Characterization of metabotropic glutamate receptors in rat C6 glioma cells

Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both L-[H-3]glutamate and [H-3](+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid ([H-3](+/-)-trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a K-d=1250+/-101 nM and B-max=12.1+/-1.8 pmol/mg protein when the assays were performed with L-[H-3]glutamate as radioligand in the presence of AMPA, kainate, NMDA and DL-threo-beta-hydroxyaspartic acid. When [H-3](+/-)-trans-ACPD was used as radioligand, the kinetic parameters obtained were K-d=2605+/-1042 nM and B-max=13.66+/-5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of L-[H-3]glutamate was sensitive to different agonists of mGlu receptors, showing a rank order of affinity L-glutamate >L-quisqualic acid >(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) >ibotenic acid much greater than(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Specific binding of L-[H-3]glutamate to mGlu receptors is regulated by guanine nucleotides. Guanylyl imidodiphosphate (Gpp(NH)p) causes an affinity shift on the L-glutamate dose-response curve, increasing the IC50 value. These results support the evidence that metabotropic glutamate receptors are present in rat C6 glioma cells and they are coupled to a G-protein.