Fujita Takuya, Kishida Takeshi, Wada Miyuki, Okada Naoki, Yamamoto Akira, Leibach Frederick H, Ganapathy Vadivel
Department of Biochemical Pharmacology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607-8414, Japan.
Brain Res. 2004 Jan 30;997(1):52-61. doi: 10.1016/j.brainres.2003.10.049.
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 [14C]glycylsarcosine (Gly-Sar), a substrate for H(+)/oligopeptide transporters PEPT1 and PEPT2, and [3H]histidine, a substrate for peptide/histidine transporters PHT1 and PHT2, was measured at 37 degrees C by a rapid filtration technique. The uptake of [14C]Gly-Sar into synaptosomes was stimulated by an inwardly directed H(+)-gradient. The uptake system exhibited a Michaelis-Menten constant (K(t)) of 110+/-20 microM 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 [14C]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-Sar uptake with the K(i) value of 29+/-14 microM. These uptake properties of the brain peptide transporter (i.e., the K(t) value for Gly-Sar uptake and the K(i) 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.
在本报告中，我们使用从大鼠大脑皮层制备的突触体研究了一种脑肽转运体的功能特性。粗制突触体（P(2) 组分）取自雄性 Wistar 大鼠的大脑皮层。通过快速过滤技术在 37℃ 下测量了 [14C]甘氨酰肌氨酸（Gly-Sar，H(+)/寡肽转运体 PEPT1 和 PEPT2 的底物）以及 [3H]组氨酸（肽/组氨酸转运体 PHT1 和 PHT2 的底物）的摄取情况。[14C]Gly-Sar 向突触体的摄取受到内向 H(+)梯度的刺激。该摄取系统对 Gly-Sar 的米氏常数（K(t)）为 110±20 μM。这个值与通过高亲和力 H(+)/肽转运体 PEPT2 摄取 Gly-Sar 的 K(t) 值相当。[14C]Gly-Sar 向突触体的 H(+) 依赖性摄取受到二肽和三肽以及 β-内酰胺抗生素的抑制，但不受氨基酸甘氨酸和组氨酸的影响。特别是，京都啡肽（Tyr-Arg）以 29±14 μM 的 K(i) 值完全抑制了 Gly-Sar 的摄取。脑肽转运体的这些摄取特性（即 Gly-Sar 摄取的 K(t) 值和京都啡肽对 Gly-Sar 摄取的 K(i) 值）与 PEPT2 的非常相似。RT-PCR 和蛋白质印迹分析表明，PEPT2 确实在大鼠大脑皮层中表达。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具有功能表达，并且该转运系统与 PEPT2 相同。
