Ueda Yuto, Doi Taku, Nagatomo Keiko, Tokumaru Jun, Takaki Mayuko, Willmore L James
Section of Psychiatry, Department of Clinical Neuroscience, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Brain Res. 2007 Jun 2;1151:55-61. doi: 10.1016/j.brainres.2007.03.021. Epub 2007 Mar 13.
Enhancement of the glutamatergic excitatory synaptic transmission efficacy in the FeCl3 induced epilepsy model is associated with changes in the levels of glutamate and GABA transporter proteins. This study examined the effect of levetiracetam (LEV) on glutamate overflow and glutamate/GABA transporters expression in rats with epileptogenesis induced by the amygdalar injection of 1.0 microl of 100 mM FeCl3 (epileptic rat) and in control rats receiving amygdalar acidic saline injection (non-epileptic rat). In amygdalar acidic saline injected rats, 40 mM KCl-evoked glutamate overflow was significantly suppressed by both 32 and 100 microM LEV co-perfusion. In unilateral amygdalar FeCl3 injected rats, 32 microM LEV was ineffective, but the 100 microM LEV statistically suppressed glutamate overflow. Western blotting was employed to determine the hippocampal expression of glutamate/GABA transporters in epileptic or non-epileptic rats. The rats were treated for 14 days with 54 mg/kg LEV or vehicle intraperitoneally injection. Following 14 days of treatment, the ipsilateral hippocampus was removed for a Western blot analysis. In non-epileptic rats, the expression increased for all of the glutamate and GABA transporters (GLAST, GLT-1, EAAC-1, GAT-1 and GAT-3) while the glutamate transporter regulating protein (GTRAP3-18) decreased in comparison to those of normal rats that were treated with the vehicle. In epileptic rats receiving LEV, the EAAC-1 and GAT-3 levels increased while GTRAP3-18 (89%) decreased in comparison to those of the epileptic rats treated with the vehicle. GTRAP3-18 inhibitor regulates glutamate-binding affinity to EAAC-1. The anti-epileptic action of LEV may be partially due to a reduction of glutamate-induced excitotoxicity and an enhancement of the GABAergic inhibition as observed with the inhibitory effect on the 40 mM KCl-evoked glutamate overflow. These conclusions are supported by the increase in the expression of glial glutamate transporters (GLAST and GLT-1), and the increase in the expression of EAAC-1 and GAT-3 associated with a decrease in GTRAP3-18. The increased expression of EAAC-1 and the decreased expression of GTRAP3-18 in association with the up-regulation of GAT-3 due to such continual LEV administration was thus found to enhance GABA synthesis and reverse the transport of GABA both in non-epileptic and epileptic rats. The suppression of glutamate excitation and the enhancement of GABA inhibition in the rats with continual LEV administration is a result of the up-regulation of glutamate and GABA transporters with the down-regulation of GTRAP3-18. These observations together demonstrated the critical molecular mechanism of the anti-epileptic activity of LEV.
在氯化铁诱导的癫痫模型中，谷氨酸能兴奋性突触传递效能的增强与谷氨酸和GABA转运蛋白水平的变化有关。本研究检测了左乙拉西坦（LEV）对经杏仁核注射1.0微升100 mM氯化铁诱导癫痫发作的大鼠（癫痫大鼠）和接受杏仁核酸性生理盐水注射的对照大鼠（非癫痫大鼠）中谷氨酸溢出及谷氨酸/GABA转运蛋白表达的影响。在注射杏仁核酸性生理盐水的大鼠中，32和100 microM的LEV共灌注均显著抑制了40 mM氯化钾诱发的谷氨酸溢出。在单侧杏仁核注射氯化铁的大鼠中，32 microM的LEV无效，但100 microM的LEV在统计学上抑制了谷氨酸溢出。采用蛋白质免疫印迹法测定癫痫或非癫痫大鼠海马中谷氨酸/GABA转运蛋白的表达。大鼠腹腔注射54 mg/kg的LEV或赋形剂，连续治疗14天。治疗14天后，取出同侧海马进行蛋白质免疫印迹分析。在非癫痫大鼠中，与接受赋形剂治疗的正常大鼠相比，所有谷氨酸和GABA转运蛋白（GLAST、GLT-1、EAAC-1、GAT-1和GAT-3）的表达均增加，而谷氨酸转运调节蛋白（GTRAP3-18）减少。在接受LEV治疗的癫痫大鼠中，与接受赋形剂治疗的癫痫大鼠相比，EAAC-1和GAT-3水平升高，而GTRAP3-18（89%）降低。GTRAP3-18抑制剂调节谷氨酸与EAAC-1的结合亲和力。LEV抗癫痫作用可能部分归因于谷氨酸诱导的兴奋性毒性的降低以及GABA能抑制作用的增强，如对40 mM氯化钾诱发的谷氨酸溢出的抑制作用所示。这些结论得到了胶质细胞谷氨酸转运蛋白（GLAST和GLT-1）表达增加以及EAAC-1和GAT-3表达增加且GTRAP3-18减少的支持。因此，发现由于持续给予LEV，EAAC-1表达增加、GTRAP3-18表达减少以及GAT-3上调，从而增强了非癫痫和癫痫大鼠中GABA合成并逆转了GABA转运。持续给予LEV的大鼠中谷氨酸兴奋的抑制和GABA抑制的增强是谷氨酸和GABA转运蛋白上调以及GTRAP3-18下调的结果。这些观察结果共同证明了LEV抗癫痫活性的关键分子机制。
