Hu Yongjun, Ocheltree Scott M, Xiang Jianming, Keep Richard F, Smith David E
Department of Pharmaceutical Sciences, The University of Michigan, Ann Arbor, Michigan 48109, USA.
Pharm Res. 2005 Aug;22(8):1281-6. doi: 10.1007/s11095-005-5261-0. Epub 2005 Aug 3.
The purpose of this research was to determine the polarity and directionality of the PEPT2-mediated uptake and transepithelial transport of the neuropeptide glycyl-L-glutamine (GlyGln) in choroid plexus.
The transport kinetics of [3H]GlyGln was studied in neonatal rat choroid plexus epithelial cells in primary culture grown on laminin-coated Transwell filter inserts. Using a bicarbonate artificial cerebrospinal fluid (CSF) buffer (pH 7.4) at 37 degrees C, GlyGln studies were performed as a function of time, substrate concentration, and the presence of potential inhibitors (at 1 mM).
GlyGln (2 microM) accumulation was about three to four times greater when introduced from the apical (CSF-facing) as opposed to the basal (blood-facing) side of the cell monolayer, and transepithelial transport was about two times greater in the apical-to-basal direction. The apical uptake of radiolabeled GlyGln (2 microM) was inhibited significantly by dipeptides (i.e., unlabeled GlyGln and cysteinylglycine) and some neuropeptides (i.e., carnosine, N-acetylaspartylglutamate, kyotorphin), but was unaffected by amino acids (i.e., glycine, glutamine) as well as by [D-Arg2]-kyotorphin and glutathione. The concentration-dependent apical uptake of GlyGln (2-1000 microM) was characterized by a high-affinity process (i.e., Vmax of 72 pmol/mg/min; Km of 136 microM), consistent with the properties of PEPT2. The intracellular hydrolysis of GlyGln was extensive, however, with only 40% of the dipeptide remaining intact after 1 h.
The results demonstrate that PEPT2 plays an important role in regulating the apical uptake of GlyGln at the blood-CSF interface. Once inside the cell, GlyGln is rapidly degraded to its constitutive amino acids for further processing.
本研究旨在确定肽转运体2（PEPT2）介导的神经肽甘氨酰-L-谷氨酰胺（GlyGln）在脉络丛中的摄取极性和方向性以及跨上皮转运情况。
在包被层粘连蛋白的Transwell滤器小室上原代培养的新生大鼠脉络丛上皮细胞中研究[³H]GlyGln的转运动力学。在37℃下使用碳酸氢盐人工脑脊液（CSF）缓冲液（pH 7.4），将GlyGln的研究作为时间、底物浓度以及潜在抑制剂（1 mM）存在情况的函数进行。
当从细胞单层的顶端（面向CSF）而非基底（面向血液）侧引入时，GlyGln（2 μM）的积累量大约高3至4倍，并且跨上皮转运在顶端到基底方向大约高2倍。放射性标记的GlyGln（2 μM）的顶端摄取受到二肽（即未标记的GlyGln和半胱氨酰甘氨酸）和一些神经肽（即肌肽、N-乙酰天冬氨酰谷氨酸、脑啡肽）的显著抑制，但不受氨基酸（即甘氨酸、谷氨酰胺）以及[D-Arg²]-脑啡肽和谷胱甘肽的影响。GlyGln（2 - 1000 μM）的浓度依赖性顶端摄取具有高亲和力过程的特征（即Vmax为72 pmol/mg/min；Km为136 μM），与PEPT2的特性一致。然而，GlyGln的细胞内水解广泛，1小时后只有40%的二肽保持完整。
结果表明PEPT2在调节血液 - CSF界面处GlyGln的顶端摄取中起重要作用。一旦进入细胞内，GlyGln会迅速降解为其组成氨基酸以进行进一步处理。
