Biomedical subjects

B Hamprecht

Publications and source records attributed to B Hamprecht.

At least 37 records · Page 2Linked to original sources

PubMed1998

Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH.

Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, D,L-3-hydroxybutyrate, acetoacetate, alpha-oxoisohexanoate and alpha-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor alpha-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.

Animals↗

PubMed1998

Malic enzyme isoforms in astrocytes: comparative study on activities in rat brain tissue and astroglia-rich primary cultures.

Anion exchange chromatography on diethylaminoethyl cellulose was optimized to separate the cytosolic and mitochondrial isoforms of malic enzyme from rat brain. Extracts of adult rat brain and of astroglia-rich primary cultures derived from the brains of newborn rats were analyzed for their content of the two isozymes. In the case of brain tissue 45% of malic enzyme activity was due to the cytosolic isoform. In contrast, in extracts from astroglia-rich primary cultures more than 95% of the total activity was associated with the cytosolic isozyme. From these data it is concluded that the cytosolic rather than the mitochondrial isoform of malic enzyme has prominent functions in astroglial metabolism.

Animals↗

PubMed1998

Rapid clearance of tertiary butyl hydroperoxide by cultured astroglial cells via oxidation of glutathione.

The ability of astroglial cells to detoxify exogenously applied tertiary butyl hydroperoxide (tBHP) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. If 200 microM tBHP was applied, this compound disappeared from the incubation buffer with an apparent half-life of about 5 min. After 20 min incubation tBHP was not detectable any more. A decay of tBHP was found even in the absence of cells. Therefore, half-times for the cell-dependent tBHP clearance were corrected for the cell-independent decay of tBHP. The cell-dependent half-time of tBHP in the incubation buffer was found strongly elevated i) with increasing concentration of tBHP, ii) after decrease of the glutathione content of the cells by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, iii) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and iv) in the absence of glucose, the precursor for the generation of NADPH. Incubation of astroglial cells with 200 microM tBHP in the absence of glucose led to a 46% oxidation of the cellular glutathione within 30 s. Under these conditions the cells were unable to restore the original high ratio of the concentrations of GSH to GSSG within 30 min of incubation. In contrast, if glucose was present the level of GSSG encountered on incubation with tBHP was lower (32% of total glutathione after 30 s) and the original ratio of the levels of GSH to GSSG was essentially reestablished within 10 min. In the presence of 3 mM mercaptosuccinate oxidation of glutathione was almost completely inhibited. These results demonstrate that an exogenous hydroperoxide is detoxified rapidly by astroglial cells via the glutathione system.

Animals↗

PubMed1998

Anethole dithiolethione, a putative neuroprotectant, increases intracellular and extracellular glutathione levels during starvation of cultured astroglial cells.

Astroglial cells protect neurons against oxidative damage. The antioxidant glutathione plays a pivotal role in the neuroprotective action of astroglial cells which is impaired following loss of glutathione. Anethole dithiolethione (ADT), a sulfur-containing compound which is used in humans as a secretagogue, increases glutathione levels in cultured astroglial cells under "physiological" conditions and is thought thereby to protect against oxidative damage. Presently, we report the effect of ADT (3-100 microM) on glutathione content of and efflux from rat primary astroglia-rich cultures under "pathological" conditions, i.e., extended deprivation of glucose and amino acids. Although cellular viability was not affected significantly, starvation of these cultures for 24 h in a bicarbonate buffer lacking glucose and amino acids led to a decrease in glutathione and protein content of approximately 43% and 40%, respectively. Although no effect on the protein loss occurred, the presence of ADT during starvation counteracted the starvation-induced loss of intracellular glutathione in a concentration-dependent way. At a concentration of 100 microM ADT even a significant increase in astroglial glutathione content was noted after 24 h of starvation. Alike intracellular glutathione levels, the amount of glutathione found in the buffer was elevated substantially if ADT was present during starvation. This ADT-mediated, apparent increase in glutathione efflux was additive to the stimulatory effect on extracellular glutathione levels of acivicin (100 microM), an inhibitor of extracellular enzymatic glutathione breakdown. However, the ADT-induced elevation of both intra- and extracellular glutathione content during starvation was prevented completely by coincubation with buthionine sulfoximine (10 microM), an inhibitor of glutathione synthesis. These results demonstrate that, most likely through stimulation of glutathione synthesis, ADT enables astroglial cells to maintain higher intra- and extracellular levels of glutathione under adverse conditions. Considering the lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of the compound is warranted.

Amino Acids↗

PubMed1998

Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.

Peroxides are often applied to cultured brain cells to investigate functions of these cells under oxidative stress. However, little is known about the ability of brain cells to detoxify peroxides. In order to investigate peroxide clearance of adherent cultured cells, the peroxide assay originally described for the determination of hydrogen peroxide production during experimental protein glycation by Jiang et al. [Z.-Y. Jiang, A.C.S. Woollard, S.P Wolff, Hydrogen peroxide production during experimental protein glycation, FEBS Lett. , 268 (1990) 69-71.] was adapted to microtiter plates. Besides hydrogen peroxide, with this assay organic hydroperoxides such as tertiary butylhydroperoxide (tBHP), and cumene hydroperoxide (CHP) can also be quantified. Up to an amount of 2.5 nmol of each peroxide per well of a plate the absorption measured was proportional to the concentration of the peroxide. Using the assay described the ability of astroglia-rich primary cultures to detoxify peroxides was monitored by measuring the peroxide content in 10 microliter samples collected at several time points from the peroxide-containing incubation buffer of one dish. If peroxides were applied at a concentration of 100 muM, hydrogen peroxide, tBHP, and CHP disappeared from the incubation buffer in reactions following first order kinetics with apparent half-times of 3.1 min, 2.9 min, and 4.2 min, respectively. In the absence of cells H2O2 and CHP were stable in the incubation buffer for at least 30 min, whereas tBHP decayed slowly in a spontaneous reaction. In conclusion, the method presented allows the determination of the rapid detoxification of various peroxides by cultured cells.

Animals↗

PubMed1998

Immunocytochemical localization of glycogen phosphorylase kinase in rat brain sections and in glial and neuronal primary cultures.

The physiological function of brain glycogen and the role of phosphorylase kinase as a regulatory enzyme in the cascade of reactions associated with glycogenolysis in the brain have not been fully elucidated. As a first step toward elucidating such a function, we studied the localization of phosphorylase kinase in glial and neuronal primary cell cultures, and in adult rat brain slices, using a rabbit polyclonal antibody against skeletal muscle glycogen phosphorylase kinase. Immunocytochemical examination of rat astroglia-rich primary cultures revealed that a large number of cells were positive for glycogen phosphorylase kinase immunoreactivity. These cells were also positive for vimentin, a marker for immature glia, while they were negative for glial fibrillary acidic protein, a marker for mature astroglia, and for galactocerebroside, an oligodendroglial marker. Neurons in rat neuron-rich primary cultures did not show any kinase-positive staining. In paraformaldehyde-fixed adult rat brain sections, phosphorylase kinase immunoreactivity was detected in glial-like cells throughout the brain, with relatively high staining found in the cerebral cortex, the cerebellum, and the medulla oblongata. Phosphorylase kinase immunoreactivity could not be detected in neurons, with the exception of a group of large neurons in the brain stem, most likely belonging to the mesencephalic trigeminal nucleus. Phosphorylase kinase was also localized in the choroid plexus and to a lesser degree in the ependymal cells lining the ventricles. Phosphorylase kinase thus appears to have the same cellular distribution in nervous tissue as its substrates, i.e. glycogen phosphorylase and glycogen, which suggests that the physiological role of brain phosphorylase kinase is the mobilization of glycogen stores to fuel the increased metabolic demands of neurons and astrocytes.

Animals↗

PubMed1998

Metabolism of glycine in primary astroglial cells: synthesis of creatine, serine, and glutathione.

The metabolism of [2-13C]glycine in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13C NMR spectroscopy. After a 24-h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2-13C]glycine, cell extracts and incubation media were analyzed for 13C-labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2-13C]glycine, the amount of de novo synthesized [2-13C]creatine was two-fold increased, and in addition, 13C-labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2-13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13C-labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2-13C] serine, [3-13C]serine, and [2,3-13C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.

Animals↗

PubMed1998

The peptide transporter PepT2 mediates the uptake of the glutathione precursor CysGly in astroglia-rich primary cultures.

The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cultures to utilize cysteinylglycine (CysGly) for glutathione synthesis. After a 24-h starvation period in the absence of glucose and amino acids, CysGly was able to substitute for cysteine plus glycine in the restoration of glutathione. Glutathione restoration from CysGly plus glutamate was only slightly affected by the dipeptides carnosine or serylglycine in a 200-fold excess. Captopril, a substrate of the peptide transporter PepT1, had almost no effect on glutathione restoration. In contrast, with increasing concentrations of alanylalanine or cefadroxil, known substrates of the peptide transporter PepT2, the amount of glutathione restored in the presence of CysGly and glutamate was strongly reduced. Cefadroxil in a 200-fold excess totally prevented the utilization of CysGly for glutathione restoration. The presence of mRNA for PepT2 in astroglia-rich primary cultures was demonstrated by application of RT-PCR. These results demonstrate that PepT2 is expressed in astroglia-rich primary cultures and that this transporter is highly likely to be responsible for the uptake of CysGly in these cultures.

Animals↗

PubMed1998

Metabolism of cysteine in astroglial cells: synthesis of hypotaurine and taurine.

The synthesis of hypotaurine and taurine was investigated in astroglia-rich primary cultures obtained from brains of neonatal Wistar rats using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3-(13)C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with 13C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3-(13)C] cysteine. The presence of [1-(13)C]hypotaurine and [1-(13)C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3-(13)C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3-(13)C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.

Animals↗

PubMed1998

Mitochondrial malic enzyme: purification from bovine brain, generation of an antiserum, and immunocytochemical localization in neurons of rat brain.

To elucidate the cellular location of mitochondrial malic enzyme in brain, immunocytochemical studies were performed. For this purpose, mitochondrial malic enzyme was purified to apparent homogeneity from bovine brain and used for the immunization of rabbits. Subjecting the antiserum to affinity purification on immobilized antigen as an absorbent yielded a purified immunoreactive antibody preparation, which was characterized by probing cytosolic and mitochondrial fractions of bovine and rat brain in western blotting. As neither cross-reactivity with cytosolic malic enzyme nor immunoreactivity against other proteins could be observed, the antibody preparation was found suitable for immunocytochemistry. By using sections of perfusion-fixed rat brain, considerable resolution was achieved at the light-microscopic level. Distinct and specific staining of neurons was observed; in contrast, no staining of astrocytes and possibly unspecific staining within the nuclei of oligodendrocytes were obtained. From these data, it is concluded that mitochondrial malic enzyme is located in neurons; however, in astrocytes, the enzyme appears to be either lacking or present at a much lower level. A protective role against oxidative stress in neurons is proposed for mitochondrial malic enzyme.

Animals↗

PubMed1998

Glutathione restoration as indicator for cellular metabolism of astroglial cells.

The restoration of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used to indicate metabolic properties of astroglial cells. At a culture age of 14-21 days these cultures contain an average total glutathione content of 32.8 +/- 3.2 nmol/mg protein and a cytosolic volume, estimated with the 3-O-methylglucose method, of 4.1 +/- 0.1 microl/mg protein. Therefore, cells of astroglial cultures have a cytosolic glutathione concentration of about 8 mM. In order to investigate glutathione synthesis in astroglial cultures the cellular glutathione content was reduced by starvation in a minimal medium lacking glucose and amino acids. Resynthesis of glutathione depended on the presence of glucose and the three constituent amino acids glutamate, cysteine and glycine. Absence of glucose reduced the amount of net glutathione restoration found after 4 h of incubation by about 50%. Of known substrates of astroglial energy metabolism, mannose could fully and fructose, lactate, pyruvate or sorbitol could partially replace glucose during glutathione restoration. In contrast to these compounds, galactose, 5-thioglucose and 2-deoxyglucose failed to substitute for glucose during glutathione restoration. Astroglial cells are able to use as precursors for the three constituent amino acids of glutathione a variety of amino acids and dipeptides. The results presented demonstrate that glutathione restoration can be used as an indicator for amino acid as well as energy metabolism of astroglial cells.

Amino Acids↗

PubMed1998

Utilization of cysteine and cysteine precursors for the synthesis of glutathione in astroglial cultures: preference for cystine.

The ability of astroglia-rich primary cultures derived from the brains of neonatal rats to take up and metabolize various sulfur containing compounds to cysteine was investigated using the content of intracellular glutathione as an indicator. Astroglial cells were partially depleted of glutathione by starvation for 24 h. Subsequent feeding for 4 h with glucose, glycine, and glutamate resulted in a restoration of the glutathione level, if cysteine was present. Substitution of cysteine by cystine during resynthesis of glutathione led to a glutathione content which exceeded that of cysteine-refed cells by 41%. Half-maximal content of glutathione was found at a concentration of about 12 microM cysteine and a maximal content at a concentration of at least 50 microM cysteine. In contrast, no plateau in the glutathione level was reached with increasing concentrations of cystine. The cystine effect could not be due to a contamination, since it was abolished after reduction of cystine by dithiothreitol. Since the cystine effect was not affected by inhibiting gamma-glutamyl transpeptidase, a promotion of cystine uptake by formation of gamma-glutamylcystine can also be excluded. Of the potential cysteine precursors tested, N-acetylcysteine was able to replace cysteine half-maximally at a concentration of 1 mM and fully at 5 mM. Feeding 2-oxothiazolidine-4-carboxylic acid at a concentration of 5 mM resulted in 64% of the glutathione level found in the presence of cysteine. A half-maximal glutathione content was attained at 50 microM 2-oxothiazolidine-4-carboxylic acid. While cystathionine could partially replace cysteine, methionine and homocysteine were not at all able to substitute for cysteine. These results demonstrate that astroglial cells prefer cystine from cysteine for glutathione synthesis and express uptake systems for N-acetylcysteine, 2-oxothiazolidine-4-carboxylic acid, and cystathionine, as well as the enzymes N-deacetylase, 5-oxoprolinase, and cystathionine gamma-lyase.

Animals↗

PubMed1997

Comparison of lactate transport in astroglial cells and monocarboxylate transporter 1 (MCT 1) expressing Xenopus laevis oocytes. Expression of two different monocarboxylate transporters in astroglial cells and neurons.

The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia. Lactate transport in primary cultures of astroglial cells was shown to be mediated by a single saturable transport system with a Km value for lactate of 7.7 mM and a Vmax value of 250 nmol/(min x mg of protein). Transport was inhibited by a variety of monocarboxylates and by compounds known to inhibit monocarboxylate transport in other cell types, such as alpha-cyano-4-hydroxycinnamate and p-chloromercurbenzenesulfonate. Using reverse transcriptase-polymerase chain reaction and Northern blotting, the presence of mRNA coding for the monocarboxylate transporter 1 (MCT1) was demonstrated in primary cultures of astroglial cells. In contrast, neuron-rich primary cultures were found to contain the mRNA coding for the monocarboxylate transporter 2 (MCT2). MCT1 was cloned and expressed in Xenopus laevis oocytes. Comparison of lactate transport in MCT1 expressing oocytes with lactate transport in glial cells revealed that MCT1 can account for all characteristics of lactate transport in glial cells. These data provide further molecular support for the existence of a lactate shuttle between astrocytes and neurons.

Animals↗

PubMed1997

Involvement of glutathione peroxidase and catalase in the disposal of exogenous hydrogen peroxide by cultured astroglial cells.

The ability of astroglial cells to detoxify exogenously applied hydrogen peroxide (H2O2) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. Incubation of astroglial cells with 100 microM H2O2 in the absence of glucose led to a 66% oxidation of the cellular glutathione within 30 s. Under these conditions, the cells were unable to re-establish the original high ratio of GSH/GSSG within 30 min of incubation. In contrast, if glucose was present the amount of GSSG produced on incubation with H2O2 was smaller (45% of total glutathione after 30 s) and the original ratio of GSH/GSSG was almost completely re-established within 10 min. If 100 microM H2O2 was applied, H2O2 disappeared from the incubation buffer with an apparent half-life of approximately 4 min. After 15 min of incubation, no H2O2 was detectable any more. The apparent half-life of H2O2 in the incubation buffer increased slightly but significantly with increasing concentration of H2O2 or when the cells were starved of glucose. A small reduction in the capacity of the cells to detoxify H2O2 was also observed after depletion of the glutathione content to 14% of control level by a 24 h pre-incubation of the cells in culture medium containing buthionine sulfoximine, an inhibitor of glutathione synthesis. Incubation of astroglial cells with mercaptosuccinate or 3-aminotriazole, inhibitors of glutathione peroxidase and catalase, respectively, only marginally reduced the rate of disappearance of H2O2 from the incubation buffer. In contrast, the rate of H2O2 clearance was strongly reduced in the presence of both inhibitors. These results demonstrate that glutathione peroxidase and catalase are involved in the detoxification of H2O2 by astroglial cells and that both enzymes are able to substitute for each other in the detoxification of H2O2.

Animals↗

PubMed1997

Expression of the surface antigen 4F2hc affects system-L-like neutral-amino-acid-transport activity in mammalian cells.

Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems of non-epithelial cells. By expression cloning we have recently demonstrated that the surface antigen 4F2hc (CD98) is a necessary component for expression of system-L-like amino acid-transport activity in C6-BU-1 rat glioma cells [Bröer, Bröer and Hamprecht (1995) Biochem. J. 312, 863-870]. 4F2hc mRNA was detected in CHO cells, COS cells, activated lymphocytes isolated from mouse spleen and primary cultures of astrocytes. In all these cell types, Na+-independent isoleucine transport was mediated by system L. No contribution of system y+L to isoleucine or arginine transport was detected in C6-BU-1 cells. In lymphocytes, both system-L-like amino acid-transport activity and 4F2hc mRNA levels increased after treatment with phorbol ester plus ionomycin. Antisense oligonucleotides caused modest inhibition of Na+-independent isoleucine transport in C6-BU-1 cells and primary cultures of astroglial cells, whereas arginine transport was unaffected. Overexpression of 4F2hc cDNA in CHO cells resulted in an increase in Na+-independent isoleucine transport.

Amino Acid Sequence↗

PubMed1997

Metabolic pathways for glucose in astrocytes.

Cultured astroglial cells are able to utilize the monosaccharides glucose, mannose, or fructose as well as the sugar alcohol sorbitol as energy fuel. Astroglial uptake of the aldoses is carrier-mediated, whereas a non-saturable transport mechanism is operating for fructose and sorbitol. The first metabolic step for all sugars, including fructose being generated by enzymatic oxidation of sorbitol, is phosphorylation by hexokinase. Besides glucose only mannose may serve as substrate for build-up of astroglial glycogen. Whereas glycogen synthase appears to be present in astrocytes as well as neurons, the exclusive localization of glycogen phosphorylase in astrocytes and ependymal cells of central nervous tissue correlates well with the occurrence of glycogen in these cells. The identification of lactic acid rather than glucose as degradation product of astroglial glycogen appears to render the presence of glucose-6-phosphatase in cultured astrocytes an enigma. The colocalization of pyruvate carboxylase, phosphenolpyruvate carboxykinase and fructose-1,6-bisphosphatase points to astrocytes as being the gluconeogenic cell type of the CNS.

Animals↗

PubMed1997

The gamma-glutamyl transpeptidase inhibitor acivicin preserves glutathione released by astroglial cells in culture.

The release of glutathione from astroglial cells was investigated using astroglia-rich primary cultures prepared from the brains of newborn rats. These cells release glutathione after onset of an incubation in a glucose-containing minimal medium. The amount of extracellular glutathione increased with the time of incubation, although the accumulation slowed down gradually. An elevated rate of increase of the glutathione concentration in the incubation medium was found if the astroglial ectoenzyme gamma-glutamyl transpeptidase was inhibited by acivicin. The activity of gamma-glutamyl transpeptidase in astroglia-rich primary cultures, which was found to be 1.9 +/- 0.3 nmol/(min x mg protein), was markedly reduced if the cells had been incubated in the presence of acivicin. After 2 h of incubation with acivicin half-maximal and maximal inhibition of gamma-glutamyl transpeptidase activity was found at concentrations of about 5 microM and 50 microM, respectively. In the presence of acivicin at a concentration above 10 microM the glutathione content found released from astroglial cells apparently increased almost proportional to time for up to 10 h. Under these conditions the average rate of release was 2.1 +/- 0.3 nmol/(h x mg protein) yielding after a 10 h incubation an extracellular glutathione content three times that of the medium of cells incubated without inhibitor. Half-maximal and maximal effects on the level of extracellular glutathione were found at 4 microM and 50 microM acivicin, respectively. After a 10 h incubation with acivicin the intracellular content of glutathione was reduced to 75% of the level of untreated astroglial cultures. These results suggest that glutathione released from astroglial cells can serve as substrate for the ectoenzyme gamma-glutamyl transpeptidase of these cells.

Animals↗

PubMed1997

Use of dipeptides for the synthesis of glutathione by astroglia-rich primary cultures.

The intracellular content of glutathione in astroglia-rich primary cultures derived from the brains of newborn rats was used as an indicator for the ability of these cells to use dipeptides for glutathione synthesis. For restoration of the glutathione level, after a 24-h starvation period in the absence of glucose and amino acids, glucose, glutamate, cysteine, and glycine have to be present in the incubation buffer. The dipeptides CysGly and gammaGluCys were able to substitute for cysteine plus glycine and glutamate plus cysteine, respectively. Half-maximal contents of glutathione were found at 20 microM CysGly and 3 mM gammaGluCys. In addition, the oxidized forms of the dipeptides CysGly and GlyCys could replace cysteine plus glycine for glutathione restoration, and the glycine-containing dipeptides GlyGly, GlyLeu, GlyGlu, GlyGln, and gammaGluGly could partially substitute for the glycine necessary for the replenishment of glutathione. The glutathione resynthesis in the presence of CysGly plus glutamate was totally inhibited in the presence of buthionine sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase. In contrast, glutathione restoration from gammaGluCys at a concentration of 10 mM in the presence of glycine was not influenced by the inhibitor. The use of CysGly or gammaGluCys was not affected by the presence of the dipeptidase inhibitors cilastatin or bestatin. In addition, carnosine and several other dipeptides applied in a 50-fold excess only slightly prevented the use of CysGly, hinting at the existence in astroglial cells of a transport system specific for CysGly. The results demonstrate that astroglial cells can use dipeptides for intracellular glutathione synthesis and that the dipeptides most likely are taken up as intact molecules into astroglial cells before intracellular hydrolysis occurs.

Animals↗