Endogenous Expression of Adenosine A(1), A (2 ) and A (3) Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [3H]DPCPX, selectiveA1Rantagonist, revealed a single binding site with a KD = 9.4 ± 1.4 nM and Bmax = 62.7 ± 8.6 fmol/mg protein. Binding of [3H]DPCPX in intact cell revealed a KD = 17.7 ± 1.3 nM and Bmax = 567.1 ± 26.5 fmol/mg protein. On the other hand, [3H]ZM241385 binding experiments revealed a single binding site population of receptors with KD = 16.5 ± 1.3 nM and Bmax = 358.9 ± 52.4 fmol/mg protein in intact cells, and KD = 4.7 ± 0.6 nM and Bmax = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A2A receptor in C6 cells. A1, A2A, A2B and A3 adenosine receptors were detected by Westernblotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Gia and Gsa proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A1R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A1 and A2 receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptorsin tumoral cells.