Functional characterization of brain peptide transporter in rat cerebral cortex:: identification of the high-affinity type H+/peptide transporter PEPT2
Functional characterization of brain peptide transporter in rat cerebral cortex:: identification of the high-affinity type H+/peptide transporter PEPT2
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DOI：
10.1016/j.brainres.2003.10.049
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发表时间：
2004-01-30
期刊：
影响因子：
2.9
通讯作者：
Ganapathy, V
中科院分区：
文献类型：
--
作者：
Fujita, T;Kishida, T;Ganapathy, V
在这份报告中，我们研究了脑肽转运蛋白的功能特性，使用突触体从大鼠大脑皮层制备。从雄性Wistar大鼠的大脑皮质制备粗突触体（P-2级分）。在37 ℃下，通过快速过滤技术测量[C-14]甘氨酰肌氨酸（Gly-Sar）（H +/寡肽转运蛋白PEPT 1和PEPT 2的底物）和[H-3]组氨酸（肽/组氨酸转运蛋白PHT1和PHT2的底物）的摄取。[14 C] Gly-Sar进入突触体的摄取由向内定向的W-梯度刺激。该摄取系统显示Gly-Sar的Michaelis-Menten常数（K-t）为110 +/-20 μ M。该值与通过高亲和力H +/肽转运蛋白PEPT 2摄取Gly-Sar的K-t值相当。[C-14] Gly-Sar进入突触体的H+依赖性摄取被二肽和三肽以及β-内酰胺抗生素抑制，但不受氨基酸甘氨酸和组氨酸的影响。特别地，kyotorphin（Tyr-Arg）完全抑制Gly-Sar摄取，Ki值为29 +/-14 μ M。脑肽转运蛋白的这些摄取特性（即，Gly-Sar摄取的K-t值和Kyotorphin摄取Gly-Sar的Ki值）与PEPT 2的那些非常相似。RT-PCR和Western blotting分析表明，PEPT 2在大鼠大脑皮层中确实表达。这些结果表明，H +-偶联的高亲和力肽转运系统在大脑皮层中功能性表达，并且该转运系统与PEPT 2相同。(C)2003 Elsevier B.V.保留所有权利。
In this report, we studied the functional characteristics of a brain peptide transporter using synaptosomes prepared from rat cerebral cortex. Crude synaptosomes (P-2 fraction) were prepared from cerebral cortices in male Wistar rats. Uptake of [C-14]glycylsarcosine (Gly-Sar), a substrate for H+/oligopeptide transporters PEPT1 and PEPT2, and [H-3]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degreesC by a rapid filtration technique. The uptake of [14 C]Gly-Sar into synaptosomes was stimulated by an inwardly directed W-gradient. The uptake system exhibited a Michaelis-Menten constant (K-t) of 110 +/- 20 muM for Gly-Sar. This value is comparable to the K-t value for Gly-Sar uptake via the high-affinity H+/peptide transporter PEPT2. The H+-dependent uptake of [C-14]Gly-Sar into synaptosomes was inhibited by di- and tripeptides and beta-lactam antibiotics, but was unaffected by amino acids glycine and histidine. In particular, kyotorphin (Tyr-Arg) completely inhibited Gly-Saruptake with the K-i value of 29 +/- 14 muM. These uptake properties of the brain peptide transporter (i.e., the K-t value for Gly-Sar uptake and the Ki value of kyotorphin for Gly-Sar uptake) are very similar to those of PEPT2. RT-PCR and Western blotting analyses revealed that PEPT2 is actually expressed in the cerebral cortex in rat. These results indicate that a H+-coupled high affinity peptide transport system is functionally expressed in the cerebral cortex and that this transport system is identical to PEPT2. (C) 2003 Elsevier B.V. All rights reserved.