Localization and functional regulation of H^+/oligopptide transporter(s) in brain

H^/寡肽转运蛋白在大脑中的定位和功能调节

• 批准号: 13672404
• 负责人: FUJITA Takuya
• 金额: $ 2.18万
• 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: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672404/
• 关键词: Peptide transporter(s); PEPT2; Na^+; H^+ exchanger; Amino acid transporters; Gabapentin; voltage-gated Ca^<2+> channels; Glial cells; cyclic GMP; ペプチドトランスポータ; H^+exchanger; アミノ酸トランスポータ; H^+; peptide cotransporter; 小脳; PCR法; RACE法; 過剰発現系

In this research project, I investigated functional characterization of H^+/oligopeptide transport system in primary cultures of neurons and astrocytes from mouse cerebral cortex. RT-PCR revealed that the low affinity-type H^+/oligopeptide transporter PEPT1 mRNA was not found in both neurons and astrocytes, whereas the high affinity-type H^+/oligopeptide transporter PEPT2 mRNA was found in astrocytes but not neurons. In addition, expression of PEPT2 protein was confirmed by Western blotting using anti-PEPT2 antibody. Uptake of [^3H]glycylsarcosine, a model dipeptide, in mouse astrocytes was pH-dependent and saturable, and a Michaelis-Menten constant (K_t) value was 110 μM, which is comparable to those values in PEPT2-stable transfectant cells and our previous report using synaptosomal membrane prepared from rat cerebral cortex. These results, therefore, indicate that PEPT2 works **tionally in mouse astrocytes. Furthermore, I demonstrated that mouse astrocytes express Na^+/H^+ exchanger … More s (NHEs), suggesting that NHE(s) was responsible for generating inward H^+ gradient which is a driving force of PEPT1/2, in mouse astrocytes as well as renal and intestinal epithelial cells.In parallel, I investigated the functional characteristics of gabapentin transport and its interaction with voltage-gated Ca^<2+> channel (VGCC) in normal human astrocytes. The uptake of leucine (Leu) and phenylalanine (Phe) in these cells was predominantly Na^+-independent, was stimulated by lowering the extracellular pH, and was inhibited by hydrophobic neutral amino acids. These transport processes were saturable with Michaelis constants (K_t) of 51 ± 7 μM and 24 ± 9 μM for Leu and Phe uptake, respectively. GBP inhibited the Na^+-independent uptake of Leu and Phe in a concentration-dependent manner, and its K_i values were 80 μM and 93 μM for Leu and Phe uptake, respectively. These K_i values were consisted with a previous report by Su et al. and were comparable to the K_t value for GBP in rat astrocytes. GBP decreased K^+ (50 mM)-stimulated [Ca^<2+>]_i increase and cyclic GMP formation, which reflects the activation of nNOS, in a concentration-dependent manner (IC_<50>=111 ± 19 μM). BCH, a specific inhibitor of system L transporter, and Leu were found to recover the inhibitory action of GBP on K^+-stimulated [Ca^<2+>]_i in human astrocytes. However, both compounds per se were failed to decrease the K^+-stimulated [Ca^<2+>]_i. On the other hand, lysine, cationic amino acid, and MeAIB, which is a selective substrate for system A, had no effect. Less

在本研究项目中，我研究了小鼠大脑皮层神经元和星形胶质细胞原代培养中H^+/寡肽运输系统的功能特征。RT-PCR结果显示，在神经元和星形胶质细胞中均未发现低亲和型H^+/寡肽转运体PEPT1 mRNA，而在星形胶质细胞中均未发现高亲和型H^+/寡肽转运体PEPT2 mRNA。此外，使用抗PEPT2抗体进行Western blotting证实PEPT2蛋白的表达。小鼠星形胶质细胞对[^3H]glycylsarcos（一种模型二肽）的摄取是ph依赖性和可饱和的，Michaelis-Menten常数（K_t）值为110 μM，这与pept2稳定的转染细胞和我们之前报道的使用大鼠大脑皮层制备的突触体膜的值相当。因此，这些结果表明，PEPT2在小鼠星形胶质细胞中起作用。此外，我证明小鼠星形胶质细胞表达Na^+/H^+交换器…More s (NHEs)，这表明NHE(s)负责在小鼠星形胶质细胞以及肾脏和肠上皮细胞中产生向内的H^+梯度，这是PEPT1/2的驱动力。同时，我研究了加巴喷丁转运的功能特征及其与正常人星形胶质细胞电压门控Ca^<2+>通道（VGCC）的相互作用。这些细胞对亮氨酸（Leu）和苯丙氨酸（Phe）的摄取主要是Na^+无关的，通过降低细胞外pH来刺激，并被疏水中性氨基酸抑制。输运过程中，Leu和Phe摄取的Michaelis常数（K_t）分别为51±7 μM和24±9 μM。GBP对Leu和Phe的吸收呈浓度依赖性，其对Leu和Phe的K_i分别为80 μM和93 μM。这些K_i值与Su等人先前的报告一致，与大鼠星形胶质细胞GBP的K_t值相当。GBP降低了K^+ (50 mM)刺激的[Ca^<2+>]_i的增加和环状GMP的形成，反映了nNOS的激活，并呈浓度依赖性（IC_<50>=111±19 μM）。系统L转运蛋白特异性抑制剂BCH和Leu可恢复GBP对人星形胶质细胞K^+刺激的[Ca^<2+>]_i的抑制作用。然而，这两种化合物本身都不能降低K^+刺激的[Ca^<2+>]_i。另一方面，赖氨酸、阳离子氨基酸和系统a的选择性底物MeAIB没有影响。少

项目成果

Ruan Ling, et al.: "Involvement of transporter recruitment as gene expression in the substrate-induced adaptive regulation of amino acid transport system A"Biochimica Biophysica Acta. 1512. 15-21 (2001)

Ruan Ling 等人：“转运蛋白募集作为基因表达参与底物诱导的氨基酸转运系统 A 的适应性调节”Biochimica Biophysicala Acta。

Michiko Oka, et al.: "Gabapentin blocks L-type and P/Q-type Ca^<2+> channels involved in depolarization-stimulated nitric oxide synthase activity in primaryi cultures of neurons from mouse cerebral cortex"Pharmaceutical Research. 20. 897-899 (2003)

Michiko Oka等人：“加巴喷丁阻断参与小鼠大脑皮层神经元原代培养物中去极化刺激的一氧化氮合酶活性的L型和P/Q型Ca 2+ 通道”药物研究。

Satoru Takahashi, et al.: "FR167653, a p38 mitogen-activated protein kinase inhibitor, prevents Helicobacter pylori-induce d gastritis in Monglian gerbils"J. Pharmacol. Exp. Ther.. 296. 48-56 (2001)

Satoru Takahashi 等人：“FR167653，一种 p38 丝裂原激活蛋白激酶抑制剂，可预防蒙古沙鼠幽门螺杆菌诱发的胃炎”J.

Ruan Ling et al.: "Involvememnt of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A"Biochimica Biophysica Acta : Biomembranes. 1512. 15-21 (2001)

Ruan Ling 等人：“氨基酸转运系统 A 的底物诱导适应性调节中转运蛋白募集和基因表达的参与”《生物化学生物物理学学报：生物膜》。

Michiko Oka, et al.: "Gabapentin blocks L-type and P/Q type Ca^<2+> channels involved in depolarization-stimulated nitric oxide synthase activity in primary cultures of neurons from mouse cerebral cortex"Pharmaceutical Research. 20. 897-899 (2003)

Michiko Oka等人：“加巴喷丁阻断参与小鼠大脑皮层神经元原代培养物中去极化刺激的一氧化氮合酶活性的L型和P/Q型Ca 2+ 通道”药物研究。