LENS GLUTATHIONE TRANSPORTERS

描述：谷胱甘肽（GSH）已知可保护透镜免受氧化剂的影响 并保持透镜透明度。 GSH水平随着年龄的增长而下降 年龄使老化的透镜易受活性氧代谢物的影响， 损伤 我们以前已经表明，GSH是运输完整的一个在 原位豚鼠灌注眼模型。 随后的研究表明，当牛 将透镜mRNA注入非洲爪蟾卵母细胞， 表达，牛和大鼠晶状体表达转录本和蛋白质 最近克隆的一种肝脏GSH转运蛋白，大鼠小管GSH 转运蛋白（RcGshT）。 最近，有证据表明， 大鼠晶状体中另外的新型Na+依赖性GSH转运蛋白 还使用卵母细胞表达系统获得上皮。 大成糖业 透镜上皮细胞mRNA表达的转运蛋白对BSP-GSH敏感（不像 RcGshT），这一发现得到了原位抑制研究的证实。 眼灌注模型 本提案的目标是， 谷胱甘肽转运蛋白，并研究其发育调控， 分子水平。 具体目的是：1）克隆Na +/GSH 透镜中的协同转运蛋白。 PI将通过大小分级实现这一点 晶状体上皮mRNA，排除或鉴定RcGshT， 正弦GSH转运蛋白（RsGshT）、γ谷氨酰转肽酶和 克隆具有Na+依赖性GSH摄取的级分，然后测序 分析. 新克隆的谷胱甘肽转运蛋白的器官分布， 还将研究转运的动力学和特异性。 （二） 在透镜中RcGshT的表征。 我们将学习发展和 RcGshT转录物和基因产物的年龄依赖性变化及定位 用原位杂交和免疫组织化学方法检测RcGshT在大鼠透镜中的表达。 还将继续使用苯巴比妥作为增加RcGshT的工具。 和 最后，3）将利用囊泡作为模型研究大鼠GSH转运 透镜皮质质膜。 具体来说，他将确定 摄取并测定多组分的Km和Vmax，研究跨膜刺激 的摄取，抑制剂特异性和识别驱动力（膜 电位、Na+依赖性、pH依赖性）。 从小说中获得的知识 谷胱甘肽转运蛋白将有助于更好地了解谷胱甘肽稳态 在透镜中，它们的发育调节， 预防与老年相关的透镜损伤的方式。

Description: Gluthathione (GSH) is known to protect the lens from oxidant stress and maintain lens transparency. GSH levels decrease with advancing age rendering the aged lens susceptible to reactive oxygen metabolites and injury. We have previously shown that GSH is transported intact in an in situ guinea-pig perfused eye model. Subsequent work showed that when bovine lens mRNA is injected into Xenopus laevis oocytes, GSH transport is expressed and that bovine and rat lenses express the transcript and protein for one of the recently cloned hepatic GSH transporters, rat canalicular GSH transporter (RcGshT). More recently, evidence for the presence of an additional, novel Na+-dependent GSH transporter in the rat lenticular epithelium was also obtained using the oocyte expression system. The GSH transport expressed by lens epithelial mRNA was sensitive to BSP-GSH (unlike RcGshT), a finding that was confirmed by inhibitory studies in the in situ eye perfusion model. The goal in the present proposal is to characterize the GSH transporters, and to study their developmental regulation at the molecular level. The specific aims are to: 1) clone the Na+/GSH co-transporter in the lens. The PI will achieved this by size fractionation of lenticular epithelial mRNA, exclusion or identification of RcGshT, the sinusoidal GSH transporter (RsGshT), gamma glutamyltranspeptidase and cloning the fraction with Na+-dependent GSH uptake followed by sequence analysis. Organ distribution of the newly cloned GSH transporter and kinetics and specificity of transport will also be studied. 2) characterization of RcGshT in the lens. We will study developmental and age-dependent changes in RcGshT transcript and gene product and localization of RcGshT in the rat lens by in situ hybridization and immunohistochemistry. Use of phenobarbital as a tool to increase RcGshT will also be pursued. And finally, 3)he will use vesicles as a model to study GSH transport in rat lens cortical plasma membranes. Specifically, he will determine kinetics of uptake and determine Km and Vmax of multicomponents, study transstimulation of uptake, inhibitor specificity and identify driving force (membrane potential, Na+-dependence, pH dependence). Knowledge gained on the novel GSH transporters will be of value in better understanding of GSH homeostasis in the lens, their developmental regulation, and in designing therapeutic modalities to prevent lens damage associated with old age.