LENS GLUTATHIONE TRANSPORTERS

晶状体谷胱甘肽转运蛋白

• 批准号: 2711144
• 负责人: RAM KANNAN
• 金额: $ 25.11万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2711144
• 关键词: aging; cell membrane; egg /ovum; glutamyltransferase; glutathione; high performance liquid chromatography; homeostasis; immunocytochemistry; in situ hybridization; laboratory rat; lens; membrane potentials; membrane transport proteins; molecular biology; molecular cloning; northern blottings; oxidative stress; polymerase chain reaction; protein sequence; protein transport; scintillation counter; sodium potassium exchanging ATPase; statistics /biometry; vesicle /vacuole

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.

说明：谷胱甘肽(GSH)可以保护晶状体免受氧化剂的伤害。 强调并保持镜头的透明度。GSH水平随着进展期的延长而降低 年龄使老化的晶状体易受活性氧代谢物和 受伤。我们以前已经证明，GSH在体内完好无损地被运输 豚鼠原位灌注眼模型。后来的研究表明，当牛 将晶状体mRNA注射到非洲爪哇卵母细胞中，GSH转运被抑制 牛和大鼠的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 转运体(RcGshT)。最近，有证据表明 大鼠晶状体中新的钠依赖谷胱甘肽转运蛋白 用卵母细胞表达系统获得上皮细胞。谷胱甘肽 晶状体上皮细胞表达的转运蛋白对BSP-GSH敏感(不像 RcGshT)，这一发现得到了原位抑制研究的证实 眼部血流灌注模型。本提案的目标是将 GSH转运蛋白，并研究它们的发育调节 分子水平。具体目标是：1)克隆Na+/GSH 晶状体中的联合传送器。PI将通过分级来实现这一点 晶状体上皮细胞的mRNA，RcGshT的排除或鉴定， 正弦GSH转运体(RsGshT)、γ-谷氨酰转肽酶和 钠离子依赖的GSH摄取片段的克隆及序列分析 分析。新克隆的谷胱甘肽转运蛋白的器官分布 此外，还将研究转运的动力学和特异性。2) RcGshT在晶状体中的表征。我们将研究发展和 RcGshT转录本和基因产物的年龄相关性变化及定位 用原位杂交和免疫组织化学方法检测RcGshT在大鼠晶状体中的表达。 还将继续使用苯巴比妥作为增加RcGshT的工具。和 最后，他将使用囊泡作为模型来研究大鼠体内的谷胱甘肽转运 晶状体皮质质膜。具体地说，他将确定 摄取并测定多组分的Km和Vmax，研究跨刺激 摄取、抑制剂专一性和识别驱动力(膜 电位、钠离子依赖性、pH依赖性)。从小说中获得的知识 GSH转运体对更好地理解GSH稳态将是有价值的 在晶状体中，它们的发育调节，以及在设计治疗 预防与老年相关的晶状体损伤的方法。