MOLECULAR CHARACTERIZATION OF BRAIN GSH TRANSPORTERS

描述(研究人员摘要)：虽然谷胱甘肽(GSH)是 在防御氧化应激方面至关重要，在有氧运动中普遍存在 细胞，大脑可以利用谷胱甘肽来实现其他特殊功能，如存储 半胱氨酸的转移和神经兴奋性传递的调节。我们 一直对完整的谷胱甘肽跨血浆转运感兴趣 膜。在大鼠肝脏中克隆了两个GSH转运蛋白，其中一个是 它，RcGshT，也在整个大脑、孤立的毛细血管和 培养的星形胶质细胞，苯巴比妥在脑内以及 肝脏。此外，我们还发现GSH可以在血脑屏障和GSH处被提取出来 与RcGshT不同，转运是由钠依赖机制介导的 (不依赖于钠)。非洲爪哇卵母细胞表达豚鼠卵母细胞 牛毛细血管mRNA表现出双向GSH转运，这可能是 通过大小分级(牛)分解成至少3种不同的GSH 转运蛋白--一个是RcGshT(牛同源基因)，另一个是新的转运蛋白(其中一个是 这是高度依赖钠的)。我们的第一个明确目标是表达， 大鼠脑毛细血管GSH转运蛋白的克隆和鉴定 非洲爪哇卵母细胞表达系统。我们将i)执行大小 MRNA的分级和卵母细胞内微量注射检测 转运体的多样性并比较它们的离子需求，II)研究 RcGshT和γ-谷氨酰转肽酶对基因表达的影响 利用反义技术研究谷胱甘肽在相关粒度组分中的转运 寡核苷酸，III)GSH转运蛋白的克隆和序列测定 RcGshT，IV)研究表达三个克隆的卵母细胞对GSH的摄取和外排 谷胱甘肽转运体：动力学、离子需求、分子形式和抑制剂 特异性，v)对不同器官的RNA进行Northern印迹分析 与新克隆的GSH转运蛋白进行了探索，并，vi)进行了Northern和 毛细血管的蛋白质印迹与对照组和对照组的毛细血管耗竭的脑 苯巴比妥治疗的大鼠。在我们的第二个目标中，我们将研究 GSH在培养的星形胶质细胞和脑内皮细胞中的表达 转运体：我们将i)确定GSH在体内的摄取和外排 星形胶质细胞和内皮细胞是饱和的，其特异性和离子 要求，以及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mRNAs和基因产物。 这些研究将有助于更好地理解动态平衡和 谷胱甘肽在正常生理和病理生理状态下的脑内转运 将在开发潜在的治疗策略方面起到关键作用 谷胱甘肽缺乏的神经状态下的谷胱甘肽释放。

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.