Endogenous expression of adenosine A1, A2 and A3 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 [(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.
Castillo
Carlos Alberto
CA
Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Químicas, Centro Regional de Investigaciones Biomédicas, Universidad de Castilla-La Mancha, Ciudad Real, Spain.
Albasanz
José Luís
JL
Fernández
Mercedes
M
Martín
Mairena
M
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D005910:Glioma / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D043682:Receptor, Adenosine A1 / Q000096:biosynthesis*; D043684:Receptor, Adenosine A3 / Q000096:biosynthesis*; D043683:Receptors, Adenosine A2 / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014227:Triazines / Q000378:metabolism; D014230:Triazoles / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D009419:Nerve Tissue Proteins; D043682:Receptor, Adenosine A1; D043684:Receptor, Adenosine A3; D043683:Receptors, Adenosine A2; D014227:Triazines; D014230:Triazoles; D014970:Xanthines; C097270:ZM 241385; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs; D000262:Adenylyl Cyclases