CELLULAR MECHANISMS OF DRUG TRANSPORT IN CHOROID PLEXUS

脉络丛药物转运的细胞机制

• 批准号: 2181281
• 负责人: KATHLEEN M GIACOMINI
• 金额: $ 17.58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181281
• 关键词: Xenopus oocyte; basolateral membrane; brain metabolism; brush border membrane; choroid plexus; drug metabolism; human tissue; laboratory rabbit; membrane transport proteins; molecular cloning; neuronal transport; nucleobase; nucleoside analog; protein sequence; serotonin

Nucleosides, nucleobases and their analogs (e.g., ganciclovir, acyclovir, dideoxyinosine, azidothymidine) have rapidly become important therapeutic agents in the treatment of a number of central nervous system viral infections including herpes-simplex encephalitis, cytomegalovirus retinitis and AIDS-related dementia complex. To exert a biological effect in the central nervous system, these compounds must penetrate either the blood brain barrier (BBB) or the blood-cerebrospinal fluid barrier (i.e., choroid plexus), the two barriers that provide a protected environment for the brain. Although the BBB has received considerable attention in the literature, it is becoming increasingly apparent that the choroid plexus plays an essential role in mediating the transport of many important compounds into the central nervous system. Notably, during the past granting period, we demonstrated the presence of a novel, broadly- selective Na+-nucleoside cotransporter (N3) in the choroid plexus. In addition, we recently obtained exciting preliminary data suggesting that there is a novel Na+-dependent transporter for nucleobases in the choroid plexus. The overall goal of studies in this competitive renewal application is to understand the mechanisms by which nucleosides, nucleobases and their structural analogs are transported in the choroid plexus. In the proposed studies, we plan to: (1) determine the structure of N3 in rabbit choroid plexus using an expression cloning strategy; (2) determine the tissue distribution in the rabbit of the mRNA transcript encoding N3; (3) determine the location (i.e., basolateral or brush border membrane) of N3 in the choroid plexus; (4) determine the mechanisms by which purine and pyrimidine nucleosides are transported in human choroid plexus; and (5) determine the mechanisms by which nucleobases are transported in the choroid plexus. Methods for determining the structure of N3 will involve expression cloning in Xenopus laevis oocytes, determining the primary sequence of the cDNA encoding N3 and deducing its amino acid sequence. To identify the location of N3 as well as of equilibrative nucleoside transporters in the choroid plexus, kinetic studies using isotopic uptake procedures will be carried out in cultured choroid plexus monolayers grown on porous filters and in isolated membrane vesicles. Isotopic uptake studies in ATP-depleted tissue slices will be used in characterizing nucleoside transport in human choroid plexus and nucleobase transport in rabbit choroid plexus. In particular, we will elucidate the interactions of clinically relevant nucleoside analogs with these nucleoside and nucleobase transporters. The information gained in these studies is critical in the rational design, delivery and targeting of clinically relevant nucleosides, nucleobases and their analogs to the central nervous system.

核苷，核碱基及其类似物（例如Ganciclovir，Acyclovir， 二甲氧氨酸，偶氮苷）已迅速成为重要的治疗 治疗许多中枢神经系统病毒的药物 感染包括疱疹刺激性脑炎，巨细胞病毒 视网膜炎和与艾滋病相关的痴呆症复合物。发挥生物学作用 在中枢神经系统中，这些化合物必须穿透 血脑屏障（BBB）或血经脊髓液屏障（即 脉络丛），这是为保护环境提供保护环境的两个障碍 大脑。尽管BBB在 文学，脉络丛越来越明显 在调解许多重要的运输方面起着至关重要的作用 化合物进入中枢神经系统。值得注意的是，过去 在给予时期，我们证明了一个新颖的存在，广泛的 脉络丛中的选择性Na+ - 核苷共转运蛋白（N3）。在 此外，我们最近获得了令人兴奋的初步数据，表明 脉络膜中有一个新型的Na+依赖性转运蛋白用于核碱基 丛。在这种竞争更新中研究的总体目标 应用是了解核苷的机制， 核碱基及其结构类似物被转运在脉络膜中 丛。在拟议的研究中，我们计划：（1）确定结构 使用表达克隆策略在兔脉络丛中的N3； （2） 确定mRNA转录本的兔中的组织分布 编码N3; （3）确定位置（即基底外侧或刷子边框 脉络丛中N3的膜； （4）通过 嘌呤和嘧啶核苷在人脉络膜中转运 丛（5）确定核碱基的机制 在脉络丛中运输。确定结构的方法 N3的表达将克隆在Xenopus laevis卵母细胞中， 确定编码N3的cDNA的主要序列并推论其 氨基酸序列。确定N3的位置以及 脉络丛中的平衡性核苷转运蛋白，动力学 使用同位素摄取程序的研究将在培养中进行 在多孔过滤器和孤立的膜上生长的脉络丛单层 囊泡。 ATP耗尽组织切片中的同位素摄取研究将是 用于表征人类脉络丛中的核苷转运和 兔脉络膜中的核碱酶转运。特别是，我们会 阐明临床相关的核苷类似物与 这些核苷和核酶转运蛋白。获得的信息 这些研究对于理性设计，交付和定位至关重要 临床相关的核苷，核碱基及其类似物 中枢神经系统。