Energy-linked cardiac transport system for glutathione disulfide

The relationship between the rate of glutathione disulfide (GSSG) export and the energy state was studied in isolated perfused rat heart. The intracellular GSSG level was maintained at saturation for transport (7.5 nmol GSSG X min-1 X g heart-1) by continuous perfusion with 20 microM t-butyl hydroperoxide. GSSG release was substantially restricted upon the addition of inhibitors of mitochondrial respiration such as KCN, antimycin A or rotenone. In contrast, no effect was observed on GSSG release during potassium-induced cardiac arrest, although changes in oxygen consumption and coronary flow were similar to those observed with KCN. The dependence of the GSSG transport rate on the cytosolic free ATP/ADP ratio reveals that GSSG transport is half-maximal at (ATP/ADP)free approximately equal to 10. The capacity of GSSG transport was unchanged by infusion of epinephrine, norepinephrine or dibutyryl cyclic AMP.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

Inhibition of biliary taurocholate excretion during menadione metabolism in perfused rat liver

In perfused rat liver menadione elicits substantial oxidation in both the NADPH and GSH redox systems. Biliary excretion of GSSG is increased several-fold. Menadione derivatives appear in the bile predominantly as the menadione-S-glutathione conjugate, thiodione (60%), or as conjugates derived therefrom (17%). About 10% appear as menadione glucuronides. The excretion of taurocholate into bile is strongly inhibited upon menadione infusion. The inhibition of taurocholate excretion is small in livers with a low content of Se-GSH-peroxidase and in glutathione-depleted livers. In these livers intracellular GSSG and biliary GSSG release remain at low values, although menadione still imposes oxidative stress as indicated by an oxidation of intracellular NADPH. Under anoxic conditions menadione has little influence on both the NADPH and GSH redox systems and also on biliary taurocholate excretion. The amount of thiodione released into bile is similar to that found under normoxia, whereas the amount of glucuronidated products almost doubled. We conclude (a) that intracellular formation of GSSG by menadione occurs via the generation of hydrogen peroxide; (b) that the inhibition of biliary taurocholate excretion by menadione is related to the increased formation of glutathione disulfide; and (c) that menadione derivatives show little, if any, contribution to the inhibition of taurocholate excretion.     

Rat lung glutathione release: response to oxidative stress and selenium deficiency

We performed experiments to characterize the glutathione-dependent metabolism occurring during tert-butyl hydroperoxide infusion in isolated perfused rat lungs and to examine the effect of selenium deficiency on this metabolism. Selenium deficiency resulted in decreased lung glutathione peroxidase activity but normal glutathione reductase activity and glutathione content. Infusion of the hydroperoxide into control lungs caused a proportional increase in tissue glutathione disulfide (GSSG) concentration and release of GSSG into the perfusate up to an infusion rate of 250 nmol of tert-butyl hydroperoxide X min-1 X 100 g body wt-1. Infusion rates greater than this resulted in continued rise of tissue GSSG concentrations but GSSG release into the perfusate plateaued. Infusion of tert-butyl hydroperoxide into selenium-deficient rat lungs resulted in much lower concentrations of tissue GSSG and GSSG release into the perfusate; however, release in the selenium-deficient rat lung was also found to be saturable at infusion rates of 450 nmol of tert-butyl hydroperoxide X min-1 X 100 g of body wt-1. Selenium deficiency in the rat decreases the rate of reduction of infused tert-butyl hydroperoxide by glutathione and may predispose the lung to free radical damage.     

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.     

A comparison of protein S-thiolation (protein mixed-disulfide formation) in heart cells treated with t-butyl hydroperoxide or diamide

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide, and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30-45 min. Cells stopped beating 25 min after the addition of t-butyl hydroperoxide, and beating did not resume. t-Butyl hydroperoxide caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42 and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment. S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by t-butyl hydroperoxide or diamide, and that the specificity of the response depends on the agent used.     

Effects of t-butyl hydroperoxide on NADPH, glutathione, and the respiratory burst of rat alveolar macrophages

The effects of t-butyl hydroperoxide on glutathione and NADPH and the respiratory burst (an NADPH-dependent function) in rat alveolar macrophages was investigated. Alveolar macrophages were exposed for 15 min to t-butyl hydroperoxide in the presence or absence of added glucose. Cells were then assayed for concanavalin A-stimulated O2 production or for NADPH, NADP, reduced glutathione, glutathione disulfide, glutathione released into the medium and glutathione mixed disulfides. Exposure of rat alveolar macrophages to 1 X 10(-5) M t-butyl hydroperoxide causes a loss of concanavalin A-stimulated superoxide production (the respiratory burst) that can be prevented or reversed by added glucose. Cells incubated without glucose had a higher oxidation state of the NADPH/NADP couple than cells incubated with glucose. With t-butyl hydroperoxide, NADP rose to almost 100% of the NADP + NADPH pool; however, addition of glucose prevented this alteration of the NADPH oxidation state. Cells exposed to 1 X 10(-5) M t-butyl hydroperoxide in the absence of glucose showed a significant increase in the percentage GSSG in the GSH + GSSG pool and increased glutathione mixed disulfides. These changes in glutathione distribution could also be prevented or reversed by glucose. With 1 X 10(-4) M t-butyl hydroperoxide, changes in glutathione oxidation were not prevented by glucose and cells were irreversibly damaged. We conclude that drastic alteration of the NADPH/NADP ratio does not itself reflect toxicity and that significant alteration of glutathione distribution can also be tolerated; however, when oxidative stress exceeds the ability of glucose to prevent alterations in oxidation state, irreversible damage to cell function and structure may occur.     

Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.     

Effect of bile salts on the biliary excretion of glycodihydrofusidate in the rat

The biliary elimination of glycodihydrofusidate (GDHF), a structural analogue of bile salts, was studied in bile fistula rats. GDHF was excreted in bile with a maximal excretory rate (Tm = 0.80 mumol min-1 kg-1) which is much lower than bile salts Tm. The effects of dehydrocholate and taurocholate on GDHF biliary secretion suggest a stimulatory effect of bile salts on canalicular excretion of the drug. (a) When a bolus intravenous injection of 3 mumol of GDHF was followed after 2 min by a continuous dehydrocholate perfusion (10 mumol min-1 kg-1), biliary excretion of GDHF was increased in comparison with control rats. (b) Upon attaining the biliary Tm by continuous perfusion of GDHF at a rate of 1.35 mumol min-1 kg-1, infusion with either taurocholate or dehydrocholate increased its Tm to a similar degree. These results are similar to those previously obtained with the effects of bile salt infusions on the Tm of bromosulfophthalein. They suggest therefore that hepatic transport of GDHF and bile salts occurs by routes which are distinct for canalicular transport in spite of the striking structural similarities between GDHF and bile salts.     

Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress

t-Butyl hydroperoxide was utilized to alter the thiol homeostasis in rat brain mitochondria. Following exposure to t-butyl hydroperoxide (50-500 microM), intramitochondrial GSH content decreased rapidly and irreversibly with a major portion of the depleted GSH being accounted for as protein-SS-Glutathione mixed disulfide. Formation of GSSG was not observed nor was efflux of GSSG or GSH from the mitochondria detected in the incubation medium. The loss of intramitochondrial GSH was accompanied by loss of protein thiols. Unlike liver mitochondria, which can reverse t-butyl hydroperoxide induced formation of GSSG, addition of 50 microM t-butyl hydroperoxide resulted in irreversible loss; indicating greater susceptibility of brain mitochondria to oxidative stress than liver mitochondria.     

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl)glutathione and N-ethylmaleimide showed intermediate inhibitory effects.     

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.     

Oxidative stress stimulates the synthesis of the eosinophil chemoattractant 5-oxo-6,8,11,14-eicosatetraenoic acid by inflammatory cells

5-Oxo-ETE (5-oxo-6,8,11,14-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that acts through a selective G-protein coupled receptor. It is formed by oxidation of the 5-lipoxygenase product 5-HETE (5S-hydroxy-6,8,11,14-eicosatetraenoic acid) by 5-hydroxyeicosanoid dehydrogenase (5-HEDH). Although leukocytes and platelets display high microsomal 5-HEDH activity, unstimulated intact cells do not convert 5-HETE to appreciable amounts of 5-oxo-ETE. To attempt to resolve this dilemma we explored the possibility that 5-oxo-ETE synthesis could be enhanced by oxidative stress. We found that hydrogen peroxide and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U937 monocytic cells. This was dependent on the GSH redox cycle, as it was blocked by depletion of GSH or inhibition of glutathione reductase and mimicked by oxidation of GSH to GSSG by diamide. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, as its effect was reversed by the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-butyl hydroperoxide. Oxidative stress could act by depleting NADPH, resulting in the formation NADP+, the cofactor for 5-HEDH. This is opposed by the pentose phosphate pathway, which converts NADP+ back to NADPH. Oxidative stress could be an important mechanism for stimulating 5-oxo-ETE production in inflammation, promoting further infiltration of granulocytes into inflammatory sites.     

Biliary excretion of inorganic electrolytes: its role in hepatic bile formation

To define the role of inorganic electrolyte secretion in hepatic bile formation, the effects of secretin, glucagon, and differently structured bile acids on bile flow and composition were studied in the dog, guinea pig, and rat. In the dog and guinea pig, secretin (2.5-10 clinical units X kg-1 X 30 min-1) increased bile flow and bicarbonate concentration in bile, a finding consistent with the hypothesis that the hormone stimulates a bicarbonate-dependent secretion possibly at the level of the bile ductule-duct. In the rat, secretin (5-15 CU X kg-1 X 30 min-1) failed to increase bile secretion. Glucagon (1.25-300 micrograms X kg-1 X 30 min-1) increased bile flow in all the three species, and produced no changes in biliary bicarbonate concentrations in the dog and rat. In the guinea pig, however, glucagon choleresis was associated with an increase in bicarbonate concentration in bile, similar to that observed with secretin. The choleretic activities of various bile acids (taurocholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, and ursodeoxycholic acid, infused at 30-360 mumol X kg-1 X 30 min-1) were similar in the rat (6.9-7.2 microL/mumol), but different in the guinea pig (11-31 microL/mumol). In the latter species, the more hydrophobic the bile acid, the greater was its choleretic activity. In all instances, bile acid choleresis was associated with a decline in the biliary concentrations of chloride, but with no major change in bicarbonate levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment of canalicular membrane with cholesterol and sphingomyelin prevents bile salt-induced hepatic damage

These studies were undertaken to characterize the role of plasma membrane cholesterol in canalicular secretory functions and hepatocyte integrity against intravenous taurocholate administration. Cholesterol and sphingomyelin concentrations and cholesterol/phospholipid ratios were significantly increased in canalicular membranes of diosgenin-fed rats, suggesting a more resistant structure against solubilization by taurocholate. During taurocholate infusion, control rats had significantly decreased bile flow, whereas diosgenin-fed animals maintained bile flow. Maximal cholesterol output increased by 176% in diosgenin-fed rats, suggesting an increased precursor pool of biliary cholesterol in these animals. Maximal phospholipid output only increased by 43% in diosgenin-fed rats, whereas bile salt output remained at control levels. The kinetics of glutamic oxalacetic transaminase, lactic dehydrogenase, and alkaline phosphatase activities in bile showed a significantly faster release in control than in diosgenin-fed rats. After 30 min of intravenous taurocholate infusion, necrotic hepatocytes were significantly increased in control animals. Preservation of bile secretory functions and hepatocellular cytoprotection by diosgenin against the intravenous infusion of toxic doses of taurocholate was associated with an increased concentration of cholesterol and sphingomyelin in the canalicular membrane. The increase of biliary cholesterol output induced by diosgenin was correlated to the enhanced concentration of cholesterol in the canalicular membrane.     

Influence of selenium deficiency on glutathione disulfide metabolism in isolated perfused rat heart

Selenium deficiency causes a fall in rat cardiac glutathione peroxidase activity. As a consequence, isolated perfused selenium-deficient heart does not release increased amounts of GSSG when hydroperoxide is infused. However, the total amount of glutathione measured as intracellular GSH, intracellular GSSG and GSSG released from the heart when hydroperoxide is infused does not equal the total glutathione measured in these pools in untreated hearts (Xia, Y., Hill, K.E. and Burk, R.F. (1985) J. Nutr. 115, 733-742). GSSG can react with protein sulfhydryl groups to form glutathione-protein mixed disulfides (PrS-SG). PrS-SG were measured in perfused selenium-deficient and control hearts infused with t-butylhydroperoxide and were found to account for the previously unmeasured glutathione. The ability of the selenium-deficient heart to transport GSSG was also examined. GSSG was produced non-enzymatically by infusing diamide. The diamide-treated selenium-deficient heart formed GSSG and released it at the same rate as similarly-treated control heart. Thus although selenium deficiency decreases GSSG formation by glutathione peroxidase, it does not affect cardiac GSSG transport.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Glutathione oxidation and activation of pentose phosphate cycle during hydroperoxide metabolism. A comparison of livers from fed and fasted rats

Perfusion of livers from fed and fasted rats with 0.07--0.1 mM t-butyl hydroperoxide for 15 min decreased the levels of reduced glutathione (GSH) by 1.5 mumol/g liver in both nutritional states. Glutathione disulfide (GSSG) was increased by 70 and 140 nmol/g liver and glutathione mixed disulfides enhanced by 45 and 150 nmol/g liver in livers from fed and fasted animals, respectively. The ratio of GSH/GSSG was decreased from 243 to 58 in fed animals, and from 122 to 8 in fasted animals. The increase of GSSG and the mixed disulfides was nearly parallel until an apparently critical low GSH content of 1.5 mumol/g was reached. Only in livers from fasted rats 14CO2-production from [1-14C]glucose was stimulated upon t-butyl hydroperoxide infusion at the employed rates. Flux of glucose through pentose phosphate cycle rose from 8 to 12% of glucose utilization via glycolysis, whereas in livers from fed animals this portion remained unchanged at 8% Dithio-erythritol reversed pentose phosphate cycle activity as well as GSSG and protein-bound glutathione contents to the original levels. In livers from fasted rats the activity of glucose-6-phosphate dehydrogenase was increased by 34% by t-butyl hydroperoxide infusion.     

Factors influencing the inhibition of biliary glutathione efflux induced by biliary obstruction

The aim of this study was to investigate mechanisms responsible for the inhibition of biliary glutathione efflux in rats with secondary biliary cirrhosis. Rats were studied after bile duct obstruction for 28 days. The biliary secretion of reduced glutathione (GSH), oxidised glutathione (GSSG) and cysteine were completely inhibited in biliary obstructed rats. Hepatic gamma glutamyltranspeptidase (gamma-GT) activity increased significantly, but following its inhibition by acivicin administration GSH, GSSG and cysteine were still absent in bile. Biliary obstruction resulted in a significant increase of the permeability of the paracellular pathway, as shown by the higher bile/plasma ratio and hepatic clearance of [14C]sucrose. GSH and GSSG were, however, significantly lower in the carotid artery and hepatic vein of obstructed animals and the arteriovenous difference across the liver was reduced. The concentration of GSH was significantly reduced and that of GSSG increased in the liver of obstructed rats. Biliary obstruction induced an increase in the hepatic concentration of cysteine and an inhibition of both gamma glutamylcysteine synthetase and methionine adenosyl transferase activities. Dichlorofluorescein (DCF) and the GSSG/GSH ratio and thiobarbituric acid reactive substances (TBARS) concentration, markers of reactive oxygen species production and lipid peroxidation, respectively, were significantly increased. Our data indicate that increased degradation or blood reflux of glutathione do not participate in the disruption of its secretion into bile and support the view that impairment of glutathione synthesis and oxidative stress could contribute to the decline in biliary glutathione output.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.