Glutathione changes occurring after S-adenosylhomocysteine hydrolase inhibition

Freshly isolated rat hepatocytes, which metabolize methionine through the cystathionine pathway, and cultured L5178Y cells, which do not, were compared for their response to the inhibition of S-adenosylhomocysteine (SAH) hydrolase (EC 3.3.1.1). When cells were incubated in Fischer's medium lacking cystine but containing 0.67 mM methionine and 10% serum, the addition of periodate-oxidized adenosine (POA), an inhibitor of SAH hydrolase, increased the level of SAH approximately 4-fold in L5178Y cells (5 mM POA) and 30-fold in hepatocytes (1 mM POA). POA treatment also decreased the amount of intracellular glutathione (GSH) in hepatocytes by 6-fold, and in L5178Y cells by 3-fold. Incubation of hepatocytes with adenosine plus homocysteine, 2-chloroadenosine, or 2',3'-acyclic adenosine increased intracellular SAH and also lowered GSH levels. Neither GSH oxidation nor efflux of GSH or GSH conjugates appeared to account for the GSH loss. Intracellular GSH, covalently bound to proteins as mixed disulfides, increased when hepatocytes were incubated with POA, but the increase was insufficient to account for the total GSH loss. In hepatocytes with prelabeled [35S]GSH, POA caused the cellular GSH content to decrease while the specific activity of [35S]GSH remained constant, suggesting that inhibitor treatments that caused elevated SAH levels may have increased the degradation of GSH while GSH synthesis was inhibited.     

Effect of 2-mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells.

The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-mercaptoethanol for survival in tissue culture. The effect of 2-mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.     

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.     

Studies on the biosynthesis of sulfolipids in the Diatom Nitzschia alba.

Labeling of sulfolipids in Nitzschia alba was studied after growth of the cells in media containing L-[35S]cystine, L-[35S], L-[35S]cysteine, L-[35S]-methionine or a mixture of L-[Me-3H]methionine and L-[35S]methionine, [35S]Cysteine or [35S]cystine labeled the deoxyceramide sulfonate and the sulfonium analog, phosphatidylsulfocholine (and its lyso derivative) but not the sterol sulfate nor the sulfoquinovosyl diglyceride; [35S]methionine labeled only the phosphatidylsulfocholine and its lyso derivative. With the [35S]- and [Me-3H]methionine mixture (3H/35S ratio 1.0) the phosphatidylsulfocholine had a 3H/35 S ratio of 1.5 indicating that both sulfonium methyl groups were derived from methionine. Probable biosynthetic pathways for these novel sulfolipids are discussed.     

Glutathiolation of the proteasome is enhanced by proteolytic inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.     

The transsulfuration pathway: a source of cysteine for glutathione in astrocytes

Astrocyte cells require cysteine as a substrate for glutamate cysteine ligase (γ-glutamylcysteine synthase; EC 6.3.2.2) catalyst of the rate-limiting step of the γ-glutamylcycle leading to formation of glutathione (l-γ-glutamyl-l-cysteinyl-glycine; GSH). In both astrocytes and glioblastoma/astrocytoma cells, the majority of cysteine originates from reduction of cystine imported by the x_c⁻ cystine-glutamate exchanger. However, the transsulfuration pathway, which supplies cysteine from the indispensable amino acid, methionine, has recently been identified as a significant contributor to GSH synthesis in astrocytes. The purpose of this review is to evaluate the importance of the transsulfuration pathway in these cells, particularly in the context of a reserve pathway that channels methionine towards cysteine when the demand for glutathione is high, or under conditions in which the supply of cystine by the x_c⁻ exchanger may be compromised.     

Cysteine transport in melanosomes from murine melanocytes

The synthesis of pheomelanin requires the incorporation of thiol-containing compound(s) during the process of mammalian melanogenesis. Since melanins are produced only in specialized, membrane-bound organelles, known as melanosomes, such thiol donor(s) must cross the membrane barrier from the cytosol to the melanosome interior. Cysteine and/or glutathione (GSH) were proposed as suitable thiol donors, although uptake of these compounds into melanosomes was not previously characterized. In this study, we show that cysteine is transported, in a temperature- and concentration-dependent manner, across membranes of melanosomes derived from murine melanocytes. Additional proof that cysteine uptake results from a carrier-mediated process and is not due to simple diffusion or to a membrane channel, was obtained in countertransport experiments, in which melanosomes preloaded with cysteine methyl ester took up significantly more [35S]cysteine than did unloaded controls. In contrast, we were unable to detect any significant uptake of [35S]GSH over a wide concentration range, in the presence or in the absence of reducing agent. This study is the first demonstration of melanosomal membrane transport of cysteine, and it strongly suggests that free cysteine is the thiol source utilized for pheomelanin synthesis in mammalian melanocytes.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

Mouse lymphoma L1210 cells can be arrested in the G1 phase by adjusting cellular cysteine and glutathione

Mouse lymphoma L1210 cells (NCI line) that have low ability to take up cystine became deficient in cellular cysteine and glutathione in normal culture media. The cells entered the resting state during culture when they were seeded at high cell densities. They remained viable and were mostly present in the G1 or G0 phase. In the growth-arrested state, the cellular glutathione content was one order of magnitude lower than in the exponentially growing phase in the presence of 2-mercaptoethanol. In the arrested state, DNA synthesis was almost inhibited, and RNA and protein synthesis decreased markedly. Transfer of the cells to medium containing 2-mercaptoethanol, which improves the utilization of cystine by these cells, produced the rapid recovery of RNA and protein synthesis. DNA synthesis slowly increased, reaching a maximum after a lag period.     

Increased loss and decreased synthesis of hepatic glutathione after acute ethanol administration. Turnover studies.

The effect of acute ethanol administration on rates of synthesis and utilization of hepatic glutathione (GSH) was studied in rats after a pulse of [35S]cysteine. A 35% decrease in hepatic GSH content 5h after administration of 4 g of ethanol/kg body wt. was accompanied by a 33% increase in the rate of GSH utilization. The decrease occurred without increases in hepatic oxidized glutathione (GSSG) or in the GSH/GSSG ratio. The rate of non-enzymic condensation of GSH with acetaldehyde could account for only 6% of the rate of hepatic GSH disappearance. The increased loss of [35S]GSH induced by ethanol was not accompanied by an increased turnover; rather, a 30% inhibition of GSH synthesis balanced the increased rate of loss, leaving the turnover rate unchanged. The rate of acetaldehyde condensation with cysteine in vitro occurred at about one-third of the rate of GSH loss in ethanol-treated animals. However, ethanol induced only a minor decrease in liver cysteine content, which did not precede, but followed, the decrease in GSH. The characteristics of 2-methylthiazolidine-4-carboxylic acid, the condensation product between acetaldehyde and cysteine, were studied and methodologies were developed to determine its presence in tissues. It was not found in the liver of ethanol-treated animals. Ethanol administration led to a marked increase (47%) in plasma GSH in the post-hepatic inferior vena cava, but not in its pre-hepatic segment. Data suggest that an increased loss of GSH from the liver constitutes an important mechanism for the decrease in GSH induced by ethanol. In addition, an inhibition of GSH synthesis is observed.     

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells

Abstract. Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 µ m cysteine and/or 200–400 µ m cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 m m BSO treatment significantly increased cell proliferation as measured by ³H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G₁ phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G₁-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.     

Biosynthesis of thiazole from thiamine in Escherichia coli]

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.     

Elevation of glutathione in phenylalanine mustard-resistant murine L1210 leukemia cells

Murine L1210 leukemia cells resistant to the antineoplastic agent L-phenylalanine mustard have a 1.5-2.0-fold elevation in their cellular GSH and GSSG content as compared to drug-sensitive cells. Cellular uptake of L-[U-14C]cystine and its incorporation into GSH of the resistant tumor are correspondingly elevated. Synthesis of gamma-glutamylcysteine, GSH, and GSSG is elevated 1.5-2.0-fold in cell-free preparations of the resistant tumor. This increased synthesis of GSH is attributed to increased cellular content (1.6-fold) of gamma-glutamylcysteine synthetase. GSH synthetase activity is equivalent in both drug-sensitive and -resistant cells. Investigation into the hydrolysis of selected peptides by cell-free preparations of both sensitive and resistant tumors suggest that aminopeptidase M participates in the formation of L-cysteine from L-Cys-Gly. This is supported by the observation that these preparations readily degrade L-Leu-p-nitroanilide and L-Ala-L-Ala-L-Ala, known substrates for aminopeptidase M, but not dipeptidase. The failure of the tumors to degrade Gly-D-Ala, a dipeptidase substrate, and the marked inhibition of L-Ala-Gly, L-Cys-Gly, and L-Ala-L-Ala-L-Ala hydrolysis by Bestatin further support a role for aminopeptidase M in the generation of L-cysteine from L-Cys-Gly. These results suggest that the drug-resistant tumor cell has developed an efficient mechanism for maintenance of elevated GSH which involves both gamma-glutamyl transpeptidase-initiated catabolism of GSH to cysteine and its reutilization by gamma-glutamylcysteine synthetase.     

Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation.

The uptake and metabolism of 35S-labelled sulphur amino acids were compared in periportal (PP) and perivenous (PV) rat hepatocytes, isolated by digitonin/collagenase perfusion, to identify the factors underlying the previously observed [Kera, Penttilä & Lindros, Biochem. J. (1988) 254, 411-417] higher rate of GSH replenishment in PP cells. The buthionine sulphoximine-inhibitable synthesis of GSH was faster in PP than in PV hepatocytes with both cysteine (6.1 versus 5.0 mumol/h per g of cells) and methionine (4.5 versus 3.3 mumol/h per g) as well as with endogenous precursors and L-2-oxo-4-thiazolidinecarboxylate as substrates. However, the uptake of cysteine by PP cells was slower than by PV cells (8.6 versus 10.3 mumol/h per g of cells), whereas methionine was taken up at similar rates. The activity of gamma-glutamylcysteine synthetase (GCS) was slightly higher in digitonin lysates from the PP than from the PV zone. Production of sulphate, the major catabolite of [35S]cysteine sulphur, as well as incorporation of the label into protein occurred at similar rates in PP and PV cells. Taurine, on the other hand, was produced from [35S]cysteine much faster by PV than by PP cells (0.7 versus 0.1 mumol/h per g of cells). Accordingly, the taurine content of PV hepatocytes tended to be higher and to increase faster during incubation with methionine. These results imply that metabolism of taurine is highly zonated within the acinus. They also suggest that both the slightly lower GCS activity and the fast metabolism of cysteine to taurine limit the capacity of PV hepatocytes to synthesize GSH.     

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.     

Determination of the metabolic origin of the sulfur atom in thiamin of Escherichia coli by mass spectrometry

In this study cells were grown in 34S-sulfate or L-[sulfane-34S]thiocystine, and the effects of unlabeled methionine and cystine on incorporation of sulfur into methionine, cystine and thiamin were determined. Unlabeled methionine effectively suppresses the incorporation of 34S into methionine but not into cysteine or thiamin. In contrast, cystine blocks incorporation of 34S only to approximately the relative ratio of 32S to 34S indicating, that cysteine is closely related to the origin of the sulfur in thiamin, and therefore the sulfane sulfur of thiocystine is also an effective source of the thiamin sulfur.     

Low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-1-infected patients

Blood plasma samples from HIV-1-infected persons contain elevated glutamate concentrations up to 6-fold the normal level and relatively low concentrations of acid-soluble thiol (i.e. decreased cysteine concentrations). The intracellular glutathione concentration in peripheral blood-mononuclear cells (PBMC) and monocytes from HIV antibody-positive persons are also significantly decreased. Therapy with azidothymidine (AZT) causes a substantial recovery of the plasma thiol levels; but glutamate levels remain significantly elevated and intracellular glutathione levels remain low. Cell culture experiments with approximately physiological amino-acid concentrations revealed that variations of the extracellular cysteine concentration have a strong influence on the intracellular glutathione level and the rate of DNA synthesis [( 3H]thymidine incorporation) in T cell clones and human and murine lymphocyte preparations even in the presence of several-fold higher cystine and methionine concentrations. Cysteine cannot be replaced by a corresponding increase of the extracellular cystine or methionine concentration. These experiments suggest strongly that the low cysteine concentration in the plasma of HIV-infected persons may play a role in the pathogenetic mechanism of the acquired immunodeficiency syndrome.     

The origin of the sulfur atom of thiamin

The incorporation of the sulfur atom of 35S-labeled amino acids into thiamin in Escherichia coli and Saccharomyces cerevisiae was studied. The specific radioactivity of the S atoms was incorporated at similar levels into thiamin and cysteine residues in cell proteins. However, the specific radioactivity of the S atoms from [35S]methionine was not incorporated into thiamin but into methionine residues in cell proteins. Thus, the origin of the S atom of thiamin was established as being the S atom of cysteine. No activity from [U-14C]cysteine was recovered in thiamin, proving that the carbon skeleton of this amino acid was not utilized in synthesizing the thiazole moiety of thiamin.     

gamma-Glutamyl transpeptidase (gamma-GT) and maintenance of thiol pools in tumor cells resistant to alkylating agents

Previous studies from this laboratory have established that acquired resistance of murine L1210 leukemia cells to L-phenylalanine mustard (L-PAM) and other alkylating agents is accompanied by a two-to threefold elevation in their glutathione (GSH) concentration (Biochem. Pharm. 31:121). In an attempt to gain insight into the mechanism by which resistant tumor cells maintain their increased GSH content, we have assessed the possible role of gamma-glutamyl transpeptidase (gamma-GT), a membrane bound enzyme involved in GSH metabolism. These results indicate that the enzyme is present in both sensitive and resistant murine L1210 leukemia cells but that the cellular content of gamma-GT is elevated two-to threefold in L-PAM resistant cells as compared to their sensitive counterparts. This elevation in enzymatic activity correlates well with the increased cellular GSH content in resistant cells. The results of a detailed kinetic analysis of gamma-GT activity indicate that there is no difference, between cell types, in the apparent Km of the enzyme for the gamma-glutamyl donor (L-gamma-glutamyl-p-nitroanilide) or the acceptor (glycylglycine). However, the apparent Vmax is increased two-to threefold in L-PAM resistant tumor cells. Investigation into the role of gamma-GT in the extracellular metabolism of GSH indicates that resistant tumor cells metabolize two-fold more GSH than do sensitive cells and that such metabolism results in a similar difference in the intracellular concentration of cysteine. Results of studies with cellular lysates also indicate a role for the enzyme in the supply of cysteine to the glutathione precursor pool of the tumor cell and in the maintenance of elevated GSH concentrations in cells resistant to alkylating agents.