Functional characterization of sodium-dependent multivitamin transporter in MDCK-MDR1 cells and its utilization as a target for drug delivery
Functional characterization of sodium-dependent multivitamin transporter in MDCK-MDR1 cells and its utilization as a target for drug delivery
复制标题
DOI：
10.1021/mp0500768
复制
发表时间：
2006-05-01
影响因子：
4.9
通讯作者：
Mitra, Ashim K.
中科院分区：
文献类型：
--
作者：
Luo, Shuanghui;Kansara, Viral S.;Mitra, Ashim K.
本研究的目的是在MDCK-MDR1细胞(转染人MDR1基因的Madin-Darby犬肾细胞)中鉴定一种钠依赖的多维生素转运体(SMVT)，并探讨利用MDCK-MDR1细胞作为体外模型研究生物素偶联的抗HIV蛋白酶抑制剂前药的通透性的可行性。阐明了生物素[H-3]吸收和转运机制。还研究了生物素偶联前药，即生物素-沙奎那韦的跨血管通透性。逆转录-聚合酶链式反应(RT-PCR)证实MDCK-MDR1细胞中存在SMVT。生物素的吸收依赖于Na+、pH和温度，但不依赖于能量。生物素的吸收是饱和的，K-m为13.0µM，V-max为21.5pmolmin(-1)(蛋白质毫克)，K-d为0.12mU L min(-1)(蛋白质mg)(-1)。[H-3]生物素的顶端和基础摄取及跨上皮转运均表明SMVT主要定位于MDCK-MDR1细胞的顶膜。[H-3]过量的未标记生物素及其结构类似物，即去硫生物素和戊酸，以及其他维生素，如硫辛酸和泛酸，可抑制生物素的摄取，但不能被醋酸、苯甲酸、生物素甲酯和生物细胞素抑制。生物素-沙奎那韦引起[H-3]生物素摄取降低，这表明它被SMVT所识别。在过量生物素或生物素-沙奎那韦存在的情况下，生物素从顶端到底端的转运也受到显著抑制。生物素-沙奎那韦在MDCK-MDR1、野生型MDCK和Caco-2细胞中的跨皮转运研究表明，生物素-沙奎那韦在所有三种细胞系中的通透性都是相似的。RT-PCR检测到SMVT基因在862bp处有一条带。在MDCK-MDR1细胞中发现了一种钠依赖的多种维生素转运体，SMVT负责生物素的摄取和转运，并对其进行了功能鉴定。因此，MDCK-MDR1细胞系可作为研究HIV蛋白酶抑制剂等生物素偶联前药通透性的体外模型。
The objective of this research is to characterize a sodium-dependent multivitamin transporter (SMVT) in MDCK-MDR1 cells (Madin-Darby canine kidney cells transfected with the human MDR1 gene) and to investigate the feasibility of utilizing the MDCK-MDR1 cell line as an in vitro model to study the permeability of biotin-conjugated prodrugs of anti-HIV protease inhibitors. Mechanism of [H-3]biotin uptake and transport was delineated. Transepithelial permeability of the biotin-conjugated prodrug, i.e., biotin-saquinavir, was also studied. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the existence of SMVT in MDCK-MDR1 cells. Biotin uptake was Na+, pH, and temperature dependent, but energy independent. Uptake of biotin was found to be saturable with a K-m of 13.0 mu M, V-max 21.5 of pmol min(-1) (mg of protein)(-1), and K-d of 0.12 mu L min(-1) (mg of protein)(-1). Both apical and basal uptake and transepithelial transport of [H-3]biotin showed that SMVT localized predominantly on the apical membrane of MDCK-MDR1 cells. [H-3]Biotin uptake was inhibited by excess unlabeled biotin and its structural analogues, i.e., desthiolbiotin and valeric acid, and other vitamins such as lipoic acid and pantothenic acid, but not by acetic acid, benzoic acid, biotin methyl ester, and biocytin. Biotin-saquinavir caused lowering of [H-3]biotin uptake, which indicates that it is recognized by SMVT. Apical to basal transport of [H-3]biotin was also significantly inhibited in the presence of excess biotin or biotin-saquinavir. Transepithelial transport studies of biotin-saquinavir in MDCK-MDR1, wild type MDCK, and Caco-2 cells revealed that permeability of biotin-saquinavir was similar in all three cell lines. A band of SMVT mRNA at 862 bp was identified by RT-PCR. A sodium-dependent multivitamin transporter, SMVT, responsible for biotin uptake and transport, was identified and functionally characterized in MDCK-MDR1 cells. Therefore, the MDCK-MDR1 cell line may be utilized as an in vitro model to study the permeability of biotin-conjugated prodrugs such as HIV protease inhibitors.