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

离子药物的肠道吸收和肾小管分泌是一种载体跨细胞转运过程，它是由这些上皮细胞刷缘或基底膜上的转运蛋白介导和调节的。本项目利用PCR技术及在爪蟾卵母细胞和哺乳动物转染物中的表达系统，研究了药物转运体的cDNA克隆、结构-功能关系和组织分布。(1)大鼠H^+/多肽共转运体cDNA克隆、功能表征及组织分布：采用基于兔寡肽转运体PEPT1氨基酸序列的退化引物PCR技术，分离出大鼠H^+/多肽共转运体PEPT1的cDNA。大鼠PEPT1蛋白由710个氨基酸组成，具有12个跨膜结构域，与兔PEPT1和人PEPT1的氨基酸同源性分别为77%和83%。Northern blot和逆转录偶联PCR分析显示，大鼠PEPT1信使RNA主要在小肠中表达，在肾皮质中表达较少。采用抗大鼠PEPT1抗体进行Western blot分析，大鼠十二指肠、空肠和回肠均检测到PEPT1，结肠和直肠未检测到PEPT1。此外，大鼠PEPT1被证明定位于小肠和肾近端小管细胞的刷状边界膜，而不是这些上皮的基底外膜。将合成的大鼠PEPT1 RNA注入卵母细胞和转染大鼠PEPT1 cDNA的哺乳动物上皮细胞进行转运研究表明，大鼠PEPT1可识别并转运口服活性β -内酰胺类抗生素，如头孢布烯（阴离子）和头孢定（两性离子）。这些发现表明，大鼠PEPT1介导小肠和肾脏对肽样药物的吸收。肾特异性有机阴离子转运蛋白的cDNA克隆与表征：利用基于大鼠肝脏有机阴离子转运蛋白（oatp）氨基酸序列的退化引物，采用PCR技术，分离出一种在肾脏中特异性表达的新型大鼠有机阴离子转运蛋白（OAT-K1）的cDNA编码。转染大鼠OAT-K1 cDNA的培养上皮细胞功能研究表明，OAT-K1定位于肾近端小管基底外膜，参与肾内一些阴离子药物的分布和分泌。总之，本项目获得的发现将为预测药物的组织分布和开发药物输送系统提供有用的信息。少

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