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

在该研究项目中，我研究了小鼠大脑皮层的神经元和星形胶质细胞原发性培养物中H^+/寡肽转运系统的功能表征。 RT-PCR表明，在神经元和星形胶质细胞中未发现低亲和力H^+/寡肽转运蛋白转运蛋白pept1 mRNA，而高亲和型H^+/寡肽转运蛋白转运蛋白转运蛋白pept2 mRNA在星形胶质细胞中被发现，但没有神经元。另外，使用抗肽2抗体的蛋白质印迹证实了pept2蛋白的表达。 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 transparent cells and our previous report using synaptosomal membrane prepared from rat cerebral cortex.因此，这些结果表明PEPT2在小鼠星形胶质细胞中起作用。此外，我证明了小鼠星形胶质细胞表达Na^+/h^+交换器…更多的S（nhes），这表明NHE（S）负责产生内向H^+梯度，这是PEPT1/2的驱动力，在鼠标星形胶质细胞以及肾脏和深刻的上皮细胞中，并与IT型相互作用，我调查了Gabapeltions。正常人星形胶质细胞中的Ca^<2+>通道（VGCC）。这些细胞中亮氨酸（LEU）和苯丙氨酸（PHE）的摄取主要是Na^+ - 独立的，通过降低细胞外pH值刺激，并被疏水性中性氨基酸抑制。这些运输过程分别具有51±7μm的Michaelis常数（K_T）和Leu和Phe摄取的24±9μm。 GBP以浓度依赖性方式抑制了Leu和Phe的Na^+ - 独立吸收，其K_I值分别为Leu和Phe摄取的80μm和93μm。这些K_I值与Su等人的先前报告一致。并且与大鼠星形胶质细胞中GBP的K_T值相当。 GBP降低了K^+（50 mm）刺激的[Ca^<2+>] _ I增加，并以浓度依赖性方式（IC_ <50> = 111±19μm）反映了NNOS激活的环状GMP形成。 BCH是系统L转运蛋白的特定抑制剂和LEU，可以恢复人类星形胶质细胞中GBP对K^+刺激的[Ca^<2+>] _ I的抑制作用。但是，两种化合物本身都无法减少k^+ - 刺激的[Ca^<2+>] _ I。另一方面，赖氨酸，阳离子氨基酸和MEAIB是系统A的选择性底物，没有作用。较少的

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。