MOLECULAR CHARACTERIZATION OF BRAIN GSH TRANSPORTERS

脑谷胱甘肽转运蛋白的分子特征

• 批准号: 2734790
• 负责人: RAM KANNAN
• 金额: $ 22.21万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734790
• 关键词: Xenopus oocyte; antisense nucleic acid; astrocytes; biological transport; brain; glutathione; laboratory rat; molecular cloning; protein glutamine gamma glutamyltransferase; tissue /cell culture; transport proteins; vascular endothelium

DESCRIPTION (Investigator's Abstract): Although glutathione (GSH) is critical in defense against oxidative stress and is ubiquitous in aerobic cells, the brain may utilize GSH for other special functions such as storage and transfer of cysteine and modulation of neuroexcitatory transmission. We have been interested in the translocation of intact GSH across plasma membranes. Two GSH transporters have been cloned in rat liver, one of which, RcGshT, is also found in whole brain, isolated capillaries, and cultured astrocytes, and is phenobarbital inducible in brain as well as liver. In addition, we showed that GSH can be extracted at the BBB and GSH transport is mediated by sodium-dependent mechanism distinct from RcGshT (sodium-independent). Xenopus laevis oocytes expressing guinea-pig or bovine capillary mRNA exhibit bidirectional GSH transport which could be resolved by size fractionation (bovine) into at least 3 different GSH transporters--one being RcGshT (bovine homologue) and two novel ones (one of which is highly sodium dependent). Our first specific aim is to express, clone and characterize rat brain capillary GSH transporters using the Xenopus laevis oocyte expression system. We will i) perform size fractionation of mRNA and microinjection into oocytes to determine multiplicity of transporters and compare their ion requirements, ii) study contribution of RcGshT and gamma-glutamyl transpeptidase, GGT, to expression of GSH transport in the relevant size fractions using antisense oligonucleotides, iii) clone and sequence GSH transporters distinct from RcGshT, iv) study GSH uptake and efflux in oocytes expressing three cloned GSH transporters: kinetics, ion requirements, molecular forms and inhibitor specificity, v) perform Northern blot analysis of RNA from various organs probed with newly cloned GSH transporters and, vi) perform Northern and Western blots of capillaries vs capillary-depleted brain from controls and phenobarbital-treated rats. In our second aim, we will study transport of GSH in cultured astrocytes and brain endothelial cells and characterize the transporters: we will i) determine whether GSH uptake and efflux in astrocytes and endothelial cells is saturable and its specificity and ion requirements, and ii) determine the presence or absence of cloned GSH transporter mRNAs and gene products in astrocytes and endothelial cells. These studies will lead to a better understanding of the homeostasis and transport of GSH in the brain in normal physiology and pathophysiology and will be critical in the development of potential therapeutic strategies of GSH delivery in GSH-deficient neurological states.

描述（研究者的摘要）：尽管谷胱甘肽（GSH）是 防御氧化应激的防御至关重要，并且有氧无处不在 细胞，大脑可以将GSH用于其他特殊功能，例如存储 半胱氨酸的转移和神经兴奋性传播的调节。 我们 对整个等离子体的完整GSH的易位感兴趣 膜。 两个GSH转运蛋白已被克隆在大鼠肝脏中，其中之一 在整个大脑中，rcgsht，孤立的毛细血管和 培养的星形胶质细胞，并且在大脑以及 肝。 此外，我们表明可以在BBB和GSH处提取GSH 转运是由钠依赖性机制介导的，不同于RCGSHT （独立于钠）。 Xenopus laevis卵母细胞表达豚鼠或 牛毛细血管mRNA表现出双向GSH转运 通过尺寸分馏（牛）至少3种不同的GSH解决 转运蛋白 - 一个是RCGSHT（牛同源物）和两个新颖的（其中之一） 这是高度钠的）。 我们的第一个具体目的是表达 克隆并使用大鼠脑毛细管GSH转运蛋白表征 爪蟾Laevis卵母细胞表达系统。 我们会）执行尺寸 将mRNA和显微注射分解为卵母细胞以确定 转运蛋白的多样性并比较其离子要求，ii）研究 RCGSHT和GAMMA-谷氨酰基肽酶GGT的贡献对表达 使用反义的相关尺寸分数中的GSH运输 寡核苷酸，iii）克隆和序列GSH转运蛋白不同 RCGSHT，iv）在表达三个克隆的卵母细胞中研究GSH摄取和外排 GSH转运蛋白：动力学，离子需求，分子形式和抑制剂 特异性，v）对来自各种器官的RNA进行北印迹分析 用新克隆的GSH转运蛋白探测，vi）进行北部和 毛细血管的蛋白质印迹与控制毛细管的大脑来自对照和 苯巴比妥治疗的大鼠。 在我们的第二个目标中，我们将研究运输 培养的星形胶质细胞和脑内皮细胞中的GSH，并表征 运输者：我们将）确定GSH的吸收和排出是否在 星形胶质细胞和内皮细胞可饱和，其特异性和离子 要求，ii）确定克隆GSH的存在或不存在 星形胶质细胞和内皮细胞中的转运蛋白mRNA和基因产物。 这些研究将使人们更好地了解体内平衡和 正常生理学和病理生理学中GSH在大脑中的运输以及 对于发展潜在的治疗策略至关重要 GSH缺乏神经系统状态的GSH递送。