Molucular analysis of tissue distribution and structure-function relationship of transporters responsible for the regulation of drug transport

负责药物转运调节的转运蛋白的组织分布和结构功能关系的分子分析

基本信息

批准号：
08457620
负责人：
INUI Ken-ichi
金额：
$ 5.57万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Tissue distribution and structure-function relationship of membrane transporters responsible for absorption and excretion of drugs have been studied, and then following results were obtained.1.Structure-function relationship of H^+-coupled peptide transportersWhen stable transfectans expressing rat peptide transporters PEPT1 and PEPT2 were treated with DEPC, a histidine-modifying agent, glycylsarcosine uptake by both transfectants were decreased. Interactions of dipeptides and beta-lactam antibiotics were analyzed, and then it became clear that alpha-amino moiety of beta-lactam antibiotics should interact with histidine residues, and may participate in substrate recognition.2.Tissue distribution and functional characteristics of renal organic ion transporters.(1)Organic anion transporter, OAT-K1 : Expression of mRNA along nephron segments was analyzed by RT-PCR.OAT-K1 mRNAs were mainly expressed in proximal straight tubules of superficial and juxtamedullary nephrons. Western blot analysis revealed that OAT-K1 is expressed only in brush-border membranes of renal tubules. Methotrexate transport by the transfectants stably expressing OAT-K1 was significantly inhibited in the presence of nonsteroidal anti-inflammatory drugs (NSAID).(2)Organic cation transporter, OCT2 : Because tetraethylammonium transport by OCT2 was H^+-gradient independent and was affected by membrane potential, OCT2 is deduced to be basolateral-type organic cation transporter. By constructing stable transfectants expressing OCT1 or OCT2, transport characteristics were further analyzed, and then it became clear that both OCT1 and OCT2 are basolateral-type organic cation transporters with broad substrate specificity and have similar substate recognition in each other.

对负责药物吸收和排泄的膜转运蛋白的组织分布和结构-功能关系进行了研究，得到了以下结果：1.H^+偶联肽转运蛋白的结构-功能关系稳定转染子表达大鼠肽转运蛋白PEPT1和PEPT2 用组氨酸修饰剂 DEPC 处理，两种转染子对甘氨酰肌氨酸的摄取均减少。通过分析二肽与β-内酰胺类抗生素的相互作用，明确β-内酰胺类抗生素的α-氨基部分应与组氨酸残基相互作用，并可能参与底物识别。2.肾脏有机离子的组织分布和功能特征(1)有机阴离子转运蛋白OAT-K1：通过RT-PCR分析沿肾单位节段mRNA的表达。OAT-K1 mRNA主要在近端直链表达浅肾小管和近髓肾单位。 Western blot 分析显示，OAT-K1 仅在肾小管刷状缘膜中表达。在非甾体抗炎药 (NSAID) 存在下，稳定表达 OAT-K1 的转染子的甲氨蝶呤转运受到显着抑制。 (2)有机阳离子转运蛋白 OCT2 ：因为 OCT2 的四乙铵转运不依赖于 H^+ 梯度，因此受到影响通过膜电位推测OCT2是基底外侧型有机阳离子转运蛋白。通过构建表达OCT1或OCT2的稳定转染子，进一步分析转运特性，发现OCT1和OCT2都是基底外侧型有机阳离子转运蛋白，具有广泛的底物特异性，并且彼此具有相似的底物识别能力。