The relationship between GSH, GSSG and non-GSH thiol in GSH-deficient erythrocytes from Finnish landrace and Tasmanian merino sheep.

1. Two automated colorimetric methods have been developed for assaying the GSH and total thiol in protein-free extracts of erythrocytes. They employ as chromogens 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and alloxan. 2. The concentrations of GSH, GSSG and total non-protein thiol have been estimated in high and low GSH erythrocytes from Finnish Landrace and Tasmanian Merino sheep. 3. In both breeds of sheep low GSH cells were found to have low concentrations of total non-protein thiol and GSSG as well as of GSH. 4. Nevertheless high and low GSH cells have similar values for the oxidation-reduction potential of the GSH : GSSG couple.     

Close linkage between the C blood group locus and the locus controlling amino acid transport in sheep erythrocytes

Data from 838 Finnish Landrace or Finnish Landrace crossbred sheep showed a highly significant correlation between phenotypes of the C blood group system and erythrocyte amino acid transport variants. Erythrocytes with normal amino acid transport properties (GSH high, Ly- type) were Cb-positive or Cb-negative. Erythrocytes with the amino acid transport lesion (GSH low, Ly +) were never Cb-negative. Sheep erythrocytes homozygous for C^bshowed stronger lysis reactions with anti-Cb than heterozygous cells. Ly + sheep were nearly always homozygous for C^b, whereas most Ly- sheep were heterozygous or Cb-negative. Inheritance studies provided strong evidence that this association is due to close genetic linkage.     

Relationship between cell age, glutathione and cation concentrations in sheep erythrocytes with a normal and a defective transport system for amino acids

Percoll density gradients were used to separate sheep erythrocytes according to cell age. Erythrocytes with low intracellular levels of glutathione (GSH) caused by an inherited deficiency of the System C amino acid transporter exhibited large age-related decreases in GSH and K+ content. In contrast, there was no age-related loss of intracellular GSH in normal sheep erythrocytes or in sheep erythrocytes with low GSH resulting from a diminished activity of gamma-glutamylcysteine synthetase. Loss of GSH from amino acid transport-deficient erythrocytes was paralleled by the progressive appearance of Heinz bodies in the cells, indicating an increased susceptibility to oxidative damage.     

Relationship between cell age,glutathione and cation concentrations in sheep erythrocytes with a normal and defective transport system for amino acids

Percoll density gradients were used to separate sheep erythrocytes according to cell age. Erythrocytes with low intracellular levels of glutathione (GSH) caused by an inherited deficiency of the System C amino acid transporter exhibited large age-realted decreases in GSH and K⁺ content. In contrast, there was no age-related loss of intracellular GSH in normal sheep erythrocytes or in sheep erythrocytes with low GSH resulting from a diminished activity of γ-glutamylcysteine synthetase. Loss of GSH from amino acid transport-deficient erythrocytes was parallel by the progressive appearance of Heinz bodies in the cells, indicating an increased susceptibility to oxidative damage.     

Characterization of normal, glutathione-deficient and arginase-deficient sheep erythrocytes by 1H-NMR spectroscopy

Normal sheep erythrocytes as well as glutathione- (GSH-) deficient and arginase-deficient sheep erythrocytes have been characterized by 1H nuclear magnetic resonance spectroscopy. The GSH deficiency is a result of defective amino acid transport (lesion 1), diminished gamma-glutamylcysteine synthetase activity (lesion 2), or both (lesions (1 + 2)). 1H-NMR spectra of normal sheep erythrocytes are similar to those for human erythrocytes, and consist of resonances from a number of small intracellular molecules, including GSH. In contrast, the resonances for GSH in the GSH-deficient erythrocytes are much weaker, and strong resonances are observed for lysine, threonine and ornithine or arginine, depending on the arginase activity, in erythrocytes with lesion 1 and lesions (1 + 2). A comparison of the intensity of GSH resonances in spectra for normal and GSH-deficient erythrocytes with GSH levels determined spectrophotometrically following reaction with the nonspecific thiol reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB) indicates that either not all of the GSH determined with Ellman's reagent is free and observable by 1H-NMR or that not all of the thiol determined by Ellman's reagent is GSH. If the latter is the case, the GSH levels determined with Ellman's reagent for erythrocytes with lesions (1 + 2) are most affected, which might account for their high susceptibility to oxidative stress.     

Seasonal, genetic and age variation in non-esterified fatty acid concentrations in the ewe

Plasma samples were collected at intervals of about 2 weeks from about ten 2 1/2 year old, or older, and five 1 1/2 year old ewes of each of the Scottish Blackface, Finnish Landrace and Tasmanian Merino ewes and were assayed for their non-esterified fatty acid (NEFA) concentrations. The NEFA concentrations showed an increase with time for every animal. The NEFA concentrations and their linear increase was least in the Finnish Landrace animals. The increase with time was greatest in the Merinos. There was no difference between the age groups. There was no evidence of any annual, cyclical trend.     

Presence of (2'-5')oligoadenylate synthetase in avian erythrocytes

(2'-5')Oligoadenylate synthetase (2-5A synthetase) was found in avian erythrocyte lysates from chicken, goose, and pigeon, with high levels being observed in chicken erythrocytes. No activities, however, were detected in erythrocytes from human, sheep, mouse, turtle, frog, trout, or lamprey. In chicken erythrocyte lysate, about 70% of ATP was converted to 2-5A molecules during a 20-h incubation, in which the tri- and tetra-adenylate were the major products. The tri-, tetra-, penta-, and hepta-adenylate were synthesized sequentially, but the levels of the di-adenylate were low throughout the reaction. 2-5A synthetase was also seen in erythrocytes from specific pathogen-free chickens, suggesting that the enzyme was not produced as a result of microbial infections. 2-5A synthetases from avian erythrocytes of chicken and pigeon were found not only in cytoplasms, but also in nuclei. No enzyme activity, however, was detected in the nuclear fraction of goose erythrocytes. The molecular size of 2-5A synthetase in nuclei from chicken erythrocytes was 45,000-60,000 daltons, while cytoplasms contained an 85,000- to 120,000-dalton enzyme. In addition, the synthetase was present in several types of chicken tissue including liver, intestine, bone marrow, spleen, bursa, pancreas, and thymus, but not in brain, heart, or stomach.     

Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease

Oxidative stress appears to play an important role in degeneration of dopaminergic neurons of the substantia nigra (SN) associated with Parkinson's disease (PD). The SN of early PD patients have dramatically decreased levels of the thiol tripeptide glutathione (GSH). GSH plays multiple roles in the nervous system both as an antioxidant and a redox modulator. We have generated dopaminergic PC12 cell lines in which levels of GSH can be inducibly down-regulated via doxycycline induction of antisense messages against both the heavy and light subunits of gamma-glutamyl-cysteine synthetase, the rate-limiting enzyme in glutathione synthesis. Down-regulation of glutamyl-cysteine synthetase results in reduction in mitochondrial GSH levels, increased oxidative stress, and decreased mitochondrial function. Interestingly, decreases in mitochondrial activities in GSH-depleted PC12 cells appears to be because of a selective inhibition of complex I activity as a result of thiol oxidation. These results suggest that the early observed GSH losses in the SN may be directly responsible for the noted decreases in complex I activity and the subsequent mitochondrial dysfunction, which ultimately leads to dopaminergic cell death associated with PD.     

Enalapril and captopril enhance glutathione-dependent antioxidant defenses in mouse tissues

The effect of enalapril and captopril on total glutathione content (GSSG + GSH) and selenium-dependent glutathione peroxidase (Se-GPx) and glutathione reductase (GSSG-Rd) activities was investigated in mouse tissues. CF-1 mice (4-mo-old females) received water containing enalapril (20 mg/l) or captopril (50 mg/l) for 11 wk. Enalapril increased GSSG + GSH content (P < 0.05) in erythrocytes (147%), brain (112%), and lung (67%), and captopril increased GSSG + GSH content in erythrocytes (190%) and brain (132%). Enalapril enhanced Se-GPx activity in kidney cortex (42%) and kidney medulla (23%) and captopril in kidney cortex (30%). GSSG-Rd activity was enhanced by enalapril in erythrocytes (21%), brain (21%), liver (18%), and kidney cortex (53%) and by captopril in erythrocytes (25%), brain (19%), and liver (34%). In vitro erythrocyte oxidant stress was evaluated by thiobarbituric acid-reactive substances (TBARS) production (control 365 +/- 11, enalapril 221 +/- 26, captopril 206 +/- 17 nmol TBARS x g Hb(-1) x h(-1); both P < 0.05 vs. control) and phenylhydrazine-induced methemoglobin (MetHb) formation (control 66.5 +/- 3.5, enalapril 52.9 +/- 0.4, captopril: 56.4 +/- 2.9 micromol MetHb/g Hb; both P < 0.05 vs. control). Both angiotensin-converting enzyme inhibitor treatments were associated with increased nitric oxide production, as assessed by plasma NO-(3) + NO-(2) level determination (control 9.22 +/- 0.64, enalapril 13.7 +/- 1.9, captopril 17.3 +/- 3.0 micromol NO-(3) + NO-(2)/l plasma; both P < 0.05 vs. control). These findings support our previous reports on the enalapril- and captopril-induced enhancement of endogenous antioxidant defenses and include new data on glutathione-dependent defenses, thus furthering current knowledge on the association of ACE inhibition and antioxidants.     

Transferrin types and reproduction in sheep

Transferrin types were determined for flocks of Finnish Landrace, Clun Forest, Soay and Merino sheep and gene frequencies were calculated. Analysis of ratios of transferrin types in segregating matings of Finnish Landrace and Clun Forest revealed a significant excess of heterozygotes in matings of heterozygous rams with heterozygous and with homozygous ewes. In Finnish Landrace, matings of sheep homozygous for Tf ^c to those heterozygous for Tf ^C gave a significant excess of homozygous male lambs and heterozygous female lambs. Finnish Landrace ewes of transferrin type BD had smaller litters than ewes of other types.     

Amino acid transport in normal and glutathione-deficient sheep erythrocytes.

1. Uptake rates for 23 amino acids were measured for both normal (high-GSH) and GSH-deficient (low-GSH) erythrocytes from Finnish Landrace sheep. 2. Compared with high-GSH cells, low-GSH cells had a markedly diminished permeability to D-alanine, L-alanine, alpha-amino-n-butyrate, valine, cysteine, serine, threonine, asparagine, lysine and ornithine. Smaller differences were observed for glycine and proline, whereas uptake of the other amino acids was not significantly different in the two cell types.     

Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress

Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.     

The relation between selenium, zinc and copper concentration and the trace element dependent antioxidative status.

An imbalance in the antioxidative system was connected with the development of a number of pathological processes. In order to receive values of a healthy group and to evaluate pathological changes of the trace element dependent antioxidative status in future, we investigated 99 healthy volunteers (45 male and 54 female, mean age 37.4 +/- 11.7 years). We determined the concentrations of Se, Cu and Zn, the concentrations of malondialdehyde (MDA) and the activities of the Se dependent glutathione peroxidase (GSH-Px) and the Zn/Cu dependent superoxide dismutase (SOD). The plasma concentrations (mean +/- SD) for Se, Cu and Zn were 0.84 +/- 0.10 micromol/l, 15.6 +/- 2.78 micromol/l and 12.6 +/- 1.80 micromol/l, resp., and for non protein-bound and protein bound MDA 0.27 +/- 0.07 micromol/l and 1.11 +/- 0.25 micromol/l, resp. The activity of GSH-Px in plasma and erythrocytes was 130 +/- 20.8 U/l and 19.8 +/- 4.18 U/mg Hb, resp. and of SOD in erythrocytes 3,159 +/- 847.2 U/g Hb. In plasma positive correlations have been found between Se concentrations and GSH-Px activities (p < 0.002, r = 0.31) and between GSH-Px activities and concentrations of non protein-bound MDA (p = 0.004, r = 0.28). A negative correlation has been observed between GSH-Px activities in plasma and in erythrocytes. The higher the concentrations of Cu in erythrocytes, the higher were the activities of SOD (p = 0.03, r = 0.22) and GSH-Px in erythrocytes (r = 0.26, p = 0.01), while an increasing activity of GSH-Px in these cells correlated with a decreasing concentration of non protein-bound MDA (r = -0,31, p = 0.002). An increase in BMI was connected with an increase in protein-bound MDA and a decrease in GSH-Px activities in pLasma (p = 0.002 and r = 0.23). As the results demonstrate, Se and Cu concentrations in erythrocytes can improve the trace element dependent antioxidative status.     

Adaptation of the Blood Antioxidant Defence Mechanisms of Sheep with a Genetic Lesion Resulting in Low Red Cell Glutathione Concentrations

Finnish Landrace sheep with a genetic lesion which results in restricted cysteine transport across the red cell membrane have total glutathione concentrations in their red blood cells that are approximately 40% of those in normal sheep of the same breed. However, dimethyldisulphide-challenged red blood cells from both phenotypes produce an ESR-spin adduct at similar rates. The resistance of the low glutathione phenotype red cells to oxidant challenge is reflected by increases in the activities of antioxidant enzymes. Sheep with a genotypic disorder in cysteine transport may be a suitable model for studying the genetic expression of antioxidant enzymes in response to oxidant loads.     

Aggregate formation of erythrocytes in postcapillary venules

The purpose of the present study was to obtain information on erythrocyte aggregate formation in vivo. The movements of erythrocytes in postcapillary venules of the rat spinotrapezius muscle at various flow rates were recorded with a high-speed video camera before and after infusion of dextran 500. To distinguish aggregates, the following criteria were used: 1) a fixed distance (4 microm) between the center points of two adjacent cells, 2) lack of visible separation between the adjacent cells, and 3) movement of the adjacent cells in the same direction. Without dextran 500 infusion, 11 and 5% of erythrocytes formed aggregates in low (33.2 +/- 28.3 s) and high pseudoshear (144.2 +/- 58.3 s) conditions, respectively, based on the above criteria. After dextran 500 infusion, 53% of erythrocytes satisfied the criteria in the low pseudoshear condition (26.5 +/- 17.0 s) and 13% of erythrocytes met the criteria in the high pseudoshear condition (240.0 +/- 85.9 s), indicating erythrocyte aggregation is strongly associated with shear rate. Approximately 90% of aggregate formation occurred in a short time period (0.15-0.30 s after entering the venule) in a region 15 to 30 microm from the entrance. The time delay may reflect rheological entrance conditions in the venule.     

Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-cysteine synthetase) and, particularly, GLR1 (glutathione reductase) are required for survival on DPS. DPS is uniquely thiol-specific, and we found that the cellular mechanisms for DPS detoxification differ substantially from that of the commonly used thiol oxidant diamide. In contrast to this oxidant, the full antioxidant pools of glutathione (GSH) and thioredoxin are required for resistance to DPS. We found that DPS-sensitive mutants display increases in the disulfide form of GSH (GSSG) during DPS exposure that roughly correlate with their more oxidizing GSH redox potential in the cytosol and their degree of DPS sensitivity. DPS seems to induce a specific disulfide stress, where an increase in the cytoplasmic/nuclear GSSG/GSH ratio results in putative DPS target(s) becoming sensitive to DPS.     

L-Cysteine influx and efflux: a possible role for red blood cells in regulation of redox status of the plasma

The objective of this study was to investigate if erythrocytes play a role in the maintenance of redox homeostasis of the plasma. Thus, we studied L-cysteine efflux and influx in vitro in human erythrocytes. In the present study, we exposed the erythrocytes to different concentrations of L-cysteine and then measured the intracellular free -SH concentrations. Erythrocytes treated in the same manner were later utilized for the cysteine efflux studies. The effect of temperature on the influx and the efflux processes were also evaluated. Change in the free -SH content of the buffer was evaluated as a measure for the presence of an efflux process. The effects of free -SH depletion on L-cysteine transport is also investigated. We also determined the rate of L-cysteine efflux in the presence and absence of buthionine sulfoximine (BSO) in erythrocytes that are pretreated with 1-chloro-2,4-dinitro benzene, a glutathione (GSH) depletory. Our L-cysteine influx studies demonstrated that erythrocytes can respond to increases in L-cysteine concentration in the extracellular media and influx L-cysteine in a concentration-dependent manner. Free -SH concentrations in erythrocytes treated with 1 mM L-cysteine reached to 1.64 +/- 0.06 mM in 1 h whereas this concentration reached to 4.30 +/- 0.01 mM in 10 mM L-cysteine treated erythrocytes. The L-cysteine efflux is also determined to be time-and concentration-dependent. Erythrocytes that are pretreated with higher L-cysteine concentrations displayed a higher efflux process. Outside concentration of free -SH in 1 mM L-cysteine pretreated erythrocytes reached to 0.200 +/- 0.005 mM in 1 h whereas this concentration reached to 1.014 +/- 0.002 with 10 mM L-cysteine pretreated erythrocytes. Our results also indicate that the rate of inward and outward transport of L-cysteine is affected by the oxidative status of the erythrocytes. When GSH is depleted and GSH synthesis is blocked, the L-cysteine uptake and the efflux processes are significantly decreased. Depending on our results, it could be concluded that erythrocytes play a role in the regulation of the plasma redox status and intracellular level of GSH determines the rate of the L-cysteine efflux.     

Resumption of ovarian function in autumn lambing Dorset, Rambouillet and Finnish Landrace ewes

The establishment of ovarian activity during lactation was studied in the postpartum period of Rambouillet, Dorset and Finnish Landrace ewes following lambing during the month of October (1981). The mean postpartum intervals to first ovulation and first estrus were 22.7 and 53.0 for Rambouillets, 25.2 and 51.0 for Dorsets, and 22.5 and 49.7 days for Finnish Landrace ewes. Estrus was not associated with the first ovulation postpartum in any breed. The number of silent ovulations prior to the first estrus was highest in the Rambouillet and lowest in Finnish Landrace breeds. Of the 18 ewes in the project, 14 had normal luteal phase lengths, 1 had a possible short luteal phase and 3 had prolonged luteal phases following the first ovulation postpartum. The first service conception rate of all ewes bred was 82% (14 17 ) at an average of 52 days postpartum. The lambing rate following the autumn breeding was higher (2.14 +/- 0.14) than the lambing rate which followed the previous spring breeding (1.28 +/- 0.11).     

Effects of glutathione depletion on gluconeogenesis in isolated hepatocytes

Glutathione-depleted hepatocytes, by incubation with diethylmaleate (DEM) or phorone (2,6-dimethyl-2,5-heptadiene-4-one), i.e., substrates of the GSH S-transferases (EC 2.5.1.18), showed rates of gluconeogenesis from various precursors significantly lower than controls; however the rate of glucose synthesis from fructose was similar to that of controls. Isolated hepatocytes from rats pretreated with those substrates 1 h before isolation to deplete hepatic glutathione (GSH) also showed a decrease of the rate of gluconeogenesis from lactate plus pyruvate. Incubation of hepatocytes with L-buthionine sulfoximine, a specific inhibitor of gamma-glutamyl-cysteine synthetase (EC 6.3.2.2), resulted in a decreased rate of gluconeogenesis from lactate plus pyruvate only when GSH values were lower than 1 mumol/g cells. Freeze-clamped livers from GSH-depleted rats showed a higher concentration of malate and glycerol 3-phosphate, indicating that GSH depletion probably affects phosphoenolpyruvate carboxykinase and glycerol-3-phosphate dehydrogenase activities. Several indicators of cell viability, such as lactate dehydrogenase leakage, malondialdehyde accumulation, ATP concentration, or urea synthesis from different precursors, were not affected by GSH depletion under the experimental conditions used here. Besides, the GSH/GSSG ratio remained unchanged in all cases.