Functional Linkage of Neutral amino acid transport system L and voltage-gated Ca^<2+> channels in central nervous systems.

在本研究中，我研究了Na^+独立的中性氨基酸运输系统L参与加巴喷丁在正常人星形胶质细胞和原代培养小鼠脑皮质神经元中电压门控制的Ca^<2+>通道阻断作用。特别是，没有关于神经元中氨基酸转运系统L的信息，这被认为是加巴喷丁的主要靶点。因此，我们首先利用小鼠脑皮质神经元原代培养物研究加巴喷丁转运到神经元中的功能特征。从15日龄胎鼠中分离脑皮质神经元。免疫细胞化学分别用抗神经元标记物map2抗体和抗神经胶质标记物gfap抗体证实神经元的纯度和神经胶质细胞的污染。神经元对[^3H]加巴喷丁的摄取主要不依赖于Na^+，通过降低细胞外pH来刺激，并被疏水中性氨基酸抑制。输运过程是饱和的，K_t为66±0.7 μM。RT - PCR结果显示，小鼠脑皮质神经元表达系统L氨基酸转运体LAT1和LAT2，以及细胞表面抗原重链4F2 (4F2hc/CD98)，这是LAT1和LAT2功能表达所必需的。加巴喷丁的结构相关化合物1-氨基-1-环己烷羧酸（ACHC）、1-氨基-1-环戊烷-羧酸（cycloleucine）和BCH对Na +非依赖性的加巴喷丁转运呈浓度依赖性，IC_<50>分别为±13 μM、185±24 μM和56±21 μM。此外，将各种中性氨基酸预加载到神经元中可显著增强Na^+非依赖性[^3H]加巴喷丁的摄取（反式刺激效应）。提示功能性氨基酸转运系统L在小鼠脑皮质神经元中有表达。

In the present research, I have investigated the involvement of Na^+-independent neutral amino acid transport system L in voltage-gated Ca^<2+> channels blocking action by gabapentin in normal human astrocytes and primary cultured mouse cerebrocortical neurons. In particular, there is no information available for amino acid transport system L in neurons that are thought to be a major target site for gabapentin. Therefore, we primarily investigated the functional characteristics of gabapentin transport into the neurons using primary cultures of mouse cerebrocortical neurons. Cerebrocortical neurons were isolated from 15-day-old-fetal ddY mice. The purity of neurons and contamination of glial cells were confirmed by immunocytochemistry using anti-MAP2 (neuronal marker) and anti-GFAP (glial marker) antibodies, respectively.The uptake of [^3H]gabapentin in neurons was predominantly Na^+-independent, was stimulated by lowering the extracellular pH, and was inhibited by hydrophobic neutral amino acids. The transport process was saturable with a K_t of 66±0.7 μM for gabapentin uptake. RT PCR revealed that mouse cerebrocortical neurons express the system L amino acid transporters, LAT1 and LAT2, and the heavy chain of the cell surface antigen 4F2 (4F2hc/CD98), which is required for the functional expression of LAT1 and LAT2. Structurally-related compounds of gabapentin, such as 1-amino-1-cyclohexanecarboxylic acid (ACHC), 1-amino-1-cyclopentane- carboxylic acid (cycloleucine), and BCH inhibited the Na^+-independent gabapentin transport in a concentration-dependent manner, and their IC_<50> values were±13, 185±24, and 56±21 μM, respectively. In addition, preloading of various neutral amino acids into neurons markedly enhanced Na^+-independent [^3H]gabapentin uptake (trans-stimulation effect).These results suggest that functional amino acid transport system L is expressed in mouse cerebrocortical neurons.