Elevated erythrocyte glutathione associated with elevated substrate in high- and low-glutathione sheep.

Erythrocyte glutathione concentration increases dramatically in sheep when they become anemic. To determine the mechanism of this change in glutathione control, we measured the enzymes and substrates necessary for glutathione control, we measured the enzymes and substrates necessary for glutathione synthesis after acute blood loss in both low- (gamma-glutamylcysteine synthetase deficient) and high-glutathione sheep. Erythrocyte glutamate, ATP, and glycine increased dramatically in all sheep. Erythrocyte gamma-glutamylcysteine synthetase increased slowly and seemed unrelated to changes in glutathione. Erythrocyte glutathione synthetase and cysteine and plasma cysteine, glutamate and glycine did not change significantly. Apparently substrate concentrations may be important in regulating erythrocyte glutathione levels.     

Some physiological aspects of genetic variation in the blood of sheep1

The principal genetic variants in sheep red cells and plasma are listed. Current hypotheses as to how the L blood group antigen affects active potassium transport across the red cell membrane are summarized. Recent work on an inherited defect in amino acid transport which results in a red cell GSH deficiency is also described.     

Some physiological aspects of genetic variation in the blood of sheep.

The principal genetic variants in sheep red cells and plasma are listed. Current hypotheses as to how the L blood group antigen affects active potassium transport across the red cell membrane are summarized. Recent work on an inherited defect in amino acid transport which results in a red cell GSH deficiency is also described.     

Macrophages regulate intracellular glutathione levels of lymphocytes. Evidence for an immunoregulatory role of cysteine

Macrophages consume cystine and generate approximately equivalent amounts of acid-soluble thiol. Stimulation of macrophages with bacterial lipopolysaccharide (LPS) or tumor necrosis factor (TNF) strongly augments the amount of thiol released into the culture supernatant. Cysteine constitutes most of the acid-soluble thiol. The intracellular glutathione level and the DNA synthesis activity in mitogenically stimulated lymphocytes are strongly increased by either exogenously added cysteine, or (syngeneic) macrophages. This cysteine dependency is observed even in the presence of relatively high extracellular cystine concentration as they occur in the blood plasma. The extracellular cysteine concentration also has a strong influence on the intracellular glutathione concentration, viability, and DNA synthesis of cycling T cell clones. Moreover, the cysteine concentration in the culture medium on Day 3 and Day 4 of a 5-day allogeneic mixed lymphocyte culture (i.e., in the late phase of incubation) has a strong influence on the generation of cytotoxic T cell activity, indicating that regulatory effects of cysteine are not restricted to the early phase of the blastogenic response. The inhibitory effect of cysteine starvation on the DNA synthesis of the T cell clones and on the activation of cytotoxic T lymphocytes can be explained essentially by the depletion of intracellular glutathione, since similar effects are observed after treatment with buthionine sulfoximine (BSO), a specific inhibitor of the glutathione biosynthesis. BSO has practically no influence, however, on the N alpha-benzyloxycarbonyl Ne-t-butyloxycarbonyl-L-lysine-thiobenzyl-ester (BLT)-esterase activity and hemolytic activity of the cell lysates from cytotoxic T cells against sheep red blood cells (perforin activity). Taken together, our experiments indicate that cysteine has a regulatory role in the immune system analogous to the hormone-like lymphokines and cytokines. It is released by macrophages at a variable and regulated rate and regulates immunologically relevant functions of lymphocytes in the vicinity.     

The biosynthesis of glutathione in human erythrocytes (author's transl)]

The concentrations of glutathione precursors in human erythrocytes were investigated. 300muM glutamate, 375 muM glycine, and 10muM cysteine were found by automated amino acid analysis. The concentration of 2-aminobutyrate, the precursor of ophthalmic acid, was 15muM. The influence of the activities of endogenous or added glutamyl-cysteine synthetase and glutathione synthetase on the rate of glutathione biosynthesis was measured in membrane-free hemolysates under physiological conditions. The results show that the rate of the overall biosynthesis mainly depends on the formation of the dipeptide glutamyl-cysteine. The effect of glutathione precursor concentrations on the synthesis of the tripeptide was investigated at constant (endogenous) activities of the synthesizing enzymes. The rate was not enhanced by addition of glutamate and/or glycine unless cysteine or glutamyl-cysteine was also added. It is concluded that the concentration of cysteine limits the actual rate of the glutamyl-cysteine-synthetase reaction in vivo. No cysteine or bis(glutamyl)cystine was detected in human hemolysate; however, these disulfides were converted to glutathione. This indicates that erythrocytes have an appropriate system for their reduction, since the disulfides themselves are not substrates for the glutathione-synthesizing enzymes. Studies with intact human red cells indicate that the uptake of cysteine is the rate-determining step in the biosynthesis of glutathione.     

Kinetics of uptake and deacetylation of N-acetylcysteine by human erythrocytes

Overproduction of reactive oxygen species associated with several diseases including sickle cell anaemia reduces the concentration of glutathione, a principal cellular antioxidant. Glutathione depletion in sickle erythrocytes increases their conversion to irreversible sickle cells that promote vaso-occlusion. Therapeutically, N-acetylcysteine partially restores glutathione concentrations but its mode of action is controversial. Following glutathione depletion, glutathione synthesis is limited by the supply of cysteine and it has been assumed that deacetylation of N-acetylcysteine within erythrocytes provides cysteine to accelerate glutathione production. To determine whether this is the case we studied the kinetics of transport and deacetylation of N-acetylcysteine. Uptake of N-acetylcysteine had a first order rate constant of 2.40+/-0.070min(-1) and only saturated above 10mM. Inhibition experiments showed that 56% of N-acetylcysteine transport was via the anion exchange protein. Deacetylation, measured using (1)H NMR, had a K(m) of 1.49+/-0.16mM and V(max) of 2.61+/-0.08micromolL(-1)min(-1). Oral doses of N-acetylcysteine increase glutathione concentrations in sickle erythrocytes at plasma N-acetylcysteine concentrations of approximately 10microM. At this concentration, calculated rates of N-acetylcysteine uptake and deacetylation were approximately 5% of the rate required to maintain normal glutathione production. We concluded that on oral administration, intracellular deacetylation of N-acetylcysteine supplies little of the cysteine required for accelerated glutathione production. Instead, N-acetylcysteine acts by freeing bound cysteine in the plasma that then enters the erythrocytes. To be effective, intracellular cysteine precursors must be designed to enter erythrocytes rapidly and employ enzymes with high activity within erythrocytes to liberate the cysteine.     

Effect of vitamin E on the oxidative state of glutathione in plasma

Reduced and oxidized glutathione levels in red blood cells and plasma from humans were determined after oral vitamin E treatment. The experiments confirmed that vitamin E enhances reduced glutathione levels in red blood cells. Moreover, vitamin E supplement resulted in a significant reduction of the plasma oxidized glutathione content. Thus, it seems that the effect of vitamin E on the reduced glutathione content is not exerted via direct modulation of the glutathione-synthesizing enzymes, but rather by a more general mechanism of preserving reduced glutathione consumption by reducing the burden of the glutathione system.     

Prooxidative effects of TEMPO on human erythrocytes

The prooxidative effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were observed in human erythrocytes. Incubation of red blood cells with the membrane-permeable TEMPO leads to a decrease in the concentration of intracellular reduced glutathione, accompanied by the reduction of TEMPO. Extracellular ferricyanide inhibited the loss of glutathione and reduction of TEMPO. TEMPO induced glutathione release from the cells and oxidation of hemoglobin to methemoglobin; ferricyanide prevented these effects. These results indicate that TEMPO may act as an oxidant to erythrocytes, whilst extracellular ferricyanide protects against its effects.     

High activity antioxidant enzymes protect flying-fox haemoglobin against damage: an evolutionary adaptation for flight?

Flying-foxes are better able to defend haemoglobin against autoxidation than non-volant mammals such as sheep. When challenged with the common physiological oxidant, hydrogen peroxide, haemolysates of flying-fox red blood cells (RBC) were far less susceptible to methaemoglobin formation than sheep. Challenge with 1-acetyl-2-phenylhydrazine (APH) caused only half as much methaemoglobin formation in flying-fox as in ovine haemolysates. When intact cells were challenged with phenazine methosulfate (PMS), flying-fox RBC partially reversed the oxidant damage, and reduced methaemoglobin from 40 to 20% over 2 h incubation, while ovine methaemoglobin remained at 40%. This reflected flying-fox cells’ capacity to replenish GSH fast enough that it did not deplete beyond 50%, while ovine RBC GSH was depleted to around 20%. The greater capacity of flying-foxes to defend haemoglobin against oxidant damage may be explained in part by antioxidant enzymes catalase, superoxide dismutase and cytochrome-b ₅ reductase having two- to four-fold higher activity than in sheep (P < 0.001). Further, their capacity to limit GSH depletion to 50% and reduce methaemoglobin (in the presence of glucose), despite ongoing exposure to PMS may result from having ten-fold higher activity of G6PD and 6PGD than sheep (P < 0.001), indicating the presence of a very efficient pentose phosphate pathway in flying-foxes.     

Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine

Cysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents. Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine. The uptake of cysteine was mediated mainly by the ASC system (Na+-dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine). On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities. Because of the instability of cysteine, the culture media usually contained cystine but not cysteine. Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine. The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bathocuproinedisulfonate, a nontoxic copper-specific chelator that inhibits autoxidation of cysteine. Cysteine added at 25-400 microM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes. In the lipopolysaccharide-activated cells, however, cysteine enhanced the cellular glutathione contents in a dose-dependent manner. These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.     

Dynamics of sulfur amino acids in mammalian brain: assessment of the astrocytic-neuronal cysteine interaction by a mathematical hybrid model

A mathematically hybrid model was used to analyze three mechanisms by which cysteine could be produced in the brain to be used as preferential substrate in the synthesis of neuronal glutathione. In that way, the fluxes of sulfur-compounds at the brain-blood barrier were integrated with their transport in astrocytes and neurons, and with their metabolism in astrocytes. We concluded that cysteine, in contrast with its precursor cystine, would not be taken up from the blood at the blood-brain barrier, but instead it must be lost continuously from astrocytes. Cysteine efflux is produced because the uptake of cystine in astrocytes is much greater than their cysteine demand to synthesize glutathione, hypotaurine and taurine. Once in the interstitial parenchyma, cysteine would be taken for the neurons, as backwardly by the endothelial cells. Remarkably, a close sulfur-macro balance can be maintained only if the surplus of the produced cysteine is transferred from the endothelial cells to the blood together with significant amounts of other sulfur-compounds, probably taurine and hypotaurine. In addition, the results obtained shown that alternative mechanisms of cysteine generation (i.e., nonenzymatic-thiol-disulfide exchange reaction, enzymatic cleavage of the glutathione effluxed from astrocytes) are not quantitatively significant under physiological conditions, in situ.     

Effect of hypobaric hypoxia on immune function in albino rats

 The effect of exposure to hypoxia on macrophage activity, lymphocyte function and oxidative stress was investigated. Hypoxia enhanced peritoneal macrophage activity as revealed by enhanced phagocytosis and free radical production. There was no significant change in antibody titres to sheep red blood cells in either serum or spleen during hypoxia. However, there was a considerable reduction in the delayed-type hypersensitivity response to sheep red blood cells, indicating the impairment of T-cell activity. Hypoxia decreased the blood glutathione (reduced) level and increased plasma malondialdehyde by a factor of about 2. It is therefore speculated that hypoxia imposes an oxidative stress leading to decreased T-cell acivity. Received: 1 September 1997 / Accepted: 22 May 1998     

Engineering and genetic approaches to modulating the glutathione network in plants

Reduced glutathione (GSH) is the most abundant low-molecular weight thiol in plant cells. It accumulates to high concentrations, particularly in stress situations. Because the pathway of GSH synthesis consists of only two enzymes, manipulation of cellular glutathione contents by genetic intervention has proved to be relatively straightforward. The discovery of a new bacterial bifunctional enzyme catalysing GSH synthesis but lacking feedback inhibition characteristics offers new prospects of enhancing GSH production and accumulation by plant cells, while the identification of γ-glutamyl cysteine and glutathione transporters provides additional possibilities for selective compartment-specific targeting. Such manipulations might also be used to affect plant biology in disparate ways, because GSH and glutathione disulphide (GSSG) have crucial roles in processes as diverse as the regulation of the cell cycle, systemic acquired resistance and xenobiotic detoxification. For example, depletion of the total glutathione pool can be used to manipulate the shoot : root ratio, because GSH is required specifically for the growth of the root meristem. Similarly, chloroplast γ-glutamyl cysteine synthetase overexpression could be used to increase the abundance of specific amino acids such as leucine, lysine and tyrosine that are synthesized in the chloroplasts. Here we review the aspects of glutathione biology related to synthesis, compartmentation and transport and related signalling functions that modulate plant growth and development and underpin any assessment of manipulation of GSH homeostasis from the viewpoint of nutritional genomics.     

The effect of sodium periodate treatment on the modulation of the sodium pump in low-potassium type (LK) sheep red cells by the L antigen

1. The action of sodium periodate and neuraminidase on active and passive K+ transport in low-potassium type (LK) sheep red cells was investigated in relation to the contribution of the Lp and Ll antigens. 2. Active K+ transport in LK sheep red cells was not affected by treatment with sodium periodate (2 mM), or with neuraminidase. 3. Passive K+ transport in LK sheep red cells was increased by sodium periodate treatment in a concentration-dependent manner. The increase was not Cl- dependent, and so differed from the increased passive K+ uptake resulting from N-ethylmaleimide treatment. 4. HK sheep red cells treated with sodium periodate showed small increases in passive K+ uptake, and N-ethylmaleimide treatment used sequentially with sodium periodate resulted in further small increases in passive K+ uptake. 5. In LK sheep red cells the stimulation of active K+ transport by anti-L was impaired by 50% in cells treated with sodium periodate (2 mM) and was slightly lowered in cells treated with neuraminidase. 6. In LK sheep red cells inhibition of passive K+ transport by anti-L was not impaired by sodium periodate treatment (2 mM), or by neuraminidase treatment.     

Changes in the intracellular homocysteine and glutathione content associated with aging

Since moderate hyperhomocysteinemia is an independent risk factor for vascular disease by mean of its oxidant effect and glutathione plays a main role as intracellular redox-regulating agent, we have studied for the first time the total intracellular content of homocysteine in aging. Plasma homocysteine concentration, total intracellular and plasma glutathione, and other related thiol compounds such as cysteine and the glutathione catabolite cysteinglycine were also studied. Forty three healthy elderly subjects and twenty seven healthy young ones were studied. The total intracellular peripheral blood mononuclear cell content was higher for homocysteine, cysteine and cysteinglycine, whereas that of the total glutathione was greatly decreased in elderly people with respect to young ones. Elderly subjects showed significantly higher levels than young ones of total plasma homocysteine and cysteinglycine, but not cysteine, whereas total plasma glutathione levels were increased. In addition, elderly subjects showed significantly decreased plasma vitamin E levels and increased concentrations of serum lipid peroxides measured as TBARS (reaction product of malondialdehyde with thiobarbituric acid). The intracellular glutathione content presented significantly negative correlation with serum TBARS, and intracellular and plasma homocysteine levels. These findings show an increase of homocysteine synthesis associated with aging, which in turn can produce an augmented oxidant effect on endothelium, and an impaired intracellular antioxidant capacity leading to an enhanced lipid peroxidation and decreased total intracellular glutathione content.     

L-antigen and active potassium transport in HK and LK red cells of Barbary sheep (Ammotragus lervia)

1. The potassium concentration in red cells of 21 Barbary sheep showed a bimodal distribution, with five animals of LK type (K+ conc. 30-45 mM) and 16 of HK type (K+ conc. 80-95 mM). 2. Evidence is presented that both Lp and Ll antigens are present on LK Barbary sheep red cells. 3. Active K+ transport in LK Barbary sheep red cells was stimulated 3-5 fold by sheep and goat anti-L. 4. Active K+ transport in HK Barbary sheep red cells was higher than in LK red cells. Five out of six HK animals tested showed no stimulation of active K+ transport with anti-L. One HK animal (2BA2) showed some stimulation of active K+ transport, and also absorbed some anti-L from antisera, suggesting that Lp antigen is present on these red cells. 5. Ouabain-sensitive ATPase in membranes from HK and LK Barbary sheep red cells showed kinetics characteristic of HK and LK membranes of domestic goats and sheep; the ATPase of LK Barbary sheep membranes sensitized with anti-L was stimulated 2-fold due to an alteration in the internal sodium and potassium affinities in favour of sodium.     

Blood groups and biochemical polymorphism in the Namaqua sheep breed

Summary. The Namaqua is an indigenous fat-tailed African breed of sheep which has remained relatively isolated and which at one time dwindled to near extinction. Frequency data are given for blood group antigens, red cell glutathione and potassium types, for electrophoretic variants of red cell haemoglobin, 'X' protein, nucleoside phosphorylase, NADH-diaphorase, lysine and carbonic anhydrase and of plasma esterase, transferrin and albumin. Of particular interest was the occurrence of the i blood group, a bimodal distribution in red cell glutathione concentrations and red cell potassium concentrations of around 57 mmo1/1 cells, i.e. neither typically LK nor HK type.     

Rosette formation by human thymocytes

A proportion of lymphocytes in human fetal and post-natal thymus, and in blood, formed rosettes with red blood cells from sheep and pig. The count of rosette-forming cells (RFC) among human thymocytes varied widely, from 2–216 per thousand cells, and was higher in fetal than in post-natal life. The count of RFC among human thymocytes was not reduced by specific rabbit anti-human immunoglobulin sera, indicating that the receptor was not of immunoglobulin character; the reaction was inhibited by antithymocyte serum and metabolic poisons and certain enzymes. The receptor may be equivalent to other “non-specific” glycoprotein hemagglutinins in plants and viruses.The importance of species differences in immunological assays is emphasized. Thus human thymocytes gave high counts of RFC only with red blood cells of sheep and pig; moreover thymus lymphocytes from only man and pig, but not several other species including rodents, were highly reactive with sheep red blood cells. The capacity for rosette formation could be a marker for T cells in human blood.     

In vivo targets of S-thiolation in Chlamydomonas reinhardtii

Glutathionylation is the major form of S-thiolation in cells. This reversible redox post-translational modification consists of the formation of a mixed disulfide between a free thiol on a protein and a molecule of glutathione. This recently described modification, which is considered to occur under oxidative stress, can protect cysteine residues from irreversible oxidation, and alter positively or negatively the activity of diverse proteins. This modification and its targets have been mainly studied in non-photosynthetic organisms so far. We report here the first proteomic approach performed in vivo on photosynthetically competent cells, using the eukaryotic unicellular green alga Chlamydomonas reinhardtii with radiolabeled [(35)S]cysteine to label the glutathione pool and diamide as oxidant. This method allowed the identification of 25 targets, mainly chloroplastic, involved in various metabolic processes. Several targets are related to photosynthesis, such as the Calvin cycle enzymes phosphoglycerate kinase and ribose-5-phosphate isomerase. A number of targets, such as chaperones and peroxiredoxins, are related to stress responses. The glutathionylation of HSP70B, chloroplastic 2-Cys peroxiredoxin and isocitrate lyase was confirmed in vitro on purified proteins and the targeted residues were identified.     

Inhibiting Glutathione Metabolism in Lung Lining Fluid as a Strategy to Augment Antioxidant Defense

Glutathione is abundant in the lining fluid that bathes the gas exchange surface of the lung. On the one hand glutathione in this extracellular pool functions in antioxidant defense to protect cells and proteins in the alveolar space from oxidant injury; on the other hand, it functions as a source of cysteine to maintain cellular glutathione and protein synthesis. These seemingly opposing functions are regulated through metabolism by gamma-glutamyl transferase (GGT, EC 2.3.2.2). Even under normal physiologic conditions, lung lining fluid (LLF) contains a concentrated pool of GGT activity exceeding that of whole lung by about 7-fold and indicating increased turnover of glutathione at the epithelial surface of the lung. With oxidant stress LLF GGT activity is amplified even further as glutathione turnover is accelerated to meet the increased demands of cells for cysteine. Mouse models of GGT deficiency confirmed this biological role of LLF GGT activity and revealed the robust expansiveness and antioxidant capacity of the LLF glutathione pool in the absence of metabolism. Acivicin, an irreversible inhibitor of GGT, can be utilized to augment LLF fluid glutathione content in normal mice and novel GGT inhibitors have now been defined that provide advantages over acivicin. Inhibiting LLF GGT activity is a novel strategy to selectively augment the extracellular LLF glutathione pool. The enhanced antioxidant capacity can maintain lung epithelial cell integrity and barrier function under oxidant stress.