EFFECT OF AGING ON INTERORGAN GLUTATHIONE HOMEOSTASIS

这项提案的总体目标是：(A)确定关键 肝内和脏器间动态平衡参数的降低 谷胱甘肽(GSH)和硫醇二硫化物；(B)定义年龄相关的变化 在Harlan Spraogue-Dawley大鼠的一生中[3-5周(未成熟，I)， 3-6个月(成熟，男性)，1岁(老年，O)，2岁(衰老，S)。具体目标 包括： 1.测定GSH转运动力学随年龄的变化 基侧(BLPM)和小管(CLPM)膜小泡。灌满了水 肝脏和分离的肝细胞显示谷胱甘肽呈正弦下降 流出(到M)，完全是由于Vmax下降，Km没有变化。胆道 GSH外流随年龄增长而增加。我们将研究由内而外的bLPM中的这些变化 和cLPM囊泡，以确定它们是否是真正的膜特异性事件。 2.测定肝窦和管状GSH的mRNA丰度 不同年龄段的运输者。推定的正弦和 小管型谷胱甘肽转运蛋白cDNA已被克隆。我们将研究年龄- Northern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 定量免疫印迹中基因产物的变化。 3.确定--量化与年龄有关的关键的周转动力学变化 血浆和超血浆硫醇-二硫化物。我们将注射35S标记的GSH， 半胱氨酸(Cysh)或半胱氨酸(Cyss)静脉注射。并将跟随着 通过未结合和结合的血浆硫醇二硫化物标记：GSH，CySH，Cyss， GSSG和半胱氨酸-谷胱甘肽二硫化物。我们将测量外表和 肝、肾、肺、肠、肌、脑、皮标签形式(S) 和红血球。示踪剂动力学数据将由 多室法测定血浆中与年龄相关的变化 硫醇-二硫化物周转。 4.测定血浆GSH的定量作用及其在体内的分解 其在I、M大鼠体内周转动力学的调节。如中的实验 (3)肾切除后血浆GSH处于准稳态状态：(1) (2)注射对乙酰氨基酚， 使I组大鼠血浆GSH浓度降至M组；(3) GSH和/或CySH的输注和/或注射，特别是为了提高 M组大鼠血浆GSH和/或CySH浓度高于I组。 5.测定胞外硫醇/二硫化物对正弦GSH的影响 不同日龄大鼠的肝脏外排。3中的实验，但是 等离子体扰动后等离子体池的准稳态后 硫醇-二硫化物状态(比率)：(1)输注硫代半胱氨酸和/或 谷氨酸，以增加血浆胱氨酸合成酶。(2)离体肝灌注术和 二硫苏糖醇(DTT)、N-二硫苏糖醇与新鲜分离的肝细胞孵育 乙酰半胱氨酸(NAC)和二硫代丙醇，以及谷胱甘肽、半胱氨酸和半胱氨酸。 6.研究GSH胞浆合成和转运的增龄性变化 到肝细胞中的线粒体。我们将检查合成材料中的变化 用胞浆和新鲜分离的肝细胞从CySH中提取GSH的能力。 线粒体GSH池的大小和转运速率将通过 示踪动力学方法。

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.