Lieb I, Chebib M, Cooper B, Dias L S, Balcar V J
Department of Anatomy and Histology, The University of Sydney, NSW, Australia.
Neurochem Int. 2000 Apr;36(4-5):319-27. doi: 10.1016/s0197-0186(99)00140-0.
Sodium-dependent binding of [3H]L-aspartate was studied in thaw-mounted horizontal sections of fresh-frozen (i.e. not fixed) rat brain. After the incubation with [3H]L-aspartate, the sections were exposed against a 3H-sensitive film and the resulting autoradiograms were evaluated by quantitative densitometry. Effects of several inhibitors were examined and their potency expressed as IC50 and nH. Together with previously published data, the present study supports the view that [3H]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the substrate-binding sites on glutamate transporters. The most potent inhibitors were (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4-methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxylate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle regional variations in the characteristics of inhibitors of [3H]L-aspartate binding were detected. It is not certain whether these differences reflect regional variations in the distribution of individual glutamate transporters or regional peculiarities in their pharmacological characteristics. In particular, (2S,4R)-4-methylglutamate, shown previously to differentiate between GLT-1 (principal glutamate transporter in the forebrain) and GLAST (expressed mainly in the cerebellum), did not strongly differentiate between the binding of [3H]L-aspartate in forebrain and cerebellum. Computer-assisted molecular modelling using selected glutamate analogues with restricted conformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(carboxycyclopropyl)-glycine: L- and D-CCG I, L-CCG III and L-CCG IV) identified at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [3H]L-aspartate or other transporter-specific ligands, will be a useful tool to study the pharmacology of substrate binding sites on glutamate transporters in the mammalian brain in situ.
在新鲜冷冻（即未固定）大鼠脑的解冻安装水平切片中研究了[³H]L-天冬氨酸的钠依赖性结合。用[³H]L-天冬氨酸孵育后，将切片暴露于³H敏感胶片上，并通过定量光密度测定法评估所得的放射自显影片。检查了几种抑制剂的作用，并将其效力表示为IC50和nH。与先前发表的数据一起，本研究支持以下观点：[³H]L-天冬氨酸与大鼠脑新鲜冷冻切片的结合代表放射性配体与谷氨酸转运体上底物结合位点的相互作用。最有效的抑制剂是（2S，3S，4R）-2-（羧基环丙基）-甘氨酸（L-CCG III）和（2S，4R）-4-甲基谷氨酸。相比之下，L-反式，内型-3,4-甲烷吡咯烷二羧酸酯（L-a，e-MPDC）的效力约低一个数量级。仅检测到[³H]L-天冬氨酸结合抑制剂特征的细微区域差异。尚不确定这些差异是反映单个谷氨酸转运体分布的区域差异还是其药理学特征的区域特殊性。特别是，先前已显示能区分GLT-1（前脑主要的谷氨酸转运体）和GLAST（主要在小脑中表达）的（2S，4R）-4-甲基谷氨酸，在前脑和小脑中[³H]L-天冬氨酸的结合之间并未表现出强烈的区分。使用选定的具有受限构象的谷氨酸类似物（L-反式-吡咯烷-2,4-二羧酸酯和2-（羧基环丙基）-甘氨酸的四种异构体：L-和D-CCG I、L-CCG III和L-CCG IV）进行的计算机辅助分子建模确定了至少一个不利的空间相互作用区域。我们得出结论，使用[³H]L-天冬氨酸或其他转运体特异性配体进行的定量放射自显影研究，将是研究哺乳动物脑原位谷氨酸转运体上底物结合位点药理学的有用工具。
