Department of Psychiatry, Faculty of Medicine, Saitama Medical University, Moroyama-machi, Iruma-gun, Japan.
Basic Clin Pharmacol Toxicol. 2011 Sep;109(3):175-85. doi: 10.1111/j.1742-7843.2011.00705.x. Epub 2011 May 5.
Stimulation of specific guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding by l-glutamate was pharmacologically characterized in rat cerebral cortical membranes. Optimization of the experimental conditions with respect to the concentrations of GDP, MgCl(2) and NaCl in assay buffer prompted us to adopt the incubation of rat cerebral cortical membranes with 0.2 nM [(35)S]GTPγS at 30°C for 60 min. in the presence of 20 μM GDP, 5 mM MgCl(2) and 100 mM NaCl as a standard condition. Specific [(35)S]GTPγS binding was stimulated by l-glutamate in a concentration-dependent manner but not by ionotropic glutamate receptor agonists. The stimulatory responses were also elicited by many agonists for metabotropic glutamate (mGlu) receptor, with (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) being the most potent. l-glutamate-stimulated [(35)S]GTPγS binding was inhibited by several mGlu antagonists, with (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495) being the most potent. The pharmacological properties of a series of agonists and antagonists indicated the involvement of group II mGlu receptors, especially mGlu2. Supportive of this notion was the finding that l-glutamate-stimulated specific [(35)S]GTPγS binding was augmented by 2,2,2-trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl)ethanesulphonamide hydrochloride (LY487379), a reportedly selective allosteric positive modulator for mGlu2, by means of upward and leftward shift of the concentration-response curve. In addition, LY487379 per se stimulated [(35)S]GTPγS binding, though, through a mechanism different from the stimulation by l-glutamate. Pre-treatment of the membranes with N-ethylmaleimide (NEM) cancelled l-glutamate-stimulated [(35)S]GTPγS binding in a concentration- and incubation time-dependent manner. Taken altogether, l-glutamate-stimulated [(35)S]GTPγS binding serves as a useful functional assay for the activation of NEM-sensitive G(i/o) -mediated group II mGlu receptors in rat cerebral cortical membranes.
在大鼠皮质膜中，通过 l-谷氨酸刺激特定的鸟嘌呤核苷酸-5'-O-（3-[(35)S]硫代）三磷酸 ([(35)S]GTPγS) 结合，对其药理学特征进行了研究。通过优化实验条件，即在测定缓冲液中优化 GDP、MgCl2 和 NaCl 的浓度，我们建议采用以下标准条件：在 30°C 下，用 20μM GDP、5mM MgCl2 和 100mM NaCl 孵育大鼠皮质膜 60 分钟，同时使用 0.2nM [(35)S]GTPγS。l-谷氨酸以浓度依赖的方式刺激特定的 [(35)S]GTPγS 结合，但不能刺激离子型谷氨酸受体激动剂。许多代谢型谷氨酸 (mGlu) 受体的激动剂也能引起这种刺激反应，(-)-2-氧代-4-氨基双环[3.1.0]己烷-4,6-二甲酸 (LY379268) 是最有效的。l-谷氨酸刺激的 [(35)S]GTPγS 结合被几种 mGlu 拮抗剂抑制，其中 (2S)-2-氨基-2-[(1S,2S)-2-羧基环丙基]-3-(黄嘌呤-9-基)丙氨酸 (LY341495) 是最有效的。一系列激动剂和拮抗剂的药理学特性表明涉及到 II 组 mGlu 受体，特别是 mGlu2。支持这一观点的是，发现 l-谷氨酸刺激的特异性 [(35)S]GTPγS 结合通过 2,2,2-三氟-N-[4-(2-甲氧基苯氧基)苯基]-N-(3-吡啶基甲基)乙磺酰胺盐酸盐 (LY487379) 增强，LY487379 是 mGlu2 的报道性选择性变构正调节剂，通过浓度反应曲线的向上和向左移动。此外，LY487379 本身也刺激 [(35)S]GTPγS 结合，但通过不同于 l-谷氨酸刺激的机制。用 N-乙基马来酰亚胺 (NEM) 预处理膜以浓度和孵育时间依赖的方式取消 l-谷氨酸刺激的 [(35)S]GTPγS 结合。综上所述，l-谷氨酸刺激的 [(35)S]GTPγS 结合可作为一种有用的功能性测定方法，用于测定大鼠皮质膜中 NEM 敏感的 G(i/o) 介导的 II 组 mGlu 受体的激活。
