Transport accounts for glutathione turnover in human erythrocytes.

Human erythrocytes were incubated with 3H-glycine to label the glutathione pool. These cells were then used to determine the rate of oxidized glutathione (GSSG) transport out of erythrocytes. For 6 normal individuals, the mean transport rate was 6.7 nmole GSSG/hr/ml red cells. This transport rate would suggest a half-life of 4.7 days for the erythrocytic glutathione, which is in close agreemwnt with the observed in vivo half-life of 4 days. These data demonstrate that GSSG transport can account for the observed turnover of erythrocytic glutathione.     

Shape response of human erythrocytes to altered cell pH

Alteration of red blood cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of phosphatidylcholine and phosphatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH cells. Possible roles of pH- titratable inner monolayer phospholipids were examined by assessing pH- dependent shape in cells in which their levels had been perturbed. In metabolically depleted cells and calcium-treated cells, which have altered levels of phosphatidic acid, phosphatidylinositol-4-phosphate, and/or phosphatidylinositol-4,5-bisphosphate, low cell pH was stomatocytogenic and high cell pH was echinocytogenic, as in control cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for cell pH-mediated shape change.     

Changes in glutathione and glutathione metabolizing enzymes in human erythrocytes and lymphocytes as a function of age of donor

Studies in animals have demonstrated that glutathione levels and the activities of glutathione S-transferase and glutathione reductase decrease with advanced age in various tissues. We have examined the effects of donor age on erythrocyte and lymphocyte levels of these three parameters in human subjects. The results indicate that glutathione content, and glutathione S-transferase and glutathione reductase activities, are higher in mature middle-age than young individuals but decrease markedly in the aged. These results support the hypothesis that a decrease in glutathione content and glutathione-metabolizing enzymes may contribute to changes associated with aging as well as the increased susceptibility to carcinogens, drugs and disease which occurs with advanced age.     

Release of ATP from human erythrocytes in response to a brief period of hypoxia and hypercapnia.

OBJECTIVE: The aims were, first, to detect and quantify the release of ATP from human erythrocytes in response to a brief exposure to a hypoxic/hypercapnic environment, similar to that found in vigorously exercising skeletal or cardiac muscle; and second, to explore the mechanism of ATP release in response to hypoxia. METHODS: Washed human erythrocytes suspended in Krebs-Henseleit solution were exposed for 50 s to an atmosphere of approximately 8.0 kPa PCO2 and 2.7 kPa PO2; ATP released into the suspension was assayed using the firefly luminescence technique. Samples of human blood were obtained by venepuncture of the median cubital vein from male and female volunteers ranging in age from 21 to 74 years. Anticoagulation was with EDTA. RESULTS: A background of 0.49 x 10(6) (SEM 0.037 x 10(6)) ATP molecules.cell-1 was attributed to spontaneous haemolysis of 1% of the cell population, as estimated by levels of haemoglobin measured in the suspension fluid. When the erythrocytes were exposed to the hypoxic/hypercapnic gas mixture at 37 degrees C the ATP concentration in the suspension fluid rose to 2.67 x 10(6) (0.27 x 10(6)) molecules.cell-1. An efflux rate of 276(37) molecules.mu-2.s-1 was calculated. The hypoxia induced ATP release was blocked in three different ways: first, by application of 50 microM of the specific band 3 anion channel blocking agents niflumic acid (a translocation inhibitor), DIDS (a transport site inhibitor), or dipyridamole (a channel blocker); secondly, by replacement of extracellular chloride and bicarbonate with the impermeant anion methanesulphonate; and thirdly, by application of 5 nM of the nucleoside transport blocker nitrobenzylthioinosine. None of these blocking techniques affected the background levels of ATP attributed to haemolysis. CONCLUSIONS: A situation of hypoxia/hypercapnia, such as would be found in exercising muscle, induces release of ATP from the erythrocyte via the plasma membrane protein moiety known as band 4.5 (a nucleoside transporter) and electrical balance across the erythrocyte membrane is maintained by the simultaneous influx of extracellular chloride and/or bicarbonate via the plasma membrane protein known as band 3 (anion channel). The circulation of erythrocytes into a region of hypoxia in vivo could promote an increase in local blood flow through release of endothelium dependent relaxing factor in response to released ATP.     

Impairment of glutathione metabolism in erythrocytes from patients with diabetes mellitus

The metabolism of glutathione and activities of its related enzymes were studied in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM). A decrease in the levels of the reduced form of glutathione and an increase in the levels of glutathione disulfide were found in erythrocytes of diabetics. To elucidate these changes in the levels of glutathione, synthetic and degradative processes were studied. The activity of gamma-glutamylcysteine synthetase was significantly lower in diabetics than in normal controls. The activity of glutathione synthetase of each group was the same. The rate of outward transport of glutathione disulfide in diabetics decreased to approximately 70% of that of normal controls. The activity of glutathione reductase decreased in diabetics. These data suggest that the decrease in the levels of reduced form of glutathione in erythrocytes of diabetics is brought about by impaired glutathione synthesis and that the increase in the levels of glutathione disulfide is brought about by the decreased transport activity of glutathione disulfide through the erythrocyte membrane together with a decrease in the activity of glutathione reductase. These data also suggest that the impairment of glutathione metabolism weakens the defense mechanism against oxidative stress in erythrocytes of diabetics.     

Efflux of 3H-thymidine by erythrocytes from mice infected with Trypanosoma brucei brucei

Erythrocytes from mice infected with Trypanosoma brucei brucei showed a higher rate of efflux of labelled thymidine than did control erythrocytes from uninfected mice (0.56 +/- 0.10 and 0.38 +/- 0.06 mumole min-1 ml-1 packed cells respectively). Efflux of the nucleoside from erythrocytes of normal and infected mice were inhibited to the same extent by a specific nucleoside transport inhibitor, nitrobenzylthioinosine. Enumeration of nitrobenzylthioinosine binding sites on the erythrocytes showed that both have similar numbers of sites (6.2-6.6 X 10(3) sites/erythrocyte). It is concluded that the membrane permeability of the erythrocytes from infected mice was affected by the trypanosome in such a way as to enhance the purine nucleoside transport capacity. This may result in an increased supply of vital purine bases and nucleosides to trypanosomes which depend on their hosts for these nutrients.     

Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes

Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H2O2 in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H2O2). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H2O2 in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H2O2 when H2O2 is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.     

Myosin content and distribution in human neonatal erythrocytes are different from adult erythrocytes

Neonatal erythrocytes (N-RBC) are different from adult erythrocytes (A-RBC). N-RBC are larger, less deformable, and undergo enhanced spontaneous and drug-induced endocytosis. The reticulocyte population of N-RBC is also different, consisting primarily of the youngest (R1) reticulocytes that are motile and capable of receptor-mediated endocytosis. Processes such as motility could require a contractile system. Myosin, a contractile protein, was identified in both A-RBC and N-RBC. We proposed to compare myosin content and distribution in A-RBC and N-RBC by immunofluorescence and enzyme-linked immunosorbent assay (ELISA) using a monospecific polyclonal rabbit antimyosin. There was bright immunofluorescence on 44% of N-RBC with some heterogeneity contrasting with a barely detectable fluorescence on A-RBC. ELISA measurements showed that A-RBC had 4,315 myosin copies/RBC, whereas N-RBC had 10,855 copies/RBC (or 2.5 times as much). ELISA measurements of white ghosts showed that A-ghosts contained 1,250 copies of myosin/RBC (29% of total) whereas N-ghosts contained 3.4 times as much at 4,320 copies/RBC (39% of total). Therefore, N-RBC not only have more myosin, but the amount that is membrane-associated is disproportionately increased. It is proposed that such differences in amount and distribution of myosin could account for some of the unusual properties of neonatal RBC indicated.     

Isozyme patterns in erythrocytes from human fetuses

Starch gel electrophoretic patterns of 26 enzymes (corresponding to 36 gene loci) were examined in hemolysates of erythrocytes from 11 first-trimester and midtrimester human fetuses (65-138 gestation days). The zymograms of 16 enzymes were identical in fetal and control adult red cells. Six enzymes (enolase, guanylate kinase, lactate dehydrogenase, nucleoside Phosphorylase, phosphofructokinase, hexokinase) showed differences in the staining intensity of certain isozyme zones as compared with the controls. Also, the fetal red cell zymograms, in contrast to those of adults, contained the mitochondrial forms of isocitric dehydrogenase and glutamic oxaloacetic transaminase as well as more definite zones of phosphoglucomutase-3. Finally, some of the isozymes of uridine diphosphate kinase in the fetal cells had slightly retarded mobility. These observed differences between fetal and adult red cells could reflect the expression of a different program of protein synthesis in red cells of the fetuses or the epigenetic modifications of isozymes in immature red cells.     

Low activity of superoxide dismutase and high activity of glutathione reductase in erythrocytes from centenarians

OBJECTIVE: to compare the activities of antioxidative enzymes in erythrocytes between centenarians and a younger group of elderly subjects. DESIGN: cross-sectional study. SETTING: county of Funen, Denmark. SUBJECTS: 41 centenarians aged between 100 and 105 years and 52 community control subjects aged between 60 and 79 years. MEASUREMENTS: enzyme activities of superoxide dismutase (CuZn-SOD), glutathione peroxidase, catalase and glutathione reductase (GR) in erythrocytes. Functional capacity among the centenarians was evaluated by Katz' index of activities of daily living, the Physical Performance Test and Mini-Mental State Examination. RESULTS: the mean CuZn-SOD activity was significantly lower and the mean GR activity was significantly higher in centenarians than in the group of elderly people. The centenarians with the lowest cognitive and physical functional capacity and who did not survive at least 1 year after blood sampling tended to have the lowest CuZn-SOD activities. The range of GR activity was shifted toward higher values in the centenarian group than in the younger group and those centenarians having the best functional capacity tended to have the highest GR activity. CONCLUSIONS: CuZn-SOD activity is decreased in centenarians, probably because of reduced demand for the enzyme at lower metabolic rate and oxygen consumption. Subjects with high GR activity occur more frequently among centenarians than expected, possibly due to their better survival. The role of GR in disease prevention and as a predictor for longevity deserves to be further explored.     

Differential reactivities of fetal and adult human erythrocytes to antisera directed against glycolipids of human erythrocytes

Summary. The immunological reactivities of fetal and adult erythrocytes to antisera which were directed against globoside, hematoside and lactosylceramide were compared. Fetal erythrocytes showed higher reactivity than adult erythrocytes in agglutination, hemolysis and absorption capacity of antibody when tested with anti-globoside and anti-hematoside. No differential reactivity was found when tested with anti-lactosylceramide. Treatment of adult erythrocytes by trypsin or by neuraminidase resulted in an increase in their reactivity to the same level as that of fetal erythrocytes, but the reactivity of fetal erythrocytes was not greatly affected by enzyme treatment. The results indicate that globosidie and hematosidie groupings of fetal cells are directly exposed on the periphery of cells, while those of mature erythrocytes are masked by ‘sialomucopeptidyl’ groupings, and their reactivities are hampered.     

Elevated glutathione accelerates oxidative damage to erythrocytes produced by aromatic disulfide

It has been shown that certain dogs have erythrocytes characterized by an inherited high concentration of reduced glutathione (GSH), five to seven times the normal level (high-GSH RBCs). We examined whether increased GSH in dog erythrocytes leads to increased protection against oxidative damage induced by acetylphenylhydrazine (APH) and/or 4-aminophenyl disulfide (4-AD). When erythrocytes were incubated with 30 mmol/L APH, the Heinz body count was appreciably higher in normal RBCs than in high-GSH RBCs, while there was no difference in the increase of the methemoglobin (metHb) concentration in both RBCs. In contrast, both the Heinz body count and metHb production were much higher in high-GSH RBCs than in normal RBCs when erythrocytes were incubated with 4-AD. Furthermore, the generation of the superoxide in erythrocytes treated with 4-AD, which was measured by spin trapping combined with electron spin resonance (ESR), was obviously higher in high-GSH RBCs than in normal RBCs. These results clearly indicate that erythrocyte GSH is an important defense against oxidative damage induced by certain compounds such as APH, but that, in contrast, elevated GSH appears to accelerate oxidative damage to erythrocytes produced by aromatic disulfides, such as 4-AD, which generated a superoxide in erythrocytes via its redox reaction with GSH.     

Aging changes of riboflavin concentration and glutathione reductase activity in erythrocytes

Aged erythrocytes obtained by fractionation using gradient centrifugation with Dextran 40, showed lower glutathione reductase activity and riboflavin content than young cells. However, both young and old cells displayed almost the same increase in enzyme activity upon addition of flavin adenine dinucleotide to the test hemolysate in vitro. The lipid peroxide content in the cell membranes showed no consistent changes with aging.     

Normal glutathione content and some related enzyme activities in the fetal erythrocytes

Pure fetal blood was obtained by direct-vision fetoscopy from 66 fetuses at 17-24 weeks gestation. The concentration of GSH and the activities of the enzymes gamma-glutamylcysteine synthetase (GCS), glutathione synthetase (GS), glutathione reductase (GR) and glutathione peroxidase (GPx) were analysed by established techniques to find the normal ranges for this gestational age. The ranges were relatively narrow and could serve as reference values for the prenatal diagnosis of defects in the GSH metabolism of erythrocytes. The results were compared with those obtained from 38 normal adults and with published values on neonatal blood. In the case of GR a comparison was also made with maternal blood. In comparison with adults, fetal erythrocytes showed higher GSH concentration and GCS activity and lower GS and GPx activities. This pattern resembled that found in neonatal erythrocytes except for the GCS activity, which was higher in the fetal cells. Furthermore the differences between fetal and adult erythrocytes were more pronounced than those between neonatal and adult cells. The GR activity of fetal erythrocytes was also higher than that of either normal adult or maternal blood. This difference, however, was reduced to an insignificant level when the enzyme was activated in vitro by flavin adenine dinucleotide (FAD) because of a relatively low per cent activation of the GR in the fetal erythrocytes.     

Reduced glutathione concentration in erythrocytes of patients with acute and chronic viral hepatitis

SUMMARY. Reduced glutathione (GSH), the main intracellular mechanism that protects against oxidative stress, is the subject of considerable interest in viral hepatitis. In patients with chronic hepatitis C, results reported from different centres are controversial, demonstrating either a reduction or an elevation of GSH concentration. The aim of this study was to evaluate the glutathione concentration in erythrocytes (normal range 2.45 ± 0.15 mmol l^?1) in patients with acute and chronic viral hepatitis. In 52 patients with acute viral hepatitis (hepatitis A virus (HAV), hepatitis B virus (HBV) and hepatitis C virus (HCV) infection) there was marked reduction of GSH at the beginning of the disease (0.79 ± 0.43 mmol l^?1. P < 0.001) with high alanine aminotransferase (ALT) activity (1549 ± 772.9 IU l^?1). In 37 patients with chronic HCV infection the mean value of GSH was below the normal range (1.92 ± 0.62 mmol l^?1. P < 0.001). In 60% of patients (n = 22), depletion of GSH was observed and 40% (n = 15) presented with a normal concentration of GSH. In 10 patients with chronic HBV infection the mean value of GSH was also below the normal range (1.93 ± 0.32 mmol l^?1, P < 0.001); in 80% of cases (n = 8) depletion of GSH was observed and 20% of patients (n = 2) had normal GSH concentrations. The ALT activity was not significantly different in patients with depleted and normal GSH concentrations (P > 0.05) in groups with chronic HBV and HCV infection.     

Stimulation of human cardiac adenylate cyclase by vanadate

Summary Experiments have been carried out to characterize the influence of the positive inotropic trace element vanadium (used as Na₃VO₄) on -adrenergic receptor coupled adenylate cyclase activity from human myocardium. Na₃VO₄ (10^–4 M) stimulates basal activity as well as isoprenaline (10 M)- and Mg²⁺ (20 mM)-activated enzyme activity 1.5–2.4-fold. In contrast, adenylate cyclase activity in the presence of maximally activating concentrations of Gpp(NH)p (10 M) cannot be further increased by Na₃VO₄. The results confirm the assumption (5) that vanadate stimulates adenylate cyclase by interacting with the nucleotide-binding site of this enzyme.

---

Stimulation der Adenylatzyklase in menschlichem Herzmuskelgewebe durch Vanadat

Zusammenfassung Es wurde der Einfluß des positiv inotropen Spurenelementes Vanadium (verwendet als Na₃VO₄) auf die -Rezeptor-gekoppelte Adenylatzyklaseaktivität aus menschlichem Herzmuskelgewebe untersucht. Na₃VO₄ (10^–4 M) stimuliert 1,5–2,4fach sowohl die Basalaktivität als auch die durch Isoprenalin (10 M) und durch Mg²⁺ (20 mM) aktivierte Enzymaktivität. Im Gegensatz dazu kann die durch Gpp(NH)p (10 m) maximal aktivierte Adenylatzyklaseaktivität durch Vanadat nicht weiter gesteigert werden. Die experimentellen Befunde bestärken die Annahme (5), daß Vanadat die Adenylatzyklase über eine Wechselwirkung mit der Nukleotidbindungsstelle des Enzyms stimuliert.

---

---

The data have been presented at the Symposium Cardiac Effects of Vanadate, Munich, October 26–27, 1979.

---

With 1 figure

---

This study was supported by the Deutsche Forschungsgemeinschaft (ER 65/2) and by the Wilhelm-Sander-Stiftung 14/1978-We 1.