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

离子型药物的肠道吸收和肾小管分泌是一种跨细胞的载体转运过程，由位于上皮细胞刷状缘或基底外侧膜的转运蛋白介导和调节。本课题利用PCR技术和表达系统，在非洲爪蟾卵母细胞和哺乳动物转基因细胞中研究了药物转运蛋白的cDNA克隆、结构与功能的关系及组织分布。（1）大鼠H^+/肽共转运蛋白的cDNA克隆、功能鉴定和组织分布：根据兔寡肽转运蛋白PEPT 1的氨基酸序列设计简并引物，通过PCR技术，获得了大鼠H^+/肽共转运蛋白PEPT 1的cDNA。大鼠PEPT 1蛋白由710个氨基酸组成，具有12个跨膜结构域，与兔和人PEPT 1的氨基酸同源性分别为77%和83%。北方印迹和逆转录偶联PCR分析 ...更多信息 表明大鼠PEPT 1信使RNA主要在小肠中表达，在肾皮质中表达较少。用抗大鼠PEPT 1抗体进行Western blot分析，在大鼠十二指肠、空肠和回肠中检测到PEPT 1，而在结肠和直肠中未检测到PEPT 1。此外，大鼠PEPT 1被证明是本地化的刷状缘膜的小肠和肾近端肾小管细胞，但不是在基底外侧膜的这些上皮细胞。使用注射了合成大鼠PEPT 1 RNA的卵母细胞和转染了大鼠PEPT 1 cDNA的哺乳动物上皮细胞进行的转运研究表明，大鼠PEPT 1识别并转运口服活性β-内酰胺抗生素，如头孢布烯（阴离子）和头孢拉定（两性霉素B）。这些结果表明，大鼠PEPT 1介导了肽类药物在小肠和肾脏的吸收。肾脏特异性有机阴离子转运蛋白的cDNA克隆和鉴定：根据大鼠肝脏有机阴离子转运蛋白（oatp）的氨基酸序列设计简并引物，通过PCR技术，获得了一个在肾脏特异表达的大鼠有机阴离子转运蛋白（OAT-K1）的cDNA。体外培养的上皮细胞转染大鼠OAT-K1 cDNA后的功能研究表明，OAT-K1定位于肾近曲小管的基底外侧膜，并参与某些阴离子药物在肾脏的分布和分泌，本研究的结果为预测药物在肾脏的组织分布和开发给药系统提供了有用的信息。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