MOLECULAR CHARACTERIZATION OF BRAIN GSH TRANSPORTERS

描述（研究者摘要）：虽然谷胱甘肽（GSH）是 在防御氧化应激中起关键作用，在有氧代谢中普遍存在。 细胞，大脑可以利用GSH的其他特殊功能，如存储 以及半胱氨酸的转移和神经兴奋性传递的调节。 我们 一直对完整GSH跨血浆的易位感兴趣 膜。 在大鼠肝脏中克隆了两种谷胱甘肽转运蛋白， RcGshT也存在于全脑、孤立的毛细血管中， 培养的星形胶质细胞，并且在脑中是苯巴比妥诱导的， 肝脏 此外，我们还发现，在血脑屏障处可以提取GSH， 转运由不同于RcGshT的钠依赖性机制介导 （钠非依赖性）。 非洲爪蟾卵母细胞表达豚鼠或 牛毛细血管mRNA表现出GSH的双向转运， 通过大小分级分离（牛）分离为至少3种不同的GSH 转运蛋白-一个是RcGshT（牛同源物）和两个新的（ 其是高度钠依赖性的）。 我们的第一个具体目标是表达， 利用RT-PCR技术克隆和鉴定大鼠脑毛细血管GSH转运蛋白 非洲爪蟾卵母细胞表达系统。 我们将i）执行大小 将mRNA分级分离并显微注射到卵母细胞中以确定 转运蛋白的多样性，并比较其离子需求，ii）研究 RcGshT和γ-谷氨酰转肽酶GGT对表达的贡献 GSH转运在相关的大小部分，使用反义 iii）克隆和测序不同于 RcGshT，iv）研究表达三种克隆的 谷胱甘肽转运蛋白：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RNA进行北方印迹分析 用新克隆的GSH转运蛋白探测，vi）进行北方和 毛细血管与对照组毛细血管耗竭脑的蛋白质印迹， 苯巴比妥处理的大鼠。 在我们的第二个目标中，我们将研究 谷胱甘肽在培养的星形胶质细胞和脑内皮细胞，并表征 转运蛋白：我们将i）确定是否GSH摄取和流出， 星形胶质细胞和内皮细胞是可饱和的，其特异性和离子 ii）确定是否存在克隆的GSH 星形胶质细胞和内皮细胞中的转运蛋白mRNA和基因产物。 这些研究将有助于更好地了解体内平衡， 在正常生理和病理生理学中GSH在脑中的转运， 将是关键的潜在治疗策略的发展， GSH缺乏的神经系统状态中的GSH递送。

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.