Albasanz J L, Ros M, Martín M
Area de Bioquímica, Facultad Ciencias Químicas, Universidad de Castilla-La Mancha, Ciudad Real, Spain.
Eur J Pharmacol. 1997 May 12;326(1):85-91. doi: 10.1016/s0014-2999(97)00154-4.
Metabotropic glutamate receptors in rat C6 glioma cells have been characterized by pharmacological and kinetic binding experiments, using both L-[3H]glutamate and [3H(+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (3H-trans-ACPD) radioligands. Saturation experiments revealed a single binding site with a Kd = 1250 +/- 101 nM and Bmax = 12.1 +/- 1.8 pmol/mg protein when the assays were performed with L-[3H]glutamate as radioligand in the presence of AMPA, kainate, NMDA and DL-threo-beta-hydroxyaspartic acid. When 3H-trans-ACPD was used as radioligand, the kinetic parameters obtained were Kd = 2605 +/- 1042 nM and Bmax = 13.66 +/- 5.01 pmol/mg protein. Pharmacological characterization indicated that specific binding of L-[3H]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 >>> (2S, 'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Specific binding of L-[3H]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.
通过药理学和动力学结合实验，利用L-[3H]谷氨酸和[3H(+/-)-1-氨基环戊烷-反式-1,3-二羧酸（3H-反式-ACPD）放射性配体，对大鼠C6胶质瘤细胞中的代谢型谷氨酸受体进行了表征。饱和实验显示，当以L-[3H]谷氨酸为放射性配体，在AMPA、海人酸、NMDA和DL-苏式-β-羟基天冬氨酸存在的情况下进行测定时，存在一个单一结合位点，其Kd = 1250 +/- 101 nM，Bmax = 12.1 +/- 1.8 pmol/mg蛋白质。当使用3H-反式-ACPD作为放射性配体时，获得的动力学参数为Kd = 2605 +/- 1042 nM，Bmax = 13.66 +/- 5.01 pmol/mg蛋白质。药理学表征表明，L-[3H]谷氨酸的特异性结合对代谢型谷氨酸受体的不同激动剂敏感，显示出亲和力顺序为L-谷氨酸>L-喹啉酸>(+/-)-1-氨基环戊烷-反式-1,3-二羧酸（反式-ACPD）>鹅膏蕈氨酸>>> (2S,'S,2'S)-2-(羧基环丙基)甘氨酸（L-CCG-I）。L-[3H]谷氨酸与代谢型谷氨酸受体的特异性结合受鸟嘌呤核苷酸调节。鸟苷酰亚胺二磷酸（Gpp(NH)p）导致L-谷氨酸剂量反应曲线上的亲和力发生变化，增加IC50值。这些结果支持了大鼠C6胶质瘤细胞中存在代谢型谷氨酸受体且它们与G蛋白偶联的证据。
