Molecular and Cell Biological Analyzes of Structure and Function of Drug Transporters in the Intestine and Kidney Proximal Tubule

肠和肾近端小管药物转运蛋白结构和功能的分子和细胞生物学分析

基本信息

批准号：
06454596
负责人：
INUI Ken-ichi
金额：
$ 4.54万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

Intestinal absorption and renal tubular secretion of ionic drugs are vectorial transcellular transport processes, which are mediated and regulated by transporters localized at the brush-border or basolateral membranes of these epithelia. In this project, we studied cDNA cloning, structure-function relationship and tissue distribution of drug transporters by using the PCR technique and expression system in Xenopus oocytes and mammalian transfectants.(1)cDNA cloining, functional characterization and tissue distribution of rat H^+/peptide contransporter : By PCR technique using degenerated primers based on amino acid sequence of the rabbit oligopeptide transporter PEPT1, a cDNA encoding for the rat H^+/peptide cotransporter PEPT1 has been isolated. The rat PEPT1 protein consisted of 710-amino acids with twelve membrane-spanning domains, and showed 77% and 83% amino acid identity with the rabbit and human PEPT1, respectively. Northern blot and reverse transcription-coupled PCR analyzes rev … More ealed that the rat PEPT1 messenger RNA was expressed predominantly in the small intestine, and to a lesser extent in the kidney cortex. By Western blot analysis using anti-rat PEPT1 antibody, rat PEPT1 was detected in duodenum, jejunum and ileum, but not in colon or rectum. Furthermore, rat PEPT1 was demonstrated to be localized at the brush-border membranes of both the small intestinal and renal proximal tubular cells, but not at the basolateral membranes of these epithelia. Transport studies using the oocytes injected with the synthetic rat PEPT1 RNA and the mammalian epithelial cells transfected with the rat PEPT1 cDNA indicated that the rat PEPT1 recognizes and transports orally active beta-lactam antibiotics such as ceftibuten (anion) and cephradine (zwitterion). These findings suggest that the rat PEPT1 mediates absorption of peptide-like drugs in the small intestine and kidney.cDNA cloning and characterization of kidney-specific organic anion transporter : By PCR technique using degenerated primers based on amino acid sequence of the rat liver organic anion transporter (oatp), a cDNA encoding for a novel rat organic anion transporter (OAT-K1) specifically expressed in the kidney was isolated. Functional studies by cultured epithelial cells transfected with the rat OAT-K1 cDNA demonstrated that OAT-K1 is localized at the basolateral membranes of renal proximal tubule and involved in the renal distribution and secretion of some anionic drugs.In conclusion, the findings obtained in this project will provide useful information for predicting tissue distribution of drugs and for development drug delivery systems. Less

离子药物的肠道吸收和肾小管分泌是矢量跨细胞转运过程，由位于这些上皮刷状缘或基底外侧膜的转运蛋白介导和调节。在本项目中，我们研究了cDNA克隆、结构功能关系和组织。利用PCR技术和表达系统研究非洲爪蟾卵母细胞和哺乳动物转染子中药物转运蛋白的分布。(1)大鼠cDNA克隆、功能表征和组织分布H^+/肽协同转运蛋白：通过使用基于兔寡肽转运蛋白PEPT1的氨基酸序列的简并引物的PCR技术，已分离出编码大鼠H^+/肽协同转运蛋白PEPT1的cDNA。大鼠PEPT1蛋白由710-组成。具有 12 个跨膜结构域的氨基酸，与兔和人 PEPT1 分别显示 77% 和 83% 的氨基酸一致性，Northern 印迹和逆转录偶联。 PCR rev … More 发现大鼠 PEPT1 信使 RNA 主要在小肠中表达，在肾皮质中表达较少。通过使用抗大鼠 PEPT1 抗体进行蛋白质印迹分析，在十二指肠、空肠和回肠中检测到了大鼠 PEPT1。，但不在结肠或直肠中。此外，大鼠 PEPT1 被证明位于小肠和肾近端肾小管细胞的刷状缘膜上，但不在结肠或直肠中。使用注射了合成大鼠 PEPT1 RNA 的卵母细胞和转染了大鼠 PEPT1 cDNA 的哺乳动物上皮细胞进行的转运研究表明，大鼠 PEPT1 识别并转运口服活性 β-内酰胺抗生素，例如头孢布烯（阴离子）和这些发现表明大鼠 PEPT1 介导小肠和肽类药物的吸收。肾特异性有机阴离子转运蛋白的 cDNA 克隆和表征：通过 PCR 技术，使用基于大鼠肝脏有机阴离子转运蛋白 (oatp) 氨基酸序列的简并引物，编码新型大鼠有机阴离子转运蛋白 (OAT-K1) 的 cDNA ）通过转染大鼠 OAT-K1 cDNA 的培养上皮细胞分离出特异性表达的 OAT-K1 位于基底外侧膜。肾近曲小管，参与一些阴离子药物的肾脏分布和分泌。总之，本项目获得的研究结果将为预测药物的组织分布和开发药物递送系统提供有用的信息。