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

中性氨基酸转运系统L和中枢神经系统电压门控Ca^2通道的功能联系。

基本信息

批准号：
15590143
负责人：
FUJITA Takuya
金额：
$ 1.98万
依托单位：
Kyoto Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

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.

在本研究中，我研究了Na^+非依赖性中性氨基酸转运系统L在加巴喷丁阻断正常人星形胶质细胞和原代培养的小鼠皮层神经元电压门控性Ca^<2+>通道中的作用。特别是，没有关于神经元中氨基酸转运系统L的信息，该系统被认为是加巴喷丁的主要靶位点。因此，我们主要研究了加巴喷丁转运到神经元的功能特性，使用原代培养的小鼠大脑皮层神经元。从15日龄胎儿ddY小鼠分离大脑皮质神经元。分别使用抗MAP 2（神经元标记物）和抗GFAP（胶质细胞标记物）抗体通过免疫细胞化学证实神经元的纯度和胶质细胞的污染，神经元对[^3H]加巴喷丁的摄取主要是Na^+非依赖性的，通过降低细胞外pH值来刺激，并被疏水性中性氨基酸抑制。加巴喷丁的转运过程是饱和的，其K_t为66±0.7 μM。RT PCR显示，小鼠大脑皮层神经元表达系统L氨基酸转运蛋白，LAT 1和LAT 2，以及细胞表面抗原4F 2的重链（4F 2 hc/CD 98），这是LAT 1和LAT 2功能表达所必需的。加巴喷丁的结构相关化合物如1-氨基-1-环己烷羧酸（ACHC）、1-氨基-1-环戊烷羧酸（cycloleucine）和BCH以浓度依赖性方式抑制Na^+非依赖性加巴喷丁转运，其IC<50>值分别为±13、185±24和56±21 μM。此外，神经元预加载各种中性氨基酸可显著增强Na^+非依赖性[^3H]加巴喷丁摄取（反式刺激效应），这些结果表明小鼠大脑皮层神经元表达功能性氨基酸转运系统L。

项目成果

期刊论文数量（47）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Functional expression of constitutive nitric oxide synthase regulated by voltage gated Na^+- and Ca^<2+> channels in cultured human astrocytes.

在培养的人星形胶质细胞中，由电压门控Na ^ - 和Ca ^ 2 通道调节的组成型一氧化氮合酶的功能表达。

DOI：
发表时间：
2004
期刊：
Glia 46
影响因子：
0
作者：
Kazunari Tanaka et al.;Tomomi Uchiyama et al.;Takuya Fujita et al.;Miyuki Wada et al.;Kazunari Tanaka et al.;Uchiyama Tomomi et al.;Takuya Fujita et al.;Miyuki Wada et al.;Kazunari Tanaka et al.;Miyuki Wada et al.;Takuya Fujita et al.;Tomomi Uchiyama et al.;Takuya Fujita et al.;Michiko Oka et al.
通讯作者：
Michiko Oka et al.

Functional characterization of brain peptide transporter in rat cerebral cortex:: identification of the high-affinity type H+/peptide transporter PEPT2

DOI：
10.1016/j.brainres.2003.10.049
发表时间：
2004-01-30
期刊：
BRAIN RESEARCH
影响因子：
2.9
作者：
Fujita, T;Kishida, T;Ganapathy, V
通讯作者：
Ganapathy, V

Takuya Fujita et al.: "Functional characterization of brain peptide transporter in rat cerebral cortex : identification of the high affinity type H^+/peptide transporter PEPT2."Brain Research. 997. 52-61 (2004)

Takuya Fujita 等人：“大鼠大脑皮层中脑肽转运蛋白的功能特征：高亲和力型 H^/肽转运蛋白 PEPT2 的鉴定。”大脑研究。

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

Functional expression and adaptive regulation of Na^+-dependent neutral amino acid transporter SNAT1/ATA in normal human astrocytes under amino acid starved condition.

氨基酸饥饿条件下正常人星形胶质细胞中Na + 依赖性中性氨基酸转运蛋白SNAT1/ATA的功能表达和适应性调节。

DOI：
发表时间：
2005
期刊：
Neuroscience Letters 378(2)
影响因子：
0
作者：
Kazunari Tanaka et al.;Tomomi Uchiyama et al.;Takuya Fujita et al.;Miyuki Wada et al.;Kazunari Tanaka et al.;Uchiyama Tomomi et al.;Takuya Fujita et al.;Miyuki Wada et al.;Kazunari Tanaka et al.
通讯作者：
Kazunari Tanaka et al.

Transport characteristics of N-acetyl-L-aspartate in rat astrocytes:: involvement of sodium-coupled high-affinity carboxylate transporter NaC3/NaDC3-mediated transport system