Endogenous Expression of Adenosine A1, A2 and A3 Receptors in Rat C6 Glioma Cells

Abstract

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 [(3)H]DPCPX, selective A(1)R antagonist, revealed a single binding site with a K (D) = 9.4 +/- 1.4 nM and B (max) = 62.7 +/- 8.6 fmol/mg protein. Binding of [(3)H]DPCPX in intact cell revealed a K (D) = 17.7 +/- 1.3 nM and B (max )= 567.1 +/- 26.5 fmol/mg protein. On the other hand, [(3)H]ZM241385 binding experiments revealed a single binding site population of receptors with K (D) = 16.5 +/- 1.3 nM and B (max) = 358.9 +/- 52.4 fmol/mg protein in intact cells, and K (D) = 4.7 +/- 0.6 nM and B (max) = 74.3 +/- 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A(2A) receptor in C6 cells. A(1), A(2A), A(2B) and A(3 )adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Gialpha and Gsalpha proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A(1)R 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 A(1) and A(2) receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.