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PubMed2004

Glutathione regulates telomerase activity in 3T3 fibroblasts.

Changes in telomerase activity have been associated either with cancer, when activity is increased, or with cell cycle arrest when it is decreased. We report that glutathione, a physiological antioxidant present at high intracellular concentrations, regulates telomerase activity in cells in culture. Telomerase activity increases in 3T3 fibroblasts before exponential cell growth. The peak of telomerase activity takes place 24 h after plating and coincides with the maximum levels of glutathione in the cells. When cells are treated with buthionine sulfoximine, which decreases glutathione levels in cells, telomerase activity decreases by 60%, and cell growth is delayed. Glutathione depletion inhibits expression of E2F4 and Id2, which regulate the cell cycle. When glutathione levels are restored after incubation with glutathione monoethylester, telomerase activity and the cell cycle-related proteins return to control values. To discover the effect of glutathione redox status on the telomerase multicomplex structure, we incubated protein extracts from fibroblasts with different glutathione redox buffers. Telomerase activity is maximal under reduced conditions i.e. when the reduced/oxidized glutathione ratio is high. Consequently glutathione concentration parallels telomerase activity. These results underscore the main role of glutathione in the control of telomerase activity and of the cell cycle.

3T3 Cells↗

PubMed1986

A comparative study of the regulation of cytochrome P-450 and glutathione transferase gene expression in rat liver.

A cDNA clone for the Ya subunit of glutathione transferase from rat liver was constructed in E. coli. The clone hybridized to Ya and Yc subunit messenger RNAs. On the basis of experiments involving cell-free translation and hybridization to the cloned probe, it was shown that prototype inducers of cytochrome P-450 such as phenobarbitone and 3-methylcholanthrene as well as inhibitors such as CoCl2 and 3-amino-1,2,4-triazole enhanced the glutathione transferase (Ya+Yc) messenger RNA contents in rat liver. A comparative study with the induction of cytochrome P-450 (b+e) by phenobarbitone revealed that the drug manifested a striking increase in the nuclear pre-messenger RNAs for the cytochrome at 12 hr, but did not significantly affect the same in the case of glutathione transferase (Ya+Yc). 3-Amino-1,2,4-triazole and CoCl2 blocked the phenobarbitone mediated increase in cytochrome P-450 (b+e) nuclear pre-messenger RNAs. These compounds did not significantly affect the glutathione transferase (Ya+Yc) nuclear pre-messenger RNA levels. The polysomal, poly (A)- containing messenger RNAs for cytochrome P-450 (b+e) increased by 12-15 fold after phenobarbitone administration, reached a maximum around 16 hr and then decreased sharply. In comparison, the increase in the case a glutathione transferase (Ya+Yc) messenger RNAs was sluggish and steady and a value of 3-4 fold was reached around 24 hr. Run-off transcription rates for cytochrome P-450 (b+e) increased by nearly 15 fold in 4 hr after phenobarbitone administration, whereas the increase for glutathione transferase (Ya+Yc) was only 2.0 fold. At 12 hr after the drug administration, the glutathione transferase (Ya+Yc) transcription rates were near normal. Administration of 3-amino-1,2,4-triazole and CoCl2 blocked the phenobarbitone-mediated increase in the transcription of cytochrome P-450 (b+e) messenger RNAs. These compounds at best had only marginal effects on the transcription of glutathione transferase (Ya+Yc) messenger RNAs. The half-life of cytochrome P-450 (b+e) messenger RNA was estimated to be 3-4 hr, whereas that for glutathione transferase (Ya+Yc) was found to be 8-9 hr. Administration of phenobarbitone enhanced the half-life of glutathione transferase (Ya+Yc) messenger RNA by nearly two fold. It is suggested that while transcription activation may play a primary role in the induction of cytochrome P-450 (b+e), the induction of glutathione transferase (Ya+Yc) may essentially involve stabilization of the messenger RNAs.

Amitrole↗

PubMed1987

Glutathione, a first line of defense against cadmium toxicity.

Experimental modulation of cellular glutathione levels has been used to explore the role of glutathione in cadmium toxicity. Mice treated with buthionine sulfoximine [an effective irreversible inhibitor of gamma-glutamylcysteine synthetase (EC 6.3.2.2) that decreases cellular levels of glutathione markedly] were sensitized to the toxic effects of CdCl2. Mice pretreated with a sublethal dose of Cd2+ to induce metallothionein synthesis were not sensitized to Cd2+ by buthionine sulfoximine. Mice sensitized to Cd2+ by buthionine sulfoximine were protected against a lethal dose of Cd2+ by glutathione mono isopropyl ester (L-gamma-glutamyl-L-cysteinylglycylisopropyl ester), but not by glutathione. These results are in accord with studies that showed that glutathione mono esters (in contrast to glutathione) are efficiently transported into cells and converted intracellularly to glutathione. The findings indicate that intracellular glutathione functions in protection against Cd2+ toxicity, and that this tripeptide provides a first line of defense against Cd2+ before induction of metallothionein synthesis occurs. The experimental approach used here in which cellular levels of glutathione are decreased or increased seems applicable to investigation of other types of metal toxicity and of other glutathione-dependent biological phenomena.

1-Propanol↗

PubMed1990

Glutathione turnover in cultured astrocytes: studies with [15N]glutamate.

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.

Amino Acids↗

PubMed2004

Contribution of glutathione peroxidase to the virulence of Streptococcus pyogenes.

Glutathione peroxidases are widespread among eukaryotic organisms and function as a major defense against hydrogen peroxide and organic peroxides. However, glutathione peroxidases are not well studied among prokaryotic organisms and have not previously been shown to promote bacterial virulence. Recently, a gene with homology to glutathione peroxidase was shown to contribute to the antioxidant defenses of Streptococcus pyogenes (group A streptococcus). Since this bacterium causes numerous suppurative diseases that require it to thrive in highly inflamed tissue, it was of interest to determine if glutathione peroxidase is important for virulence. In this study, we report that GpoA glutathione peroxidase is the major glutathione peroxidase in S. pyogenes and is essential for S. pyogenes pathogenesis in several murine models that mimic different aspects of streptococcal suppurative disease. In contrast, glutathione peroxidase is not essential for virulence in a zebrafish model of streptococcal myositis, a disease characterized by the absence of an inflammatory cell infiltrate. Taken together, these data suggest that S. pyogenes requires glutathione peroxidase to adapt to oxidative stress that accompanies an inflammatory response, and the data provide the first demonstration of a role for glutathione peroxidase in bacterial virulence. The fact that genes encoding putative glutathione peroxidases are found in the genomes of many pathogenic bacterial species suggests that glutathione peroxidase may have a general role in bacterial pathogenesis.

Animals↗

PubMed1988

Glutathione protects against exogenous oxidant injury to rabbit renal proximal tubules.

Glutathione, comprising a major portion of cellular nonprotein thiols, plays a central role in a diverse group of cell metabolic functions. Glutathione and related enzyme systems have been shown to protect against both toxin and oxidant-induced injury in several organ systems. The role of glutathione in protecting renal epithelia against oxidant stress has not been investigated previously. We report here the response of enriched, isolated rabbit renal proximal tubule segments to oxidant stress induced by tert-butyl hydroperoxide. In addition, the effects of glutathione depletion by various biochemical means and of exogenous glutathione supplementation on the response of tubule segments to tert-butyl hydroperoxide exposure are described. Depletion of cell glutathione by several distinct methods potentiates oxidant-induced injury. Augmentation of cellular glutathione affords significant protection against exogenous oxidant stress. The protective effect of glutathione may reside in its ability, in conjunction with glutathione peroxidase, to arrest the propagation of lipid peroxidation and, therefore, to minimize alterations in plasma membrane permeability. The results of this study do not exclude the possibility that glutathione prevents tert-butyl hydroperoxide induced oxidation of critical sulfhydryl groups of catalytic or structural proteins associated with control of cell cation homeostasis. These results confirm the important role of glutathione in protecting renal tubular epithelia against oxidant stress.

Adenosine Triphosphate↗

PubMed1998

Glutathione restoration as indicator for cellular metabolism of astroglial cells.

The restoration of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used to indicate metabolic properties of astroglial cells. At a culture age of 14-21 days these cultures contain an average total glutathione content of 32.8 +/- 3.2 nmol/mg protein and a cytosolic volume, estimated with the 3-O-methylglucose method, of 4.1 +/- 0.1 microl/mg protein. Therefore, cells of astroglial cultures have a cytosolic glutathione concentration of about 8 mM. In order to investigate glutathione synthesis in astroglial cultures the cellular glutathione content was reduced by starvation in a minimal medium lacking glucose and amino acids. Resynthesis of glutathione depended on the presence of glucose and the three constituent amino acids glutamate, cysteine and glycine. Absence of glucose reduced the amount of net glutathione restoration found after 4 h of incubation by about 50%. Of known substrates of astroglial energy metabolism, mannose could fully and fructose, lactate, pyruvate or sorbitol could partially replace glucose during glutathione restoration. In contrast to these compounds, galactose, 5-thioglucose and 2-deoxyglucose failed to substitute for glucose during glutathione restoration. Astroglial cells are able to use as precursors for the three constituent amino acids of glutathione a variety of amino acids and dipeptides. The results presented demonstrate that glutathione restoration can be used as an indicator for amino acid as well as energy metabolism of astroglial cells.

Amino Acids↗

PubMed1985

Sinusoidal efflux of glutathione in the perfused rat liver. Evidence for a carrier-mediated process.

Turnover of hepatic glutathione in vivo in the rat is almost entirely accounted for by cellular efflux, of which 80-90% is sinusoidal. Thus, sinusoidal efflux play a major quantitative role in homeostasis of hepatic glutathione. Som preliminary observations from our laboratory (1983. J. Pharmacol. Exp. Ther. 224:141-147.) and circumstantial evidence in the literature seemed to imply that the raising of the hepatic glutathione concentration above normal was not accompanied by a rise in the rate of sinusoidal efflux. Based on these observations, we hypothesized that the sinusoidal efflux was probably a saturable process and that at normal levels of hepatic glutathione the efflux behaved as a zero-order process (near-saturation). We tested our hypothesis by the use of isolated rat livers perfused in situ, single pass, with hemoglobin-free, oxygenated buffer medium at pH 7.4 and 37 degrees C. Preliminary experiments established a range of perfusion rates (3-4 ml/min per g) for adequacy of oxygenation, lack of cell injury, and minimization of variability contributed by perfusion rates. Hepatic glutathione was lowered to below normal by a 48-h fast, diethylmaleate (0.1-1.0 ml/kg i.p.), and buthionine sulfoximine (8 mmol/kg i.p.), and raised to above normal by 3-methylcholanthrene (20 mg/kg x 3 d i.p.) and cobalt chloride (0.05-0.27 g/kg-1 subcutaneously). Steady state sinusoidal efflux from each liver was measured over a 1-h perfusion, during which the coefficient of variation of glutathione in perfusates stayed within 10%. Hepatic glutathione efflux as a function of hepatic concentration was characterized by saturable kinetics with sigmoidal (non-hyperbolic) features. The data were fitted best with the Hill model and the following parameter values were estimated: Vmax = 20 nmol/min per g, Km = 3.2 mumol/g, and n = 3 binding/transport sites. The efflux could be inhibited reversibly by sulfobromophthalein-glutathione conjugate but was not affected by the addition of glutathione to the perfusion medium. The results support our hypothesis that sinusoidal efflux of glutathione is near saturation (approximately equal to 80% of Vmax) at normal (fed and fasted) liver glutathione concentrations. The phenomenon of saturability coupled with the ability to inhibit the efflux leads us to propose that sinusoidal efflux from hepatocytes appears to be a carrier-mediated process. Some recent studies by others, using sinusoidal membrane-enriched vesicles, also support these conclusions.

Animals↗

PubMed2003

Plasma creatine kinase activity and glutathione after eccentric exercise.

PURPOSE: This study examined whether plasma total glutathione levels could explain the intersubject variability in the creatine kinase (CK) response to eccentric exercise. We hypothesized that the increase in plasma CK activity after eccentric exercise would be lower for individuals with low plasma total glutathione (<2.5 micromol x L-1) compared with individuals with high total glutathione (>3.8 micromol x L-1), but other indicators of muscle damage would be the same between groups. METHODS: Resting blood samples were obtained over 2 d from 60 subjects and analyzed for plasma total glutathione. Eight subjects who had total glutathione values below 2.5 micromol x L-1 (LG), and nine who had values above 3.8 micromol x L-1 (HG) performed 50 maximal eccentric actions of the elbow flexors. Maximal voluntary isometric contraction (MVC), relaxed arm angle (RANG), and blood samples for CK, myoglobin (Mb), and total glutathione were obtained pre, post (except blood samples), 24, 48, 72, 96, and 120 h after exercise. RESULTS: There was a significant group-by-time interaction in analysis of MVC, RANG, total glutathione, CK, and Mb response to exercise. Although LG showed a smaller CK response to eccentric exercise compared with HG, LG also showed a smaller increase in plasma Mb, a faster recovery of MVC and RANG, and an increase in plasma total glutathione. CONCLUSION: Subjects with low plasma total glutathione levels had a smaller plasma CK and Mb response and a faster recovery from eccentric exercise compared with subjects having high plasma total glutathione levels. We suggest that a blunted inflammatory response in subjects with low plasma glutathione may be one explanation for these findings.

Adolescent↗

PubMed1992

Protective effects of glutathione, glycine, or alanine in an in vitro model of renal anoxia.

Both glutathione and glycine provide some protection against ischemic renal injury in a variety of experimental models. However, results have been inconsistent and there may also be model heterogeneity. The effects of glutathione, glycine, and alanine in a cell culture model of renal anoxia/reoxygenation injury were tested. When primary cultures of rat proximal tubule epithelial cells were subjected to 60 min of anoxia and 30 min of reoxygenation, glutathione (2 mM) essentially eliminated lethal cell injury as determined by lactate dehydrogenase release. Glycine or alanine, on the other hand, provided only partial protection. Glutamate did not protect, although cysteine did. The glutathione synthesis inhibitor buthionine sulfoximine blocked the protective effect of exogenous glutathione, and the glutathione transport inhibitor probenecid partially blocked glutathione protection. A combination of glycine, glutamate, plus cysteine also protected against anoxia/reoxygenation injury. The studies suggest that both glutathione degradation with intracellular resynthesis and transport of intact glutathione into the cell are involved in the protection afforded by exogenous glutathione. These results are different from those obtained in other experimental models of renal ischemia, such as freshly isolated proximal tubules, because the protective effects of glutathione were not derived solely from glycine generation. These studies also suggest the need for caution in extrapolating results from one model of renal anoxic injury to another.

Alanine↗

PubMed2004

The effect of oral folic acid on glutathione, glycaemia and lipids in Type 2 diabetes.

Plasma homocysteine is an established risk factor for vascular disease and precursor of the anti-oxidant glutathione. This study was designed to investigate the relationship of changes in homocysteine (Hcy) induced by oral folate to glutathione and measures of glycaemia and lipid metabolism in Type 2 diabetes (T2DM). Twenty-seven patients (26 male, 1 female, aged 48-68 years) with T2DM and microalbuminuria were treated with folic acid 10 mg daily for 3 months. During the study, diastolic blood pressure (p=0.04), HbA1c (p=0.04), serum triglycerides (p=0.04) and serum total/HDL-cholesterol ratio (p=0.004) all increased and serum HDL-cholesterol fell (p=0.006). The increased red cell folate correlated with a reduction in microalbuminuria (p=0.001). Overall, plasma glutathione increased (p=0.016) despite reduction in its precursor Hcy (p<0.001). Change in glutathione correlated inversely with change in HbA1c (p<0.02), total cholesterol (p=0.003) and triglycerides (p<0.02) and positively with HDL-cholesterol (p=0.033). Increase in glutathione correlated with levels of vitamin B6 (p<0.05). Metformin treatment protected against the rise in blood pressure (BP) (p=0.02), independently of changes in plasma glutathione. In summary, oral folic acid supplementation in T2DM reduced plasma Hcy and increased glutathione levels. HbA1c, triglycerides and HDL-cholesterol deteriorated during the trial: their levels correlated inversely with changes in glutathione. The increase in glutathione may depend on an adequate supply of B6, as changes in glutathione correlated with vitamin B6 levels. Reduced Hcy and increased glutathione may both mediate improvement in vascular function and outcome. Some aspects of the response to folate may be different in patients on metformin.

Adult↗

PubMed2003

[Effects of dissolved oxygen and pH on Candida utilis batch fermentation of glutathione].

The effects of dissolved oxygen (DO) and pH on glutathione batch fermentation by Candida utilis WSH-02-08 in a 7 liters stirred fermentor were investigated. It was shown that DO concentration is an important factor in glutathione production. With the initial glucose concentration of 30 g/L and a 5 L/min air flow rate, and the agitation rate less than 250 r/min, the DO concentration was not sufficient to satisfy the oxygen requirement during the fermentation. With an agitation rate of more than 300 r/min, the cell growth and glutathione production were enhanced significantly, with the dry cell mass and glutathione production were 20% and 25% higher than that at 200 r/min. When C. utilis WSH 02-08 was cultivated in a batch process without pH control, cell growth and glutathione production were inhibited, likely due to a dramatic decrease in the pH. Intracellular glutathione leakages were observed when the pH was 1.5 or less. To assess the effect of pH on glutathione production, six batch processes controlled at pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 were conducted. The yield was highest at pH 5.5, when the dry cell mass and yield were 27% and 95% respectively higher than fermentation without pH control. The maximal intracellular glutathione content (2.15 %) was also achieved at the pH. To improve our understandings on the effect of pH on the batch glutathione production, a modified Logistic equation and Luedeking-Piret equation were used to simulate cell growth and glutathione production, respectively, under different pH. Based on the parameters obtained by the nonlinear estimation, kinetic analysis was performed to elucidate the effect of pH on the batch glutathione production. The process controlled at pH 5.5 was proven to be the best due to the higher value of K(I) (substrate inhibitory constant in the Logistic equation), lower value of a and higher value of beta (slope and intercept in the Luedeking-Piret equation, respectively).

Candida↗

PubMed1989

Glutathione may inhibit sodium-dependent phosphate transport by renal brush-border membrane vesicles.

Glutathione, a biological reductant, can affect certain aspects of transport function in renal cortical slices. The influence of glutathione on the rate of sodium-dependent phosphate uptake into renal brush-border membrane (BBM) vesicles was examined. Vesicles were obtained from rat renal cortex by the conventional calcium precipitation method. The amounts of GSH and oxi-glutathione were measured by chromatography. The vesicles were incubated for 15 min with glutathione (10 mM) prior to a 1-min influx of phosphate. The incubation with glutathione reduced the phosphate uptake from 1887 +/- 217 to 1496 +/- 116 pmol/mg (n = 5, p less than 0.05). A kinetic study revealed that the incubation with glutathione resulted in an increase of Km in the sodium-dependent phosphate transporter from 85 +/- 7.9 to 159 +/- 18 microM (n = 5, p less than 0.05). Glutathione was catabolized rapidly by the incubation with BBM, and a concomitant production of oxi-glutathione was recognized. Incubation with oxi-glutathione (10 mM) for 15 min, however, did not affect the phosphate uptake, the present results suggest that incubation with reduced-form glutathione decreases the affinity of the sodium-dependent phosphate transport across the renal brush-border membrane, and that catabolism of glutathione may be linked the inhibitory mechanism.

Animals↗

PubMed1987

Stereoselective glutathione conjugation and amidase-catalyzed hydrolysis of alpha-bromoisovalerylurea enantiomers in isolated rat hepatocytes.

alpha-Bromoisovalerylurea (BIU) is used as model substrate for studies on the pharmacokinetics of glutathione conjugation in vivo. Its metabolism in isolated rat hepatocytes is presently studied. A major part of the substrate was conjugated with glutathione, but also amidase-catalyzed hydrolysis occurred, resulting in the products urea and alpha-bromoisovaleric acid (BI). The amidase activity was located in the microsomal fraction of the rat liver. The product of hydrolysis, BI, also was conjugated efficiently with glutathione. In glutathione-depleted hepatocytes, no glutathione conjugates but only urea and BI were formed. A pronounced stereoselectivity in the metabolism of the BIU enantiomers was observed: (R)-BIU was conjugated with glutathione much faster than (S)-BIU. (S)-BIU was hydrolyzed substantially in the cells and the glutathione conjugate of the hydrolytic product, (S)-BI, could be detected. At high BIU concentrations (500 microM of the racemate) intracellular glutathione was seriously depleted; then, the cosubstrate availability most likely was the rate-limiting factor in the conjugation of BIU with glutathione. More urea was formed from (racemic) BIU in isolated rat hepatocytes in the present study than in the perfused liver and the intact rat in previous studies. This in vivo-in vitro difference is tentatively assigned to differences in glutathione availability in these systems. The results suggest that BI may also be a useful model substrate to study the kinetics of glutathione conjugation in vivo and in vitro.

Amidohydrolases↗

PubMed1986

alpha-Bromoisovalerylurea as model substrate for studies on pharmacokinetics of glutathione conjugation in the rat. II. Pharmacokinetics and stereoselectivity of metabolism and excretion in vivo and in the perfused liver.

The hypnotic drug alpha-bromoisovalerylurea (BIU) has been studied in the rat with respect to its potential use as model substrate to investigate the pharmacokinetics of glutathione conjugation in vivo. The major metabolites of racemic BIU are the diastereomeric glutathione conjugates (bile) and mercapturates (urine). BIU was metabolized mainly by glutathione conjugation: after i.v. administration of [14C]BIU to freely moving rats, 89% of the dose was recovered in urine within 24 hr, mostly as mercapturates. The rate-limiting step in the clearance of BIU from blood most likely is glutathione conjugation as it was shown that rate-limitation is not due to flow-limited clearance in the liver (the initial extraction ratio of BIU in the perfused liver preparation was low: hepatic extraction ratio = 0.23), protein binding (60% was unbound in plasma) or enzyme saturation (linear pharmacokinetics in the dose range studied: 22-270 mumol/kg). Water solubility of BIU was sufficient to allow its i.v. administration, whereas the absence of toxic effects enables animal as well as human studies. Thus, BIU is a promising model substrate for studies of glutathione conjugation in vivo. In pentobarbital-anesthetized rats with a bile duct catheter, equal amounts of metabolites were excreted in bile (almost exclusively as the two diastereomeric BIU glutathione conjugates) and urine (mostly as the two diastereomeric mercapturates). Based on similar experiments with bile duct-ligated rats, it was concluded that the appearance of the mercapturates in urine could also occur without biliary excretion and subsequent gut metabolism of the BIU glutathione conjugates. The ability of the liver to metabolize BIU was studied in a hemoglobin-free, recirculating liver perfusion system. Of the recovered radioactivity 40% was excreted in bile within 2 hr, almost exclusively in the form of the two BIU glutathione conjugates. Also, glutathione conjugates were found in the perfusate (16% of the radioactivity present in the perfusate after 2 hr). A distinct stereoselectivity was observed in the metabolite excretion rates. The excretion half-lives of the two diastereomeric glutathione conjugates in bile differed 2- to 3-fold, both in anesthetized rats and in the perfused liver preparation. A similar difference in excretion half-lives was found for the urinary excretion of the diastereomeric mercapturates. Thus, BIU can be used to investigate in vivo the stereoselectivity of glutathione conjugation.

Animals↗

PubMed1979

The export of glutathione from human diploid cells in culture.

The export of glutathione from cultured human diploid fibroblasts into the surrounding medium was found by isotopic labeling experiments using [35S]cystine and by enzymatic measurements. The major part of the glutathione exported from the cells was found in normal culture medium as mixed disulfide of glutathione and cysteine. Radioactivity of 35S, mostly derived from cellular glutathione, was mainly located in glutathione moiety, not in cysteine moiety, of the mixed disulfide. Export of free glutathione was found when cystine-free medium was used. It was, therefore, concluded that mixed disulfide of glutathione and cysteine was formed in the medium by exported glutathione and medium cystine via sulfhydryl-disulfide exchange reaction. Amount of total glutathione exported from the cells was measured by enzymatic method and it was found that more than 10% of normal cellular glutathione was exported within 2 h. Apparent concentration of glutathione in the medium after a day of culture reached 3 to 4 micrometer, which was comparable to that observed in normal plasma by the same enzymatic method.

Biological Transport↗

PubMed1983

Turnover and functions of glutathione studied with isolated hepatic and renal cells.

Suspensions of freshly isolated rat hepatocytes and renal tubular cells contain high levels of reduced glutathione (GSH), which exhibits half-lives of 3-5 and 0.7-1 h, respectively. In both cells types the availability of intracellular cysteine is rate limiting for GSH biosynthesis. In hepatocytes, methionine is actively converted to cysteine via the cystathionine pathway, and hepatic glutathione biosynthesis is stimulated by the presence of methionine in the medium. In contrast, extracellular cystine can support renal glutathione synthesis; several disulfides, including cystine, are rapidly taken up by renal cells (but not by hepatocytes) and are reduced to the corresponding thiols via a GSH-linked reaction sequence catalyzed by thiol transferase and glutathione reductase (NAD(P)H). During incubation, hepatocytes release both GSH and glutathione disulfide (GSSG) into the medium; the rate of GSSG efflux is markedly enhanced during hydroperoxide metabolism by glutathione peroxidase. This may lead to GSH depletion and cell injury; the latter seems to be initiated by a perturbation of cellular calcium homeostasis occurring in the glutathione-depleted state. In contrast to hepatocytes, renal cells metabolize extracellular glutathione and glutathione S-conjugates formed during drug biotransformation to the component amino acids and N-acetyl-cysteine S-conjugates, respectively. In addition, renal cells contain a thiol oxidase acting on extracellular GSH and several other thiols. In conclusion, our findings with isolated cells mimic the physiological situation characterized by hepatic synthesis and renal degradation of plasma glutathione and glutathione S-conjugates, and elucidate some of the underlying biochemical mechanisms.

Animals↗

PubMed1984

Biliary transport of glutathione S-conjugate by rat liver canalicular membrane vesicles.

Transport of S-dinitrophenyl glutathione, a model compound of glutathione S-conjugates, was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibited time-dependent uptake of [2-3H]glycine-glutathione conjugate into an osmotically sensitive intravesicular space. Inactivation of vesicle-associated gamma-glutamyltransferase by affinity labeling with L-(alpha-S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole-acetic acid had no effect on the initial rate of transport. Chemical analysis revealed that the intact glutathione conjugate accounted for most vesicle-associated radioactivity, reflecting the low transferase activity in the liver and membrane vesicles. The initial rate of transport followed saturation kinetics with respect to conjugate concentrations; an apparent Km of 1.0 mM and Vmax of 1.7 nmol/mg of protein X 20 s were calculated. These results indicate that transport of the glutathione S-conjugate across the canalicular membranes is a carrier-mediated process. Sodium chloride in the transport medium could be replaced by KCl, LiCl, or choline chloride without any changes in transport activity. The rate of conjugate transport was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). The rate of conjugate uptake was enhanced by replacing KCl in the transport medium with K gluconate, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. These data indicate that conjugate transport is electrogenic and involves the transfer of negative charge. Transport of S-dinitrophenyl glutathione was inhibited by S-benzyl glutathione, oxidized glutathione, or reduced glutathione. This transport system in canalicular membranes may function in biliary secretion of glutathione S-conjugates of xenobiotics whose synthesis in hepatocytes requires glutathione S-transferases.

Animals↗