Chemical and pathological oxidative influences on band 3 protein anion-exchanger.

BACKGROUND/AIMS: The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl co-transport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells. METHODS: To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg2+ and tested for sulphate (SO4-) uptake, K+ efflux, G6PDH activity and glutathione (GSH) concentration. RESULTS: In normal red blood cells, the rate constants of SO4- uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg2+ in parallel to a significant increase in sulphate transport. In both cells, the oxidizing agents increased K+ efflux with depletion of GSH. CONCLUSION: The data are discussed in light of the possible opposite effects exerted by oxidative agents and Mg2+ on KCC and on the protein tyrosine kinase (PTK)-protein tyrosine phosphatase (PTP) equilibrium. The decreased sulphate uptake observed in the experimental and pathological conditions could be due to band 3 SH-groups oxidation or to oxidative stress-induced K-Cl symport-mediated cell shrinkage with concomitant band 3 tyrosine phosphorylation.     

Influence of glutathione (GSH) on net uptake of sulphate and sulphate transport in tobacco plants

Net uptake of sulphate by the roots, and its transport to theshoot was inhibited in intact tobacco plants by 1 h exposureof its roots to 0.1 or 1.0 mM GSH. The relative amount of sulphatetaken up that was transported to the shoot was not affectedby this treatment. Apparently, sulphate uptake, but not xylemloading of sulphate, was inhibited by GSH. Similar results wereobtained when L-cysteine was used instead of GSH. As L-cysteine,mediated inhibition of sulphate, net uptake was counteractedby exposure of the roots to BSO, GSH synthesis seems to be requiredfor inhibition of sulphate uptake by L-cysteine. However, exposureto 0.1 or 1.0mM GSH did not result In an enhanced GSH levelin roots or shoots. Also the cysteine contents remained unchanged.Small changes in metabolically active pools of GSH or cysteineseem to be responsible for the inhibition of sulphate uptakeand its transport to the shoot. Flap-feeding of GSH to a matureleaf resulted in an inhibition of net uptake of sulphate bythe roots and its transport to the shoot. These findings supportthe hypothesis that GSH produced in the leaves acts as a signalto control sulphur nutrition of plants. Key words: Sulphate transport, sulphate uptake, xylem loading, Nicotiana, Solanaceae, glutathione, cysteine, buthionine sulphoximine     

Antibiotic effects on SO4(2-) uptake in human erythrocytes

The erythrocyte is a cell highly exposed to oxygen pressure that, in turn, provokes oxidative stress involving loss of SH-groups, cell shrinkage by activation of K(+)-Cl(-) cotransport (KCC) and membrane destabilization which plays an important role in the premature haemolysis of red blood cells (RBCs). Oxidative stress provoked by chemicals frequently occurs in human erythrocytes. The aim of this study was to test whether the antibiotics alter the redox state and investigate their influences on band 3 protein that is involved in the facilitated electro neutral exchange of Cl(-) for HCO(3)(-) across the membrane of mammalian erythrocytes. Normal erythrocytes were treated with some antibiotics and thiol oxidizing agent N-ethylmaleimide (NEM) and tested for sulphate uptake, K(+) efflux and for glutathione (GSH) concentration as an index of oxidative stress. The rate constant of SO(4)(=) uptake measured in erythrocytes treated with antibiotics as well as NEM was decreased with respect to control cells as a result of band 3 SH-groups oxidation or the stress-induced K(+)-Cl(-) symport-mediated cell shrinkage. In fact, this hypothesis was verified by increased K(+) efflux and decreased GSH values measured in treated erythrocytes compared to controls.     

Oxygen binding by marine fish blood under hypothermic experimental conditions

Influence of temperature in the range of 1-15 degrees C on oxygen binding properties of blood of thermophilic--golden mullet (Liza aurata), anchovy (Engraulis encrasicolus), and cold-tolerant--sardelle (Clupeonella cultriventris) fishes has been investigated under experimental conditions. Heat dependence of oxygenation reaction in thermophilic fish blood at temperature below 10 degrees C considerably increases, which is evidenced by high deltaH values. That is accompanied by a substantial increase of blood oxygen affinity and complicates blood deoxygenation at the tissue level. This reaction is apparently determined by the change of hemoglobin interaction with intraerythrocyte medium. The concentration of NTP in erythrocytes increases, that partially compensates negative changes of blood oxygen affinity (parameter P50 is raised) under long-term maintenance of fishes at 5 degrees C. However this reaction is not observed at low temperatures (1-2 degrees C).     

Evidence for a role of taurine in the in vitro oxidative toxicity of neutrophils toward erythrocytes

Production of hydrogen peroxide and secretion of myeloperoxidase by stimulated neutrophils resulted in myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl), the reaction of HOCl with taurine to yield taurine monochloramine (TauNHCl), and accumulation of TauNHCl in the extracellular medium. When erythrocytes were present, the yield of TauNHCl was lower as the result of uptake of TauNHCl into erythrocytes. The zwitterion taurine was not taken up, but the anion TauNHCl and other anionic oxidants including taurine dichloramine (TauNCl2) and L-alanine chloramines were transported into erythrocytes by the anion-transport system. Oxidation of intracellular components such as glutathione (GSH) by taurine chloramines resulted in reduction of the chloramines and trapping of taurine within erythrocytes. At high oxidant:erythrocyte ratios, TauNHCl also oxidized hemoglobin (Hb) and depleted ATP, but caused little lysis. TauNCl2 was much more effective as a lytic agent. At low oxidant:erythrocyte ratios, the chloramines caused net loss of GSH when no glucose was provided, but Hb was not oxidized and GSH content returned to normal when glucose was added. Therefore, anionic chloramines may mediate oxidative toxicity when the neutrophil:erythrocyte ratio is high. Under more physiologic conditions, chlorination of taurine by neutrophils and the uptake and reduction of TauNHCl by erythrocytes prevents accumulation of oxidants and may protect blood cells, plasma components, and tissues against oxidative toxicity.     

Determination of reduced and oxidized glutathione in erythrocytes by high-performance liquid chromatography with ultraviolet absorbance detection

A method is described for simultaneous quantitation of reduced (GSH) and oxidized (GSSG) glutathione in erythrocytes by HPLC. They were determined by standard addition method. Blood samples were collected in tubes containing 1,10-phenanthroline. The separated erythrocytes were hemolyzed with water containing standard. After deproteinization, GSH and GSSG were converted to N-(2,4-dinitrophenyl) derivatives and analyzed by HPLC with UV detection. The coefficients of variation of GSH and GSSG on replicate assays were 6% and 8%, respectively. The stabilities of GSH and GSSG and of the derivatives were also examined. The present method appears to be satisfactory for determination of these physiological concentrations in erythrocytes.     

The transport of oxidized glutathione from the erythrocytes of various species in the prescence of chromate

1. Erythrocytes from normal and glucose 6-phosphate dehydrogenase-deficient humans were subjected to hydrogen peroxide diffusion to oxidize the GSH. Studies were carried out in the presence and absence of chromate to inhibit glutathione reductase and with or without the addition of glucose. 2. The GSH content of erythrocytes from other species was oxidized by subjecting them to hydrogen peroxide diffusion in the presence of chromate and glucose. 3. Chromate (1.3mm) inhibited glutathione reductase by about 80%, whereas glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase and pyruvate kinase were not inhibited. 4. The GSSG formed was transported from the erythrocytes to the medium. 5. The transport rate of GSSG from glucose 6-phosphate dehydrogenase-deficient erythrocytes subjected to hydrogen peroxide diffusion in the presence of chromate was comparable with that from normal and glucose 6-phosphate dehydrogenase-deficient erythrocytes. 6. The rate of transport of GSSG from erythrocytes of various species studied could be ranked: pigeon>rabbit>rat>donkey>man>dog>horse>sheep>chicken>fish.     

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.     

Rethinking cystic fibrosis pathology: the critical role of abnormal reduced glutathione (GSH) transport caused by CFTR mutation

Though the cause of cystic fibrosis (CF) pathology is understood to be the mutation of the CFTR protein, it has been difficult to trace the exact mechanisms by which the pathology arises and progresses from the mutation. Recent research findings have noted that the CFTR channel is not only permeant to chloride anions, but other, larger organic anions, including reduced glutathione (GSH). This explains the longstanding finding of extracellular GSH deficit and dramatically reduced extracellular GSH:GSSG (glutathione disulfide) ratio found to be chronic and progressive in CF patients. Given the vital role of GSH as an antioxidant, a mucolytic, and a regulator of inflammation, immune response, and cell viability via its redox status in the human body, it is reasonable to hypothesize that this condition plays some role in the pathogenesis of CF. This hypothesis is advanced by comparing the literature on pathological phenomena associated with GSH deficiency to the literature documenting CF pathology, with striking similarities noted. Several puzzling hallmarks of CF pathology, including reduced exhaled NO, exaggerated inflammation with decreased immunocompetence, increased mucus viscoelasticity, and lack of appropriate apoptosis by infected epithelial cells, are better understood when abnormal GSH transport from epithelia (those without anion channels redundant to the CFTR at the apical surface) is added as an additional explanatory factor. Such epithelia should have normal levels of total glutathione (though perhaps with diminished GSH:GSSG ratio in the cytosol), but impaired GSH transport due to CFTR mutation should lead to progressive extracellular deficit of both total glutathione and GSH, and, hypothetically, GSH:GSSG ratio alteration or even total glutathione deficit in cells with redundant anion channels, such as leukocytes, lymphocytes, erythrocytes, and hepatocytes. Therapeutic implications, including alternative methods of GSH augmentation, are discussed.     

Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.     

Useful agents for the study of glutathione metabolism in erythrocytes. Organic hydroperoxides

t-Butyl hydroperoxide and cumene hydroperoxide, both known to be substrates for glutathione peroxidase, were used to oxidize erythrocyte GSH. Addition of concentrations of hydroperoxides equimolar with respect to GSH in the erythrocytes or whole blood quantitatively oxidizes GSH in the erythrocytes with a half-time of 4.5s at 37 degrees C and about three times as long at 4 degrees C. In the presence of glucose, normal erythrocytes regenerate all the GSH in about 25min. However, glucose 6-phosphate dehydrogenase-deficient erythrocytes failed to regenerate GSH. Treatment of erythrocytes with hydroperoxides does not affect erythrocyte survival in rabbits. Oxidation of erythrocyte GSH with equimolar concentrations of hydroperoxides does not lead to formation of mixed disulphides of haemoglobin and GSH. The hydroperoxides do not affect erythrocyte glycolytic and hexose monophosphate-shunt-pathway enzymes. Previous studies on transport of GSSG from erythrocytes were confirmed by using t-butyl hydroperoxide to oxidize erythrocyte GSH.     

Penetration of 2,4,6-trinitrobenzenesulfonate into human erythrocytes: Consequences for studies on phospholipid asymmetry

The glutathione content of human erythrocytes rapidly diminishes when cells are exposed to 2,4,6-trinitrobenzenesulfonate (20 μmol/l cells) at 37°C. Even at 0°C a slow decrease in glutathione content is observed. The uptake of trinitrobenzenesulfonate by the cells is retarded by inhibitors of the inorganic anion exchange system, indicating that trinitrobenzenesulfonate enters the cells by this pathway.The disappearance of glutathione most probably results from the reaction: 2 GSH + trinitrobenzenesulfonate → GSSG + aminodinitrobenzenesulfonate The reaction of trinitrobenzenesulfonate with glutathione occurs prior to its covalent binding to amino groups of hemoglobin which makes this reaction a more sensitive method of detection of penetration of trinitrobenzenesulfonate into erythrocytes. Results of studies on the asymmetric distribution of phospholipids using trinitrobenzenesulfonate as the only probe should be reconsidered in the light of these new data.     

Morphofunctional peculiarities of erythrocytes in wild and farmed Coregonid fishes from Lake Baikal

Structure and cytometric indices of red blood cells (RBC) and hemoglobin content (Hb) and oxygen capacity of the blood (OCB) of omul, whitefish, and hybrids thereof captured in Lake Baikal (wild) and incubated and grown in a freshwater aquarium complex (FAC) (farmed) have been analyzed. Cytometric parameters of red blood cells of wild omul, whitefish, and hybrids thereof exceed those of the cells of fish reared in aquariums under identical conditions. The effect of aquarium rearing on the shape of red blood cells is the least pronounced in Siberian whitefish and F1 progeny of Siberian whitefish females and omul males (f Sw x m Om). The erythrocyte size in hybrids of female Lacustrine whitefish and male omul (f Lw x m Om) is determined by the size of these cells in female parents, since female Lacustrine whitefish have the largest erythrocytes. Cytometric parameters of erythrocytes of all Coregonid fishes investigated are higher in fish reared in warm aquaria than in conspecifics reared in aquaria with cold water. Erythrocyte nuclei are smaller in artificially propagated hybrids than in parent fish captured in the wild or in whitefish and omul reared in aquaria under the same conditions. A distinct pool of erythrocytes from whitefish captured in the wild have a 20–30% higher content of functionally active mitochondria than erythrocytes of whitefish reared in aquaria; a disrupted mitochondrial structure is also observed in erythrocytes from the latter population of fish. The results show that distinctive features of metabolism related to oxygen transport in the Baikal coregonid fish that were investigated are determined by adaptation to the conditions of the ecological niches occupied by the fish.     

Interaction of sulfate and glutathione transport in cultured tobacco cells

Photoheterotrophic and heterotrophic suspension cultures of tobacco (Nicotiana tabacum L.) were grown with 1 mM glutathione (reduced; GSH) as sole source of sulfur. Addition of sulfate to both cultures did not alter the rate of exponential growth, but affected the removal of GSH and sulfate in different ways. In photoheterotrophic suspensions, addition of sulfate caused a decline in the net uptake of GSH, whereas sulfate was taken up by the green cells immediately. In heterotrophic suspensions, however, addition of sulfate did not affect the net uptake of GSH and sulfate was only taken up by the cells after the GSH supply in the medium had been exhausted. Apparently, GSH uptake in photoheterotrophic cells is inhibited by sulfate, whereas sulfate uptake is inhibited by GSH in heterotrophic cells. The differences in the effect of GSH on sulfate uptake in photoheterotrophic and heterotrophic tobacco suspensions cannot be attributed to differences in the kinetic properties of sulfate carriers. In short-time transport experiments, both cultures took up sulfate almost entirely by an active-transport system as shown by experiments with metabolic inhibitors; sulfate transport of both cultures obeyed monophasic Michaelis-Menten kinetics with similar app. K_m (photoheterotrophic cells: 16.0±2.0 M; heterotrophic cells: 11.8±1.8 M) and V_max (photoheterotrophic cells: 323±50 nmol·min^-1·g^-1 dry weight; heterotrophic cells: 233±3 nmol·min^-1·g^-1 dry weight). Temperature- and pH-dependence of sulfate transport showed almost identical patterns. However, the cultures exhibited remarkable differences in the inhibition of sulfur influx by GSH in short-time transport experiments. Whereas 1 mM GSH inhibited sulfate transport into heterotrophic tobacco cells completely, sulfate transport into photoheterotrophic cells proceeded at more than two-thirds of its maximum velocity at this GSH concentration. The mode of action of GSH on sulfate transport in chloroplast-free tobacco cell does not appear to be direct: a 14-h exposure to 1 mM GSH was found to be necessary to completely block sulfate transport; a 4-h time of exposure did not affect this process. Consequently, glutathione does not seem to be a product of sulfur metabolism acting on sulfate-carrier entities by negative feedback control. When transferred to the whole plant, the observed differences in sulfate and glutathione influx into green and chloroplast-free cells may be interpreted as a regulatory device to prevent the uptake of excess sulfate by plants.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - DNP dinitrophenol - DW dry weight - FW fresh weight - GSH reduced glutathione     

Stimulation of glutathione absorption in rat small intestine by alpha-adrenergic agonists.

The alpha-adrenergic agonist, phenylephrine (1.6 microM), caused a threefold stimulation of glutathione (GSH) transport from the lumen into the vasculature in isolated, vascularly perfused rat small intestine. Stimulation of GSH transport by phenylephrine was blocked by the alpha-adrenergic antagonists, prazosin or phentolamine. Norepinephrine and epinephrine (both alpha and beta agonists) also stimulated GSH absorption but not to the same extent as phenylephrine. Isoproterenol, a strict beta-adrenergic agonist, had no effect on the rate of GSH absorption. Under physiological luminal GSH concentrations, phenylephrine stimulated GSH efflux from the lumen, accumulation in the intestinal mucosa, and transport into the mesenteric vasculature. Phenylephrine did not stimulate the transport of polyethylene glycol, a high molecular weight molecule, and stimulated uptake of cysteine and glycine by 30%. This suggests that the effect of phenylephrine on GSH transport is not due to enhanced bulk flow through paracellular pathways. Studies with isolated small intestinal epithelial cells showed that phenylephrine also stimulated the release of GSH from the cells. Oral administration of phenylephrine with GSH caused a two- to fivefold transient increase in plasma GSH concentrations in rats. Phenylephrine alone or with the amino acid constituents of GSH caused no increase in plasma GSH concentration. Thus, absorption of dietary GSH is under hormonal regulation. The physiological importance of this regulation is not known, although such regulation may function to control utilization of dietary GSH for detoxication and may have therapeutic benefits for individuals with deficient GSH or increased risk of oxidative or chemically induced injury.     

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.     

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

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

Direct evidence for the role of the membrane potential in glutathione transport by renal brush-border membranes

Transport of GSH was studied in isolated rat kidney cortical brush-border membrane vesicles in which gamma-glutamyltransferase had been inactivated by a specific affinity labeling reagent, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Transport of intact 2-3H-glycine-labeled GSH occurred into an osmotically active intravesicular space of AT-125-treated membranes. The initial rate of transport followed saturation kinetics with respect to GSH concentrations; an apparent Km of 0.21 mM and Vmax of 0.23 nmol/mg protein X 20 were calculated at 25 degrees C with a 0.1 M NaCl gradient (vesicle inside less than vesicle outside). Sodium chloride in the transport medium could be replaced with KCl without affecting transport activity. The rate of GSH uptake was enhanced by replacing KCl in the transport medium with K2SO4, providing a less permeant anion, and was reduced by replacing KCl with KSCN, providing a more permeant anion. The rate of GSH transport markedly decreased in the absence of a K+ gradient across the vesicular membranes and was enhanced by a valinomycin-induced K+ diffusion potential (vesicle-inside-positive). These results indicate that GSH transport is dependent on membrane potential and involves the transfer of negative charge. The rate of GSH transport was inhibited by S-benzyl glutathione but not by glycine, glutamic acid, and gamma-glutamyl-p-nitroanilide. When incubated with [2-3H]glycine-labeled GSH, intact untreated vesicles also accumulated radioactivity; the rate of uptake was significantly higher in a Na+ gradient than in a K+ gradient. Sodium-dependent transport, but not sodium-independent uptake, was almost completely inhibited by a high concentration of unlabeled glycine. At equilibrium, most of the radioactivity which accumulated in the intravesicular space was accounted for by free glycine. These results suggest that GSH which is secreted into the tubular lumen by a specific translocase in the lumenal membranes or filtered by the glomerulus may be degraded in situ by membranous gamma-glutamyltransferase and peptidase activities which hydrolyze peptide bonds of cysteinylglycine and its derivatives. The resulting free amino acids can be reabsorbed into tubule cells by sodium-dependent transport systems in renal cortical brush-border membranes.