EFFECT OF AGING ON INTERORGAN GLUTATHIONE HOMEOSTASIS

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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.