Proton-Coupled Oligopeptide Transporter (POT) Family Expression in Human Nasal Epithelium and Their Drug Transport Potential
Proton-Coupled Oligopeptide Transporter (POT) Family Expression in Human Nasal Epithelium and Their Drug Transport Potential
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DOI：
10.1021/mp100234z
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发表时间：
2011-05-01
影响因子：
4.9
通讯作者：
Massoud, Emad
中科院分区：
文献类型：
--
作者：
Agu, Remigius;Cowley, Elizabeth;Massoud, Emad
研究了人鼻上皮中肽转运蛋白（PEPT1 和 PEPT2、PHT1、PHT2）的分子和功能表达。使用定量/逆转录酶聚合酶链反应（qPCR/RT-PCR）、蛋白质印迹和间接免疫组织化学来研究转运蛋白的功能基因和蛋白表达。使用代谢稳定的肽 [β-丙氨酰-L-赖氨酰-N epsilon-7-氨基-4-甲基-香豆素-3-乙酸 (β-Ala-Lys-AMCA) 和 β-丙氨酰-L-组氨酸（肌肽）] 进行摄取和转运研究。研究了浓度、温度、极性、竞争肽和抑制剂对肽摄取和转运的影响。检测到对应于 PEPT1 (150 bp)、PEPT2 (127 bp)、PHT1 (110 bp) 和 PHT2 (198 bp) 的 PCR 产物。免疫组织化学和蛋白质印迹证实了 PEPT1 和 PEPT2 基因的功能表达。 β-Ala-Lys-AMCA 的摄取呈浓度依赖性且可饱和（V(max) = 4.1 +/- 0.07 mu mol/min/mg 蛋白质，K(m) = 0.6 +/- 0.07 mu M）。 β-Ala-Lys-AMCA 细胞内积累的最佳 pH 值为 6.5。二肽和羰基氰间氯苯腙 (CCCP) 显着抑制肽的摄取和转运，而 L-Phe 对肽的转运没有影响。 β-丙氨酰-L-组氨酸的渗透具有浓度、方向和温度依赖性。摄取、渗透、qPCR/RT-PCR 和蛋白质表达数据表明，人鼻上皮功能性地表达质子偶联寡肽转运蛋白。
The molecular and functional expression of peptide transporters (PEPT1 and PEPT2, PHT1, PHT2) in human nasal epithelium was investigated. Quantitative/reverse transcriptase polymerase chain reaction (qPCR/RT-PCR), Western blotting and indirect immuno-histochemistry were used to investigate the functional gene and protein expression for the transporters. Uptake and transport studies were performed using metabolically stable peptides [beta-alanyl-L-lysyl-N epsilon-7-amino-4-methyl-coumarin-3-acetic acid (beta-Ala-Lys-AMCA) and beta-alanyl-L-histidine (carnosine)]. The effects of concentration, temperature, polarity, competing peptides, and inhibitors on peptide uptake and transport were investigated. PCR products corresponding to PEPT1 (150 bp), PEPT2 (127 bp), PHT1 (110 bp) and PHT2 (198 bp) were detected. Immunohistochemistry and Western blotting confirmed the functional expression of PEPT1 and PEPT2 genes. The uptake of beta-Ala-Lys-AMCA was concentration-dependent and saturable (V(max) = 4.1 +/- 0.07 mu mol/min/mg protein, K(m) = 0.6 +/- 0.07 mu M). The optimal pH for intracellular accumulation of beta-Ala-Lys-AMCA was 6.5. Whereas dipeptides and carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly inhibited peptide uptake and transport, L-Phe had no effect on peptide transport. The permeation of beta-alanyl-L-histidine was concentration-, direction-, and temperature-dependent. The uptake, permeation, qPCR/RT-PCR and protein expression data showed that the human nasal epithelium functionally expresses proton-coupled oligopeptide transporters.