kinase

摘要: Extracellularly added P1,P3-di(adenosine-5′) triphosphate (Ap3A), P1,P4-di(adenosine-5′) tetraphosphate (Ap4A), ATP, ADP, AMP and adenosine are growth inhibitory for rat C6 glioma cells. Analysis of nucleotide hydrolysis the use nucleotidase inhibitors demonstrated that latter inhibition is due to nucleotides adenosine. Agonists P2YAC−-receptor enhance cells if their inhibited by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). In these conditions, potency stimulate cell parallels ranking receptor agonists, i.e. 2-methylthioadenosine-5′-diphosphate (2MeSADP)>Ap3A>Ap4A. ATP ADP still hydrolysed in presence PPADS have no proliferative effect on cells. The enhanced a P2YAC−-receptor-mediated activation p42/44 mitogen-activated protein kinase (MAPK) as shown immunoblotting assays active MAPK MAPK/extracellular signal-regulated (MEK) inhibitor PD98059. The UTP-induced enhancement sensitive receptor. In conclusion, determined ecto-nucleotidases receptors. Hydrolysis induces while agonists enhances MAPK. Keywords: Ap3A, Ap4A, cells, inhibition, MAPK, NPPase, receptor, proliferation, purinoceptor, P2YAC−-receptor Introduction Nucleotides, particular adenosine, been reported affect proliferation different types. The effects not always unequivocal, e.g. stimulates primary cultures astrocytes aortic smooth muscle (Abbracchio et al., 1994; Harper 1998), ineffective transformed mouse fibroblasts (Weisman 1988) has an several human tumour lines (Seetulsingh-Goorah & Stewart, 1998; Rapaport 1983). Adenosine most examined types Fishman although stimulation also (Lelievre 1998). This apparent discrepancy may be explained existence mechanisms take place at surface: firstly, extracellular degraded cascade surface-bound enzymes, ecto-ATPase, ecto-apyrase, ecto-nucleotide pyrophosphatase/phosphodiesterase I (NPPase) ecto-5′-nucleotidase, hydrolysing into (Zimmermann, 1996). Cellular uptake transporters can induce adenosine-dependent pyrimidine starvation resulting (Lasso de la Vega 1994). Secondly, breakdown products receptors, coupled phospolipase C (PLC) or adenylate cyclase (AC). addition, activate ecto-protein kinases modulate activity autocrine factors (Vilgrain Baird, 1991; Friedberg 1995). other phosphates one combination mechanisms. Two main classes P1 P2, described (Burnstock, 1978). P1-receptors mainly responsive adenosine. P1-receptor subtypes A1 A3 negatively, A2 positively AC. P2-receptors, activated ATP/ADP UTP/UDP, subdivided metabotropic P2Y- ionotropic P2X- P2Y-receptors PLC-dependent Ca2+ mobilization (PKC) AC (P2Y11, P2Y12), P2X-receptors, ligand gated ion-channels, generate Ca2+-influx (Fredholm Communi 1997; Boeynaems 2000; Hollopeter 2001). We investigated its mono- dinucleotides bipotential line often used model system astrocytes. Several purinergic receptors present cells: A2b-receptor, ‘nucleotide receptor' PLC all nucleoside triphosphates, PLC-coupled P2Y6-receptor UDP, P2Y-receptor negatively presumed P2Y12-receptor (Ohkubo 2001; Lin Chuang, Nicholas 1996; Boyer 1993; 2001). agonist AC-coupled more less same P2Y1-receptor hitherto called P2Y1-like (Albert Grobben 2000). However, contrast P2Y1-receptor, this it antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) 5′-phosphoadenosine 3′-phosphate (Boyer Although Webb al. (1996) concluded P2Y1-like- identical but second messenger system, Schachter (1997) disproved finding. To describe accurately pharmacological properties we propose name P2YAC− future reference receptor. recently cloned proposed (Hollopeter antagonist P2Y12-receptor, (Grobben 2000), suggesting P2Y12 When appears unique P2Yn nomenclature used. In study Both were observed, depending whether not. proved essential inhibition. We previously studied nucleotides, identified NPPase ecto-enzyme 1999). was PPADS, observed potent P2YAC−-receptor, except lesser extent which even PPADS. MAPK.