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

离子药物的肠道抽象和肾小管分泌是矢量性跨细胞转运过程，这些过程是由位于这些上皮的刷子或基底外侧机制的转运蛋白介导和调节的。在该项目中，我们通过在异武卵母细胞和哺乳动物转化体中使用PCR技术和表达系统研究了药物转运蛋白的cDNA克隆，结构 - 连接关系和组织分布。（1）使用PCR技术的大鼠H^+/pcr技术的cDNA克隆，功能性表征和组织分布，基于PCR技术的频率序列： Pept1是分离的大鼠H^+/肽共转运蛋白PEPT1的cDNA。大鼠PEPT1蛋白由710个氨基酸组成，该氨基酸具有十二个跨膜结构域，分别显示出与兔和人类PEPT1的77％和83％的氨基酸同一性。 Northern印迹和逆转录耦合的PCR分析REV…更肯定地说，大鼠PEPT1 Messenger RNA主要在小肠中表达，并且在肾脏皮层中的程度较小。通过使用抗RAT PEPT1抗体的蛋白质印迹分析，在十二指肠，空肠和回肠中检测到大鼠PEPT1，但在结肠或直肠中未检测到。此外，大鼠PEPT1被证明位于小肠和肾近端结节细胞的刷状膜上，但不在这些上皮的基底外侧膜上。使用与大鼠PEPT1 cDNA翻译的合成大鼠PEPT1 RNA和哺乳动物上皮细胞注入的卵母细胞的转运研究表明，大鼠PEPT1识别和转运口服活性的β-乳糖酰胺抗生素，例如Ceftibuten（Ceftibuten（Ceftibuten）（阴离子）和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)在肾脏中特别表达。用大鼠燕麦蛋白cDNA翻译的培养的上皮细胞的功能研究表明，OAT-K1位于肾近端小管的基底外侧机制上，并参与了一些阴离子药物的肾脏分布和分泌。较少的