Bruhn T, Christensen T, Diemer N H
Cerebral Ischemia Research Group, University of Copenhagen, Denmark.
J Neurosci Methods. 1995 Jul;59(2):169-74. doi: 10.1016/0165-0270(94)00179-k.
The role of glutamate as a possible mediator of neurodegeneration is well described, and the homeostasis of extracellular glutamate is considered of major importance when addressing the pathogenesis of excitatory neurodegeneration. Applying the 'indicator diffusion' method to the microdialysis technique, we present a method that is suitable for the in vivo investigation of the capacity of cellular uptake of glutamate. Using 14C-mannitol as reference, we measured the cellular extraction and the cell membrane permeability of the test substance 3H-D-aspartate in the corpus striatum of the rat brain. The cellular extraction fraction of 3H-D-aspartate was 0.29, and the cell membrane permeability 2.24 x 10(-4) cm/s. In the presence of the glutamate-uptake blocker DL-threo-beta-hydroxyaspartate (THA) the extraction of 3H-D-aspartate was completely abolished, indicating that extraction of 3H-D-aspartate was due to cellular uptake by glutamate transporters. The cell membrane permeability towards 3H-D-aspartate was reduced by approximately 98% due to THA, indicating that the cell membranes per se are highly resistant to diffusion of 3H-D-aspartate. It is concluded that the present method can be used in studying the capacity of the glutamate transporters in vivo.
谷氨酸作为神经退行性变的一种可能介质的作用已得到充分描述，在探讨兴奋性神经退行性变的发病机制时，细胞外谷氨酸的稳态被认为至关重要。将“指示剂扩散”方法应用于微透析技术，我们提出了一种适用于体内研究谷氨酸细胞摄取能力的方法。以14C-甘露醇作为参照，我们测定了大鼠脑纹状体中受试物质3H-D-天冬氨酸的细胞摄取率和细胞膜通透性。3H-D-天冬氨酸的细胞摄取率为0.29，细胞膜通透性为2.24×10(-4) cm/s。在谷氨酸摄取阻断剂DL-苏式-β-羟基天冬氨酸（THA）存在的情况下，3H-D-天冬氨酸的摄取完全被消除，这表明3H-D-天冬氨酸的摄取是由于谷氨酸转运体的细胞摄取所致。由于THA的存在，细胞膜对3H-D-天冬氨酸的通透性降低了约98%，这表明细胞膜本身对3H-D-天冬氨酸的扩散具有高度抗性。得出的结论是，本方法可用于体内研究谷氨酸转运体的能力。
