Influence of Complete and Pronounced Incomplete Cerebral Ischemia and Subsequent Recirculation on Cortical Concentrations of Oxidized and Reduced Glutathione in the Rat

Abstract: The influence of complete and pronounced incomplete cerebral ischemia on cortical concentrations of reduced (GSH) and oxidized (GSSG) glutathione was studied in lightly anaesthetized (70% N₂ O) rats. GSH was extracted with HCl-methanol-perchloric acid and GSSG with trichloroacetic acid in the presence of N-ethylmaleimide and measured fluorometrically, giving normal concentrations in cortical tissue of about 2 and 0.01 μmol.g^?1 respectively. Reversible complete ischemia was induced by increasing the intracranial pressure to above the systolic blood pressure by infusing mock CSF into the cisterna magna. Reversible pronounced incomplete ischemia was induced by bilateral carotid artery clamping combined with hypovolemic hypotension. Whether complete or incomplete, a 30-min ischemic period caused a similar decrease in cortical GSH concentration (to about 90% of control) without any concomitant accumulation of GSSG in the tissue (or in CSF). Prolongation of the ischemic period (complete ischemia) to maximally 120 min caused an almost linear decrease of the tissue glutathione concentration to 45% of the preischemic value. During subsequent recirculation following a 30 min period of either complete or pronounced incomplete ischemia, there was a further decrease in cortical GSH concentrations without a reciprocal increase in GSSG concentrations. Lipid peroxidation (verified by determination of malondialdehyde production) induced in brain cortical tissue in vitro caused oxidation of tissue GSH with accumulation of GSSG. As the observed decrease in GSH during brain ischemia in vivo was not accompanied by any reciprocal increase in GSSG the results fail to support the hypothesis that peroxidative damage occurs during or following brain ischemia. The finding of an unchanged GSSG concentration does, however, not exclude the possibility of an increased turnover rate in the glutathione reductase reaction. It is concluded that the observed decrease in tissue GSH concentration mainly reflects a decrease in the glutathione pool size, due to an imbalance between breakdown and synthesis secondary to tissue energy failure.     

CEREBRAL METABOLIC AND CIRCULATORY CHANGES INDUCED BY HYPOXIA IN STARVED RATS

In order to study cerebral metabolic and circulatory effects of hypoxia under conditions of restricted glucose supply, the arterial P_o2, was reduced to 25–30mm Hg in artificially ventilated and lightly anaesthetized rats that were starved for 24 or 48 h prior to experiments. Arterial glucose concentrations, that were initially around 6μmol g^-1, were significantly reduced after 15min of hypoxia, and decreased to 50^o of control after 30min. In animals studied after 30min of hypoxia (24 h of starvation), cerebral blood flow had increased 4-fold and there was a moderate (25%) rise in cerebral oxygen consumption. During the course of hypoxia, cerebral cortical concentrations of glucose fell to low values. In spite of this, concentrations of pyruvate and lactate rose with time, and the sum of citric acid cycle intermediates (citrate, α-ketoglutarate, fumarate. malate and oxaloacetate) increased. Changes in amino acids were dominated by a fall in aspartate and a rise in alanine concentration. There was a moderate reduction in phosphocreatine and a slight rise in ADP concentration, but concentrations at ATP and AMP were unchanged. The changes observed are similar to those previously obtained in fed animals. It is concluded that even if blood glucose concentrations fall to 3μmol g^-1, and cerebral energy flux is maintained, substrate supply is sufficient to cover the energy requirements of the tissue. Hypoxia was accompanied by increases in the lactate/pyruvate and β-hydroxybutyrate acetoacetate ratios of blood. In the tissue, NADH/NAD⁺ ratios derived from the lactate, malate and β-hydroxybutyrate dehydrogenase systems rose, while that derived from the glutamate dehydrogenase reaction fell. It is concluded that the latter system is not well suited for estimating mitochondrial redox changes in brain tissue.     

Disulfiram augments oxidative stress in rat brain following bilateral carotid artery occlusion

We examined the brain oxidative stress which accompanies 30 min of bilateral carotid artery ligation (BCAL) in terms of changes in brain levels of glutathione; reduced (GSH) and oxidized (GSSG) forms and the exacerbation of oxidative stress by disulfiram (DSF). These results indicate that BCAL alone decreases GSH content and limits glutathione reductase (GR) activity, and these changes were enhanced by DSF pretreatment. Similar observations were recorded with DSF alone. GR activity (74.3±4.0 µmol min^–1 mg^–1 tissue; p<0.001) and GSH content (1.23±0.06 µmol min^–1g^–1 tissue; p<0.001) was attenuated in rats subjected to synergistic effect of BCAL and DSF with a concomitant increase of GSSG (0.006±0.006 µmol min^–1 g^–1 tissue; p<0.001). Recovery of GSH/GSSG level and GR activity during reperfusion following 30 min BCAL was considerably delayed (96 h) in the BCAL and DSF group as compared to the recovery time of 24 h in the group subjected to BCAL-reperfusion alone. Perturbation of GSH/GSSG homeostasis as a result of BCAL was augmented by DSF. These findings clearly demonstrate central nervous system oxidative stress due to a BCAL-DSF synergistic effect. Based on the results obtained with this model, we conclude that DSF increases brain oxidative stress and this may be detrimental to alcoholics who might drink and develop an acetaldehyde-induced hypotension while taking DSF.     

High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia-Tolerant Reptiles Contrasted with Levels in Anoxia-Intolerant Species

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia-tolerant pond and box turtles, moderately tolerant garter snakes, anoxia-intolerant clawed frogs (Xenopus laevis), and intolerant Long-Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g^?1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g^?1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g^?1). Regionally distinct levels (2–4 µmol g^?1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior-to-posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia-tolerant turtles, 2–3 µmol g^?1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior-to-posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.     

Copper deficiency and tissue glutathione concentration in the rat

Copper deficiency in rats increased renal vein and arterial (heart) plasma GSH concentration by approximately 50%. There was no change in plasma GSSG concentration. Renal vein plasma GSSG/GSH ratio was decreased in copper deficiency, which is consistent with previous reports showing a copper-dependent thiol oxidase activity in the renal basement membrane. No change occurred in arterial plasma GSSG/GSH ratio. Hepatic GSH concentrations were also elevated by 50% in copper deficiency, GSSG concentrations were unaffected, but GSSG/GSH ratio was depressed. Renal and cardiac tissue GSH and GSSG were unaffected by copper deficiency. The decreased SOD activity and GSH-Px activity observed in copper deficiency may contribute to increased hepatic and plasma GSH concentrations.     

Changes in Non-Enzymatic Antioxidants in the Blood Following Anaerobic Exercise in Men and Women

Purpose

The aim of this study was to compare changes in total oxidative status (TOS), total antioxidative capacity (TAC) and the concentration of VitA, VitE, VitC, uric acid (UA), reduced (GSH) and oxidized glutathione (GSSG) in blood within 24 hours following anaerobic exercise (AnEx) among men and women.

Methods

10 women and 10 men performed a 20-second bicycle sprint (AnEx). Concentrations of oxidative stress indicators were measured before AnEx and 3, 15 and 30 minutes and 1 hour afterwards. UA, GSH and GSSH were also measured 24 hours after AnEx. Lactate and H⁺ concentrations were measured before and 3 minutes after AnEx.

Results

The increase in lactate and H⁺ concentrations following AnEx was similar in both sexes. Changes in the concentrations of all oxidative stress indicators were significant and did not differ between men and women. In both sexes, TOS, TAC, TOS/TAC and VitA and VitE concentrations were the highest 3 minutes, VitC concentration was the highest 30 minutes, and UA concentration was the highest 1 hour after AnEx. GSH concentration was significantly lower than the initial concentration from 15 minutes to 24 hour after AnEx. GSSG concentration was significantly higher, while the GSH/GSSG ratio was significantly lower than the initial values 1 hour and 24 hour after AnEx.

Conclusions

With similar changes in lactate and H⁺ concentrations, AnEx induces the same changes in TAC, TOS, TOS/TAC and non-enzymatic antioxidants of low molecular weight in men and women. Oxidative stress lasted at least 24 hours after AnEx.     

The role of glutathione in rat adipocyte pentose phosphate cycle activity

An assay for reduced and oxidized glutathione was adapted to isolated rat epididymal adipocytes in order to correlate pentose phosphate cycle activity and glutathione metabolism. In collagenase-digested adipocytes the [GSH/GSSG] molar ratio was in excess of 100. Cells incubated for 1 hr with low glucose concentrations (0.28–0.55 mm) had higher GSH contents (3.2 μg/10⁶ cells) than in the absence of glucose (2.3 μg/10⁶ cells). The glutathione oxidant diamide caused a dose-related decrease in intracellular GSH, an increase in GSSG released into the medium, but no detectable change in the low intracellular GSSG content. The intracellular content of GSH and amount of GSSG released into the medium were therefore taken to reflect the glutathione status of the adipocytes most closely. Addition of H₂O₂ to a concentration of 60 μm to adipocytes caused to decline within 5 min in GSH content, which was less severe and more rapid to recover in the presence of 1.1 mm glucose, suggesting that the concomitant stimulation of glucose C-1 oxidation induced by the peroxide in the presence of glucose provided NADPH for regeneration of GSH. Further evidence for tight coupling between adipocyte [GSH/GSSG] ratios and pentose phosphate cycle activity was that (i) lowering intracellular GSH to 35–60% of control values by agents as diverse in action as t-butyl hydroperoxide, diamide, or the sulfhydryl blocker N-ethylmaleimide resulted in optimal stimulation of glucose C-1 oxidation and fractional pentose phosphate cycle activity, and (ii) incubating adipocytes directly with 2.5 mm GSSG resulted in a slight increase in glucose C-1 oxidation and when 0.5 mm NADP⁺ was also added a synergistic effect on pentose phosphate cycle activity was found. On the other hand, electron acceptors such as methylene blue did not lower cellular GSH content, but did stimulate the pentose phosphate cycle, confirming a site of action independent of glutathione metabolism. The results show that (i) glucose metabolism by the pentose phosphate cycle contributes to regeneration of GSH and that (ii) glutathione metabolism either directly or via coupled changes in [NADPH/NADP⁺] ratios may play a significant role in short-term control of the pentose phosphate cycle.     

Glutathione Is an Endogenous Ligand of Rat Brain N-Methyl-D-Aspartate (NMDA) and 2-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionate (AMPA) Receptors

A study was made of the effects of reduced (GSH) and oxidized (GSSG) glutathione on the Na⁺-independent and N-methyl-D-aspartate (NMDA) displaceable bindings of glutamate, on the binding of kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and ligands of the brain NMDA receptor-ionophore complex: glycine, dizocilpine (MK-801) and (±)-3-(2-car-boxypiperazin-4-yl)propyl-1-phosphonate (CPP). GSH and GSSG strongly inhibited the binding of glutamate, CPP and AMPA, kainate and glycine binding being less affected. Both peptides enhanced the binding of dizocilpine in a time- and concentration-dependent manner. This activatory effect was not additive to that of saturating concentrations of glutamate or glutamate plus glycine. The activation of dizocilpine binding by GSH and GSSG was prevented by the competitive NMDA and glycine antagonists DL-2-amino-5-phosphonovalerate and 7-chlorokynurenate. GSH and GSSG may be endogenous ligands of AMPA and NMDA receptors, binding preferably to the glutamate recognition site via their -glutamyl moieties. In addition to this, at millimolar concentrations they may regulate the redox state of the NMDA receptor-ionophore complex.     

Increased efflux of oxidized glutathione (GSSG) causes glutathione depletion and potentially diminishes antioxidant defense in sickle erythrocytes

Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress.     

A Possible Role of Glutathione as an Endogenous Agonist at the N-Methyl-d-Aspartate Recognition Domain in Rat Brain

Abstract: Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of l -[³H]-glutamic acid (l -[³H]Glu) and dl -(E)-2-[³H]amino-4-propyl-5-phosphono-3-pentenoic acid ([³H]CGP-39653) in rat brain synaptic membranes, with less potent displacement of binding of dl -α-amino-3-hydroxy-5-[³H]-methylisoxazole-4-propionic and [³H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with l -Glu by <1%. Both GSH and GSSG potentiated (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) binding in a manner similar to that found with l -Glu. Pre-treatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration-response curve for l -Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [³H]MK-801 binding to a somewhat smaller extent than that found in the presence of l -Glu at a maximally effective concentration. The potentiation of [³H]MK-801 binding by GSH was invariably attenuated by addition of CGP-39653, d -2-amino-5-phosphonovaleric acid (d -AP5), and 5,7-dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP-39653, d -AP5, DCKA, and 6,7-dichloroquinoxaline-2,3-dione to inhibit [³H]MK-801 binding when determined in the presence of both l -Glu and Gly. These results suggest that glutathione may be an endogenous agonist selective for the N-methyl-d -aspartate (NMDA) recognition domain on the NMDA receptor ionophore complex.     

Circulating glutathione concentrations in marine,semiaquatic, and terrestrial mammals

An important low molecular weight antioxidant in biological systems is glutathione; its efficiency depends on the equilibrium between its reduced (GSH) and oxidized (GSSG) forms. The oxidized:total glutathione (GSSG:GSH‐Eq) ratio can be used as an indicator of oxidative stress. Previous studies suggest that marine mammals, unlike terrestrial mammals, do not show adverse effects in tissues exposed to ischemia/reperfusion during the peripheral vasoconstriction associated with breath‐hold diving. This is due, in part, to higher antioxidant enzyme activities in marine mammals compared with terrestrial mammals. The objective of this study was to compare circulating glutathione levels among mammals with different diving capacities. Circulating GSH‐Eq, GSH, and GSSG concentrations in erythrocyte samples from northern elephant seals (Mirounga angustirostris), bottlenose dolphins (Tursiops truncatus), neotropical otters (Lontra longicaudis annectens), domestic pigs (Sus scrofa), and humans were quantified using spectrophotometry. Higher GSH‐Eq and GSH concentrations and a lower GSSG:GSH‐Eq index were found in erythrocytes from northern elephant seals and bottlenose dolphins as compared to otters, domestic pigs, and humans. Results suggest that marine mammals, independent of their diving capacity, possess a highly developed antioxidant system, including GSH; continuous availability of GSH could allow these species to avoid oxidative damage and tolerate ischemia/reperfusion and hypoxia/reoxygenation events associated with diving.     

Comparison between the effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state in ex vivo culture of CD34(+) cells

Hypoxia maintained biological characteristics of CD34(+) cells through keeping lower intracellular reactive oxygen specials (ROS) levels. The effects of normoxia and hypoxia on antioxidant enzymes and glutathione redox state were compared in this study. Hypoxia decreased the mRNA expression of both catalase (CAT) and glutathione peroxidase (GPX), but not affected mRNAs expression of superoxide dismutase (SOD). While the cellular GPX activities under hypoxia were apparently less than those under normoxia, neither SOD activities nor CAT activities were affected by hypoxia. The analysis of glutathione redox status and ROS products showed the lower oxidized glutathione (GSSG) levels, the higher reduced glutathione (GSH) levels, the higher GSH/GSSG ratios, and the less O(2)- and H(2)O(2) generation under hypoxia (versus normoxia). Meanwhile more primary CD34(+)CD38(-) cells were obtained when cultivation was performed under hypoxia or with N-acetyl cysteine (the precursor of GSH) under normoxia. These results demonstrated the different responses of anti-oxidative mechanism between normoxia and hypoxia. Additionally, the present study suggested that the GSH-GPX antioxidant system played an important role in HSPCs preservation by reducing peroxidation.     

Taurine release in developing mouse hippocampus is modulated by glutathione and glutathione derivatives

Summary. Glutathione (reduced form GSH and oxidized form GSSG) constitutes an important defense against oxidative stress in the brain, and taurine is an inhibitory neuromodulator particularly in the developing brain. The effects of GSH and GSSG and glycylglycine, γ-glutamylcysteine, cysteinylglycine, glycine and cysteine on the release of [³H]taurine evoked by K⁺-depolarization or the ionotropic glutamate receptor agonists glutamate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) were now studied in slices from the hippocampi from 7-day-old mouse pups in a perfusion system. All stimulatory agents (50 mM K⁺, 1 mM glutamate, 0.1 mM kainate, 0.1 mM AMPA and 0.1 mM NMDA) evoked taurine release in a receptor-mediated manner. Both GSH and GSSG significantly inhibited the release evoked by 50 mM K⁺. The release induced by AMPA and glutamate was also inhibited, while the kainate-evoked release was significantly activated by both GSH and GSSG. The NMDA-evoked release proved the most sensitive to modulation: L-Cysteine and glycine enhanced the release in a concentration-dependent manner, whereas GSH and GSSG were inhibitory at low (0.1 mM) but not at higher (1 or 10 mM) concentrations. The release evoked by 0.1 mM AMPA was inhibited by γ-glutamylcysteine and cysteinylglycine, whereas glycylglycine had no effect. The 0.1 mM NMDA-evoked release was inhibited by glycylglycine and γ-glutamylcysteine. In turn, cysteinylglycine inhibited the NMDA-evoked release at 0.1 mM, but was inactive at 1 mM. Glutathione exhibited both enhancing and attenuating effects on taurine release, depending on the glutathione concentration and on the agonist used. Both glutathione and taurine act as endogenous neuroprotective effectors during early postnatal life. Authors’ address: Prof. Simo S. Oja, Brain Research Center, Medical School, FI-33014 University of Tampere, Finland     

External GSSG enhances intracellular glutathione level in isolated cardiac myocytes

The addition of external GSSG at concentrations in the range 50-500 microM produces in isolated adult rat heart myocytes an increase of GSH level and only a slight increase of GSSG level. On the contrary, external GSH at the above same indicated concentrations did not change the cell glutathione pool. The pretreatment of the cells with diethylamaleate depleted the myocytes of glutathione and enhanced the GSSG-induced replenishment effect on GSH level. On the contrary, the addition of GSH did not increase the concentration of cell glutathione. The level of cell GSH in diethylmaleate-treated myocytes was not increased after 30 min of incubation with cysteine, or acetylcysteine. The GSSG induced-stimulation on GSH level was not inhibited by buthionine sulfoximine, an inhibitor of glutathione synthesis. On the contrary, this stimulatory effect was inhibited by N, N-bis(2-chloroethyl)-N-nitrosourea, an inhibitor of glutathione reductase, or partially, by the remotion of glucose from the incubation medium. These results support the idea that the isolated adult rat heart myocytes are able to utilize external GSSG in order to increase the intracellular glutathione pool, probably through the reduction of the imported GSSG to GSH.     

The effect of aging on glutathione and cysteine levels in different regions of the mouse brain

A general glutathione (GSH) deficiency occurs in many tissues of the aging mouse. However, there is no information on GSH in the aging brain even though it has been involved in a number of neurobiologic reactions. To this end, C57BL/6 mice, 3-31 months old, representing the growth, maturation, and aging periods of the life-span were studied. Brain cortex, hippocampus, and stem samples were dissected, processed, and analyzed specifically for reduced and oxidized glutathione (GSH, GSSG) and cyst(e)ine using high performance liquid chromatography with dual electrochemical detection. The GSH content of each brain region varied in the order brain cortex greater than brain hippocampus greater than brainstem. However, the GSH profiles of all regions were the same through the life-span, namely, high values during growth dropping to a maturation plateau and then decreasing 30% during aging. In contrast to GSH, the order of cysteine levels was brain cortex less than brain hippocampus less than brainstem and no life-span changes occurred in any region. In addition, the brain GSSG and cystine contents of all regions were very low and did not change during the life-span. Thus, the GSH loss was not accountable by oxidation to GSSG or degradation to cyst(e)ine. Altogether these results demonstrated a GSH deficiency in brain tissues of aging mice like that found previously in other tissues. These findings suggest an increased susceptibility of the aging brain to oxidative damage.     

Glutathione redox system,GSH-Px activity and lipid peroxidation (LPO) levels in tadpoles of R.r.ridibunda and B.viridis

Total glutathione (t-GSH), reduced glutathione (GSH), glutathione disulphide (GSSG) levels, t-GSH/GSSG ratio, glutathione peroxidase (GSH-Px) activity and lipid peroxidation (LPO) levels were investigated during the development period of a predominantly aquatic amphibian R.r.ridibunda and a predominantly terrestrial amphibian B. viridis. While t-GSH and GSH showed a similar trend, GSSG concentration increased significantly (p<0.05) during the larval stages in R.r.ridibunda larvae. In contrast to R.r.ridibunda larvae, there was no significant (p>0.05) change between 1 and 5 weeks in the t-GSH and GSH concentrations of B. viridis. t-GSH and GSH concentrations of B. viridis larvae became sharply elevated after the fifth week, GSSG levels increased 3.25-fold during the metamorphosis. The t-GSH/GSSG ratio fluctuated and the lowest t-GSH/GSSG ratios were observed at the third week for both species. GSH-Px activities for both species increased significantly (p<0.05) during the growing period. The highest GSH-Px activities in R.r.ridibunda and B.viridis were observed at the eighth week and they were 3.45 +/- 0.17 and 4.1 +/- 0.21 IU mg(-1), respectively. The membrane LPO levels in the R.r.ridibunda and B. viridis tadpoles significantly (p<0.001) decreased from 206 +/- 10.3 to 146 +/- 7.3 and from 198 +/- 9.9 to 23 +/- 1.15 nmol MDA g(-1) w.w., respectively.     

Glutathione in Intact Vacuoles: Comparison of Glutathione Pools in Isolated Vacuoles,Plastids, and Mitochondria from Roots of Red Beet

Proportions between oxidized and reduced glutathione forms were determined in vacuoles isolated from red beet (Beta vulgaris L.) taproots. The pool of vacuolar glutathione was compared with glutathione pools in isolated plastids and mitochondria. The ratio of glutathione forms was assessed by approved methods, such as fluorescence microscopy with the fluorescent probe monochlorobimane (MCB), high-performance liquid chromatography (HPLC), and spectrophotometry with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The fluorescence microscopy revealed comparatively low concentrations of reduced glutathione (GSH) in vacuoles. The GSH content was 104 μM on average, which was lower than the GSH levels in mitochondria (448 μM) and plastids (379 μM). The content of reduced (GSH) and oxidized (GSSG) glutathione forms was quantified by means of HPLC and spectrophotometric assays with DTNB. The glutathione concentrations determined by HPLC in the vacuoles were 182 nmol GSH and 25 nmol GSSG per milligram protein. The respective concentrations of GSH and GSSG in the plastids were 112 and 6 nmol/mg protein and they were 228 and 10 nmol/mg protein in the mitochondria. The levels of GSH determined with DTNB were 1.5 times lower, whereas the amounts of GSSG were, by contrast, 1.5–2 times higher than in the HPLC assays. Although the glutathione redox ratios depended to some extent on the method used, the GSH/GSSG ratios were always lower for vacuoles than for plastids and mitochondria. In vacuoles, the pool of oxidized glutathione was higher than in other organelles.     

The multidrug resistance protein 1 (Mrp1), but not Mrp5, mediates export of glutathione and glutathione disulfide from brain astrocytes

Astrocytes play an important role in the glutathione (GSH) metabolism of the brain. To test for an involvement of multidrug resistance protein (Mrp) 1 and 5 in the release of GSH and glutathione disulfide (GSSG) from astrocytes, we used astrocyte cultures from wild-type, Mrp1-deficient [Mrp1(-/-)] and Mrp5-deficient [Mrp5(-/-)] mice. During incubation of wild-type or Mrp5(-/-) astrocytes, GSH accumulated in the medium at a rate of about 3 nmol/(h.mg), whereas the export of GSH from Mrp1(-/-) astrocytes was only one-third of that. In addition, Mrp1(-/-) astrocytes had a 50% higher specific GSH content than wild-type or Mrp5(-/-) cells. The presence of 50 microm of the Mrp inhibitor MK571 inhibited the rate of GSH release from wild-type and Mrp5(-/-) astrocytes by 60%, but stimulated at the low concentration of 1 microm GSH release by 40%. In contrast, both concentrations of MK571 did not affect GSH export from Mrp1(-/-) astrocytes. Moreover, in contrast to wild-type and Mrp5(-/-) cells, GSSG export during H(2)O(2) stress was not observed for Mrp1(-/-) astrocytes. These data demonstrate that in astrocytes Mrp1 mediates 60% of the GSH export, that Mrp1 is exclusively responsible for GSSG export and that Mrp5 does not contribute to these transport processes.     

Effects of Nickel on Human and Fish Red Blood Cells

The objective of this study was to assess the effects of nickel chloride on human and rainbow trout erythrocytes in vitro. The cells were incubated with 0, 0.5 and 1 mM nickel chloride for 1 h at pH 7.40 and 25°C, then K⁺ efflux, SO₄²⁻ uptake and GSH and GSSG concentrations were measured. In both kind of cells, “high concentration” nickel treatment increased KCl efflux with respect to the control. The SO₄²⁻ uptake was not significantly different at “low nickel concentration” but was lower in erythrocytes treated with 1 mM nickel chloride; the rate constant of SO₄²⁻ uptake decreased by 35% in human erythrocytes and by 44% in fish erythrocytes. Nickel chloride also acts on cellular metabolism and in particular on erythrocyte glutathione peroxidase with consequent increase in oxidative stress; the data show a significant decrease in intracellular GSH in both human (25%) and fish erythrocytes (18%) after treatment with nickel chloride, with concomitantly high GSSG concentrations and lower GSH/GSSG ratios.     

THE ACTIVITIES OF BUTYRYLCHOLINESTERASE AND CARBONIC ANHYDRASE, THE RATE OF ANAEROBIC GLYCOLYSTS, AND THE QUESTION OF A CONSTANT DENSITY OF GLIAL CELLS IN CEREBRAL CORTICES OF VARIOUS MAMMALIAN SPECIES FROM MOUSE TO WHALE

Abstract— The question of a constant density of glial cells in mammalian cerebral cortex regardless of species was examined by surveying the cortical activities of two enzymes primarily localized to dial cells. The cortical activity of butyrylcholinesterase (EC 3.1.1.8) was essentially constant at a rate of approx. 0.1 μmol of butyrylthiocholine hydrolysed min^-1 g^-1 over the range of species from rat (brain wt., 1.6 g) to fin whale and sperm whale (brain wt., 6800 and 7800 g, respectively). Over the same range the activity of cortical acetylcholinesterase, a neuronal enzyme, decreases by a factor of 7. Thus, butyrylcholinesterase ranged from < 2 per cent (in small rodent brains) to approximately 10 per cent (in whale brain) of the cortical acetylcholinesterase activity. The cortical activity of carbonic anhydrase (EC 4.2.1.1) was constant at a rate of 6.2 (± 0.25) μmol of CO₂ evolved min^-1 g^-1 over the range of species from guinea-pig (brain wt., 4.75 g) to fin whale (brain wt., 6800 g). These data obtained by assaying the dehydration reaction were confirmed by limited assays of the esterase activity of the enzyme (with p-nitrophenylacetate as substrate) and agreed with limited, previously reported data for the hydration reaction. Thus, the circumstantial evidence strongly favoured a relative constancy of cortical glial cell density regardless of species. The rates of anaerobic glycolysis in the cerebral cortex of various species were also investigated. For six species from mouse (brain wt., 0.4 g) to beef (brain wt., 380 g) cortical anaerobic glycolysis varied only slightly in the range of 50–62 μmol of CO₂ evolved h^-1 g^-l, whereas cortical oxygen consumption for the same range of species decreased by a factor of 3. Previously frozen samples of beef cortex glycolysed at 35 per Cent of the rate of fresh (unfrozen) samples. Since identical rates were obtained for previously frozen samples of fin whale cerebral cortex, we concluded that the relative constancy of cortical anaerobic glycolysis could be extended to the range from mouse to whale and that this aspect of cortical metabolism is probably primarily glial in localization. Some implications of the latter conclusion for the proposed role of astrocytes as modulators of neuronal activity have been discussed.