Department of Physiology, University of Lausanne, Switzerland.
Brain Res. 2010 Feb 26;1316:27-34. doi: 10.1016/j.brainres.2009.12.028. Epub 2009 Dec 22.
Astrocytes are responsible for the majority of the clearance of extracellular glutamate released during neuronal activity. dl-threo-beta-benzyloxyaspartate (TBOA) is extensively used as inhibitor of glutamate transport activity, but suffers from relatively low affinity for the transporter. Here, we characterized the effects of (2S, 3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (TFB-TBOA), a recently developed inhibitor of the glutamate transporter on mouse cortical astrocytes in primary culture. The glial Na(+)-glutamate transport system is very efficient and its activation by glutamate causes rapid intracellular Na(+) concentration (Na(+)(i)) changes that enable real time monitoring of transporter activity. Na(+)(i) was monitored by fluorescence microscopy in single astrocytes using the fluorescent Na(+)-sensitive probe sodium-binding benzofuran isophtalate. When applied alone, TFB-TBOA, at a concentration of 1 microM, caused small alterations of Na(+)(i). TFB-TBOA inhibited the Na(+)(i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) value of 43+/-9 nM, as measured on the amplitude of the Na(+)(i) response. The maximum inhibition of glutamate-evoked Na(+)(i) increase by TFB-TBOA was >80%, but was only partly reversible. The residual response persisted in the presence of the AMPA/kainate receptor antagonist CNQX. TFB-TBOA also efficiently inhibited Na(+)(i) elevations caused by the application of d-aspartate, a transporter substrate that does not activate non-NMDA ionotropic receptors. TFB-TBOA was found not to influence the membrane properties of cultured cortical neurons recorded in whole-cell patch clamp. Thus, TFB-TBOA, with its high potency and its apparent lack of neuronal effects, appears to be one of the most useful pharmacological tools available so far for studying glial glutamate transporters.
星形细胞负责清除神经元活动期间释放的细胞外谷氨酸。dl-threo-β-苯甲氧基天冬氨酸（TBOA）被广泛用作谷氨酸转运体活性的抑制剂，但对转运体的亲和力相对较低。在这里，我们描述了最近开发的谷氨酸转运体抑制剂（2S，3S）-3-[3-[4-（三氟甲基）苯甲酰胺基]苄氧基]天冬氨酸（TFB-TBOA）对原代培养的小鼠皮质星形细胞的影响。胶质细胞的 Na(+)-谷氨酸转运系统非常有效，其被谷氨酸激活会导致细胞内 Na(+)浓度（Na(+)(i)）迅速变化，从而能够实时监测转运体的活性。Na(+)(i)通过荧光显微镜在单个星形细胞中使用荧光 Na(+)敏感探针苯甲酰基荧光素-异羟肟酸进行监测。当单独使用时，TFB-TBOA 在 1 μM 的浓度下引起 Na(+)(i)的微小变化。TFB-TBOA 以浓度依赖的方式抑制由 200 μM 谷氨酸引起的 Na(+)(i)反应，IC(50)值为 43+/-9 nM，如 Na(+)(i)反应的幅度所示。TFB-TBOA 对谷氨酸诱导的 Na(+)(i)增加的最大抑制作用超过 80%，但仅部分可逆。在 AMPA/海人藻酸受体拮抗剂 CNQX 的存在下，残留的反应仍然存在。TFB-TBOA 还能有效抑制 d-天冬氨酸引起的 Na(+)(i)升高，d-天冬氨酸是一种转运体底物，不会激活非 NMDA 离子型受体。发现 TFB-TBOA 不影响全细胞膜片钳记录的培养皮质神经元的膜特性。因此，TFB-TBOA 具有高效力和明显缺乏神经元作用，似乎是迄今为止研究胶质细胞谷氨酸转运体最有用的药理学工具之一。
