EFFECT OF AGING ON INTERORGAN GLUTATHIONE HOMEOSTASIS

衰老对器官间谷胱甘肽稳态的影响

• 批准号: 2413307
• 负责人: MURAD OOKHTENS
• 金额: $ 25.29万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2413307
• 关键词: aging; aminoacid metabolism; aminoacid transport; biological transport; blood proteins; cysteine; glutamate transporter; glutamyltransferase; glutathione; high performance liquid chromatography; homeostasis; hydrolysis; laboratory rat; liver cells; liver metabolism; messenger RNA; methionine; mitochondria; northern blottings; radionuclides; scintillation counter; sulfides; sulfur aminoacid; thiols; western blottings

The overall objectives of this proposal are to: (a) define the key parameters of intrahepatic and interorgan homeostasis of reduced glutathione (GSH) and thiol-disulfides; (b) define age-specific changes in them over life-span of Harlan Sprague-Dawley rats [3-5 wks (immature, I), 3-6 mos (mature, M), 1 yr (old, O) and 2 yrs (senescent, S). Specific aims are: 1. Determine age-related changes in kinetics of GSH transport in basolateral (bLPM) and canalicular (cLPM) membrane vesicles. With perfused livers and isolated hepatocytes we have shown a declining sinusoidal GSH efflux (to M), due solely to a declining Vmax and no change in km. Biliary GSH efflux rose with age. We will study these changes in inside-out bLPM and cLPM vesicles to determine if they are truly membrane-specific events. 2.Determine the abundance of mRNA for sinusoidal and canalicular GSH transporters in different age groups. The putative sinusoidal and canalicular GSH transporter cDNAs have been cloned. We will study age- related changes in abundance of transcripts in livers with Northern blot analysis. As polyclonal antibodies become available, we will determine changes of the gene products in quantitative Western blots. 3.Identify-quantify key age-related changes in kinetics of turnover of plasma and extra-plasma thiol-disulfides. We will inject 35S-labeled GSH, cysteine (CYSH) or cystine (CYSS) i.v. and will follow the movement of label through unbound and bound plasma thiol-disulfides: GSH, CYSH, CYSS, GSSG and cysteine-glutathione disulfide. We will measure appearance and form(s) of label in liver, kidney, lung, intestine, muscle, brain, skin and red blood cells. Tracer-kinetic data will be analyzed by multicompartmental methods to determine age-related changes in plasma thiol-disulfide turnover. 4. Determine quantitative role of plasma GSH and its breakdown in regulation of kinetics of its turnover in I and M rats. Experiments as in 3, but under quasi-steady-states of plasma GSH after: (1) Nephrectomy (ligation of kidneys) +/- acivicin; (2) Injections of acetaminophen, specifically to lower plasma GSH concentration of I rats to that of M; (3) Infusions and/or injections of GSH and/or CYSH, specifically to raise plasma GSH and/or CYSH concentrations in M rats to those of I. 5.Determine effect of extracellular thiols/disulfides on sinusoidal GSH efflux from livers of rats of different age. Experiments as in 3, but after quasi-steady-states of plasma pools after perturbations of plasma thiol-disulfide status (ratio) with: (1) Infusions of CYSS and/or glutamate, to increase plasma CYSS. (2) Perfusions of isolated livers and incubations of freshly-isolated hepatocytes with dithiothreitol (DTT), N- acetylcysteine (NAC) and dimercaprol, in addition to GSH, CYSH and CYSS. 6. Study age-related changes in cytosolic synthesis and transport of GSH to mitochondria in hepatocytes. We will examine for changes in synthetic capacity of GSH from CYSH, using cytosol and freshly-isolated hepatocytes. Mitochondrial GSH pool sizes and rates of transport will be studied by tracer-kinetic methods.

本建议的总体目标是：（a）确定关键 肝内和器官间稳态参数减少 谷胱甘肽（GSH）和巯基二硫化物;（B）定义了 在哈兰Sprague-Dawley大鼠的寿命期间[3-5周（未成熟，I）， 3-6 mos（成熟，M），1岁（老年，O）和2岁（衰老，S）。具体目标 为： 1.确定年龄相关的GSH转运动力学变化， 基底外侧（bLPM）和小管（cLPM）膜囊泡。随着灌注 肝脏和离体肝细胞中，我们发现， 流出量（至M），仅由于Vmax下降且km无变化。胆道 GSH流出量随年龄增长而增加。我们将在由内而外的bLPM中研究这些变化 和cLPM囊泡，以确定它们是否是真正的膜特异性事件。 2.测定窦状隙和小管GSH的mRNA丰度 不同年龄段的交通工具。假定的正弦和 已经克隆了小管GSH转运蛋白cDNA。我们会研究年龄- 用北方印迹分析肝脏转录本丰度的相关变化 分析. 随着多克隆抗体的出现，我们将确定 定量Western印迹中基因产物的变化。 3.确定-量化与年龄有关的关键人员更替动力学变化， 血浆和血浆外的硫醇二硫化物。我们会注射35 S标记的谷胱甘肽 半胱氨酸（CYSH）或胱氨酸（CYSS）静脉注射，并将遵循的运动， 通过未结合和结合血浆巯基-二硫化物标记：GSH、CYSH、CYSS GSSG和半胱氨酸-谷胱甘肽二硫化物。我们将测量外观， 肝、肾、肺、肠、肌肉、脑、皮肤中的标签形式 和红细胞。 示踪剂动力学数据将通过 测定血浆中年龄相关变化的多房室方法 巯基-二硫键转换 4.确定血浆GSH的定量作用及其分解， 调节I和M大鼠的周转动力学。实验如 3，但在血浆GSH的准稳态后：（1）肾切除术后 （肾结扎）+/- acivicin;（2）注射对乙酰氨基酚， 特异性地降低I大鼠的血浆GSH浓度;（3） GSH和/或CYSH的输注和/或注射，特别是为了提高 血浆GSH和/或CYSH浓度与I. 5.测定细胞外硫醇/二硫化物对正弦GSH的影响 不同年龄大鼠肝脏的外排。实验3，但 等离子体扰动后等离子体池的准稳态后 巯基-二硫化物状态（比率）：（1）输注CYSS和/或 谷氨酸，以增加血浆CYSS。(2)离体肝脏灌注， 新鲜分离的肝细胞与二硫苏糖醇（DTT）、N- 乙酰半胱氨酸（NAC）和二巯基丙醇，以及GSH、CYSH和CYSS。 6.研究细胞内GSH合成和转运的年龄相关变化 肝细胞中的线粒体。我们将检查合成的 使用细胞质和新鲜分离的肝细胞从CYSH中提取GSH的能力。 线粒体GSH库的大小和转运速率将通过 示踪动力学方法