FUNCTIONS OF THE HEPATIC GAMMA-GLUTAMYL CYCLE

肝脏γ-谷氨酰循环的功能

• 批准号: 2518398
• 负责人: NAZZARENO BALLATORI
• 金额: $ 18.22万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518398
• 关键词: Xenopus oocyte; acetylcysteine; affinity chromatography; chemical conjugate; chemical synthesis; clearance rate; detoxification; enzyme activity; glutamyltransferase; glutathione; guinea pigs; high performance liquid chromatography; laboratory rabbit; laboratory rat; liver function; liver metabolism; membrane transport proteins; microinjections; molecular cloning; nucleic acid sequence; protein structure; sulfur aminoacid; tissue /cell culture

Glutathione plays a central role in cellular defense against reactive electrophiles and oxidants, two of the most common producers of cellular injury in both chemical toxicity and disease. Our overall objective is to identify mechanisms for disposition of glutathione S conjugates and mercapturic acids (S-substituted N-acetyl-L-cysteines) via a novel intrahepatic pathway. This pathway was recently described from our laboratory, and is thought to play an important role in hepatic detoxification of electrophiles and toxic metals such as mercury, mercapturic acid biosynthesis, bile formation, and glutathione homeostasis. The specific aims are: (1) Define the role of the intrahepatic gamma- glutamyl cycle in mercapturic acid formation. In contrast to current models of mercapturic acid biosynthesis, our recent studies demonstrated that this detoxification pathway can occur entirely within the liver. Moreover, in contrast to current thinking, a representative mercapturic acid (S.2,4-dinitrophenyl-N-acetylcysteine, or DNP-NAC) was excreted by isolated rat liver almost exclusively into bile, and not into the sinusoidal circulation. To best this model and to identify the mechanisms involved, we plan to: A.- Examine sinusoidal uptake and disposition of structurally distinct cysteine S-conjugates and mercapturic acids, both in vivo and in the perfused liver (with ethyl iodide and 1-chloro-2,4-dinitrobenzene as model xenobiotics). B. Using isolated canalicular and basolateral membrane vesicles, define the driving forces and substrate specificities for hepatic transport of mercapturic acids, and C. Test the role of sinusoidal gamma-glutamyl transferase iii hepatic clearance of GSH and glutathione S-conjugates, and in mercapturic acid biosynthesis. (2) Express the novel canalicular ATP-independent, high affinity glutathione S-conjugate carrier in Xenopus laevis oocytes using poly(A)+ RNA isolated from rat liver, and characterize the properties of the transporter in the oocyte system. With oocytes as a functional assay for transport, use expression cloning strategy to identify the cDNA for the transporter. Cloning will not only yield important structural information on this glutathione S-conjugate transporter, but will allow production of molecular probes with which to define its role in xenobiotic metabolism in liver and the whole organism. It is anticipated that these studies will ultimately provide information critical for the prevention and treatment of toxic liver cell injury and its extrahepatic complications induced by reactive electrophilic compounds.

谷胱甘肽在针对反应性的细胞防御中起着核心作用 电力和氧化剂，两个最常见的细胞生产者 化学毒性和疾病的损伤。 我们的总体目标是 确定处置谷胱甘肽的结合物的机制和 通过新颖的 肝内途径。 最近从我们的 实验室，被认为在肝脏中起着重要作用 电泳和有毒金属（例如汞）的排毒， 胃酸酸生物合成，胆汁形成和谷胱甘肽 稳态。 具体目的是：（1）定义肝内γ- 硫酸形成中的谷氨酸循环。与当前相反 我们最近的研究表明，苏联生物合成的模型 这种排毒途径可以完全发生在肝脏内。 而且，与当前的思维相反，代表性的苏联 酸（S.2,4-二苯基-N-乙酰半胱氨酸或DNP-NAC）被排泄 孤立的大鼠肝脏几乎只有胆汁，而不是 正弦循环。 为了最好的这个模型，并确定 涉及的机制，我们计划：A。-检查正弦的摄取和 在结构上不同的半胱氨酸S-偶联物和 体内和灌注肝脏中的胃酸酸（乙基 碘化物和1-氯-2,4-二硝基苯作为型号异种生物学）。 B.使用 孤立的口腔和基底外侧膜囊泡，定义 肝运输的驱动力和底物特异性 胃酸和C。测试正弦γ-谷氨酰基的作用 GSH和谷胱甘肽S-偶联物的转移酶III肝清除率， 和铜氨酸生物合成。 （2）表达新颖的管道 Xenopus中的非依赖ATP的高亲和力谷胱甘肽S偶联载体 使用从大鼠肝脏分离的poly（a）+ RNA的Laevis卵母细胞， 表征卵母细胞系统中转运蛋白的性质。 将卵母细胞作为运输的功能分析，使用表达克隆 识别转运蛋白的cDNA的策略。 克隆不仅会 在此谷胱甘肽S-偶联物上产生重要的结构信息 转运蛋白，但将允许生产分子探针 定义其在肝脏和整个生物体中的异生物代谢中的作用。 预计这些研究最终将提供信息 对预防和治疗有毒肝细胞损伤至关重要 它由反应性亲电诱导的肝外并发症 化合物。