| Project/Area Number |
06454596
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
INUI Ken-ichi Kyoto University, Department of Pharmacy, Professor, 医学研究科, 教授 (70034030)
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| Co-Investigator(Kenkyū-buntansha) |
SAITO Hideyuki Kyoto University, Department of Pharmacy, Instructor, 医学研究科, 助手 (40225727)
TAKANO Mikihisa Kyoto University, Department of Pharmacy, Associate Professor, 医学研究科, 助教授 (20211336)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1995: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1994: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | Drug Transport / beta-Lactam Antibiotics / Peptide Transporter / Intestinal Absorption / Renal Tubular Secretion / Organic Anions / cDNA Cloning / ジペプチド / 輸送担体 / 培養上皮細胞 / クローニング / セファロスポリン / 卵母細胞 |
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| Research Abstract |
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
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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
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