SEARCH

SEARCH BY CITATION

Keywords:

  • Astrocytes;
  • Neurons;
  • Glutathione;
  • Conditioned medium;
  • Oxidative stress;
  • Apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Abstract: Cysteine is the rate-limiting precursor of glutathione synthesis. Evidence suggests that astrocytes can provide cysteine and/or glutathione to neurons. However, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. In this report, we analyzed cysteine, glutathione, and related compounds in astrocyte conditioned medium using HPLC methods. In addition to cysteine and glutathione, cysteine-glutathione disulfide was found in the conditioned medium. In cystine-free conditioned medium, however, only glutathione was detected. These results suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione: glutathione + cystine [LEFT RIGHT ARROW] cysteine + cysteineglutathione disulfide. Conditioned medium from neuronenriched cultures was also assayed in the same way as astrocyte conditioned medium, and no cysteine or glutathione was detected. This shows that neurons cannot themselves provide thiols but instead rely on astrocytes. We analyzed cysteine and related compounds in rat CSF and in plasma of the carotid artery and internal jugular vein. Our results indicate that cystine is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Cysteine and glutathione are unstable and oxidized to their disulfide forms under aerobic conditions. Therefore, constant release of glutathione by astrocytes is essential to maintain stable levels of thiols in the CNS.

Glutathione (GSH) is the major cellular antioxidant and as such plays an important neuroprotective role. Cellular GSH levels are closely correlated with cell survival under adverse conditions (Meister and Anderson, 1983; Ratan et al., 1994; Drukarch et al., 1997). GSH is synthesized from glutamate, cysteine (CSH), and glycine. CSH is the rate-limiting precursor of GSH synthesis (Beutler, 1989). In vitro experiments have shown that the extracellular abundance of thiol-containing compounds substantially influences intracellular GSH levels (Meister, 1989). Adding CSH to a neuronal culture that has been temporarily deprived of amino acids can increase GSH content, whereas cystine (CSSC), the oxidized form of CSH, cannot (Kranich et al., 1996). However, CSH is very unstable owing to autoxidation under aerobic conditions.

The mechanisms by which a stable CSH level is maintained by astrocytes have been explored in recent years. Astrocytes have been shown to have profound neurosupportive effects in neuronal culture experiments (Banker, 1980; McCaffery et al., 1984; Vernadakis, 1988; Yuzaki et al., 1993; Wang and Cynader, 1999). Release of thiols by astrocytes has been reported and considered to play an important role to increase neuronal GSH synthesis (Yudkoff et al., 1990; Sagara et al., 1993, 1996; Dringen et al., 1999). However, it is still uncertain as to how thiols are provided. Yudkoff et al. (1990) found GSH in conditioned medium of astrocytes. Sagara et al. (1993) reported that glial cells can release CSH to the cultured medium to supply neurons. In a later report (Sagara et al., 1996), these investigators found GSH efflux from cultured astrocytes. Recently, Dringen et al. (1999) suggested that the ectoenzyme γ-glutamyl-transpeptidase uses GSH released by astrocytes as a substrate to generate the dipeptide CysGly, which is subsequently used by neurons as the precursor for GSH synthesis. In the present experiments, we tried to determine whether astrocytes release CSH, GSH, or both. What is the relationship of CSH and GSH? What mechanism represents the in vivo situation?

We have analyzed CSH, GSH, and other related compounds in astrocyte conditioned medium (ACM), neuron conditioned medium (NCM), CSF, and plasma of the carotid artery and internal jugular vein of the rat using HPLC methods developed by Reed et al. (1980). These methods are capable of evaluating thiols (CSH, GSH, homocysteine, and CysGly), disulfides [CSSC, GSH disulfide (GSSG), and cysteine-GSH (CSSG)], and acidic amino acids (glutamic acid, aspartic acid, cysteic acid, and homocysteic acid), among others. This advantage makes it possible to analyze the relationships among these compounds.

Our experimental results suggest that astrocytes release GSH directly and that CSH is generated by an extracellular thiol/disulfide exchange reaction: GSH + CSSC [LEFT RIGHT ARROW] CSH + CSSG. This reaction can occur nonenzymatically (Jocelyn, 1967). Our results show that astrocytes can provide small molecules to enhance GSH synthesis and therefore the antioxidative capacities of neighboring neurons.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Materials

Both pregnant and adult male Long-Evans rats were obtained from Charles River (Laval, QC). CSSG was obtained from Toronto Research Chemicals (North York, ON, Canada). Fetal bovine serum, trypsin, and Dulbecco's phosphate-buffered saline were obtained from GibcoBRL (Grand Island, NY, U.S.A.). DNase I was from Boehringer Mannheim (Mannheim, Germany). CSH, CSSC, GSH, GSSG, glutathionesulfonic acid, glutamic acid, aspartic acid, CysGly, cysteic acid, homocysteic acid, iodoacetic acid, 1-fluoro-2,4-dinitrobenzene (FDNB), Eagle's minimum essential medium (MEM), CSSC-free Eagle's MEM (M-2289), insulin, transferrin, selenium, and poly-L-lysine were obtained from Sigma (St. Louis, MO, U.S.A.). The Angiocath catheter was obtained from Becton Dickinson (Sandy, UT, U.S.A.).

HPLC was performed using the Gilson 712 gradient system from Gilson Medical Electronics (Middleton, WI, U.S.A.). The 3-aminopropyl ion-exchange column, with a particle size of 5 μm and dimensions of 4.6 × 200 mm, was obtained from CEL Associates (Houston, TX, U.S.A.).

Primary cultures of cortical astrocytes

Astrocyte cultures were prepared by a method modified from that described by McCarthy and de Vellis (1980). In brief, cerebral cortices of 2-day-old newborn rats were taken, and meninges were removed. The tissue was dissected and enzymatically digested with 0.25% trypsin and 0.1 mg/ml DNase. The dissociated cells were plated in poly-L-lysine-precoated 75-cm2 plastic flasks at a high density of 2 × 105 cells/cm2. The culture medium was Eagle's MEM, supplemented with glucose (33 mM), glutamine (2 mM), HEPES (10 mM), NaHCO3 (26 mM), and 10% fetal bovine serum. The medium was changed twice a week. The cultures were grown to confluence in 2 weeks. At 14 days in vitro (DIV), the flasks were tightly sealed and shaken at 260 rpm for 18 h. Suspended cells in the flasks were discarded after shaking, and the adherence cells were flat, polygonal astrocytes, which were identified by morphology and glial fibrillary acidic protein immunostaining. The density of confluent astrocytes is ∼ 1 × 105 cells/cm2. Astrocyte-enriched cultures 2-6 weeks old in flasks were used to make ACM.

For coculture purposes, confluent astrocytes in flasks were digested with trypsin and subcultured onto poly-L-lysine-coated glass coverslips (28 mm in diameter) in 10% fetal bovine serum-supplemented MEM. After 1 week, the subcultured astrocytes become confluent and ready for use.

Primary cultures of cortical neurons

Neuron-enriched primary cultures were prepared in a serum-free glial-neuronal coculture system. The detailed procedures are described elsewhere (Wang and Cynader, 1999). The culture medium is serum-free Eagle's MEM, supplemented with glucose (33 mM), glutamine (2 mM), HEPES (10 mM), NaHCO3 (26 mM), pyruvate (1 mM), and a mixture of insulin (10 mg/L), transferrin (5.5 mg/L), and sodium selenite (5 mg/L). Cerebral cortices of 18-day-old rat embryos were used. The tissue was enzymatically digested as described above. The dissociated cells were suspended in the serum-free medium and plated onto the glass coverslips (28 mm in diameter) at a density of 1 × 105 cells/cm2. The coverslips were coated with poly-L-lysine and dried. Before plating the dissociated cells, the coverslips were briefly coated with the serum-free ACM for 5 min and rinsed twice with Hanks' solution. The latter procedure helps cell attachment in serum-free medium. Primary neurons were cocultured with the astrocyte feeder layer facing down in 35-mm-diameter Petri dishes. The two coverslips were separated by a V-shaped glass holder 5 mm tall. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Glial cells did not proliferate in the serum-free medium, and a neuron-enriched population (>95%) was obtained as identified by immunostaining against neuron-specific enolase. At 7 DIV, the cultures were used to prepare NCM.

Procedures for obtaining CSF and plasma of artery and vein

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Three-month-old male Long-Evans rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg). Only one sample was taken from each animal, i.e., CSF, arterial plasma, or venous plasma. CSF was obtained from the cerebellomedullary cistern. The rat was first placed in a stereotaxic frame. The skin was incised along the midline over the occipital crest, and the muscles were separated. A puncture through occipital foramen magnum was made with the Angiocath catheter. About 100 μl of CSF was slowly drawn over 2 min. The CSF was centrifuged at 300 g for 2 min to deposit blood cells and was processed immediately following the procedures described below.

For obtaining the blood samples, the anesthetized rat was laid on his back. The carotid artery or internal jugular vein was carefully exposed by separating the surrounding muscles and connective tissues. A vessel puncture was made with the Angiocath catheter. About 1 ml of blood was drawn into the heparin-rinsed syringe. The blood was then centrifuged at 300 g for 2 min. Two-tenths milliliter of plasma was taken and processed following the procedures described below.

HPLC assay of thiols, disulfides, and associated compounds

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Cysteine and related compounds were analyzed using the HPLC method developed by Reed and co-workers (Reed et al., 1980; Fariss and Reed, 1983) with minor modifications. In principle, thiols of the sample are first reacted with iodoacetic acid to block free thiol groups and then with FDNB to produce N-dinitrophenyl derivatives, which are analyzable by spectrophotometric detection at 365 nm. This method is capable of evaluating thiols, disulfides, and acidic amino acids, among others. In our procedure, 200 μl of the sample was first reacted with 25 μl of 100 mM iodoacetic acid in 0.2 mMm-cresol purple and 25 μl of NaHCO3 (0.24 M)/NaOH (0.12 M) buffer for 30 min. Then, 225 μl of 1% (vol/vol) FDNB in ethanol was added, and the mixture was stored at 4°C overnight. Thereafter, 25 μl of 1 M lysine was added to eliminate unreacted FDNB, and the sample was then ready for analysis. In cases in which only a small amount of sample was available, such as CSF, the volumes of the above reactants were adjusted proportionately according to the sample amount.

HPLC solvent A was 80% methanol in water. Solvent B was 0.8 M sodium acetate in 64% methanol (for preparation of solvent B, refer to Reed et al., 1980). Twenty microliters of the samples was injected into the HPLC column. The mobile phase was maintained at 80% A/20% B for 5 min, followed by a gradient elution to 1% A/99% B over 10 min, and held for 5 min. The flow rate was 1.5 ml/min. The retention values for several important compounds in our experiments are shown in Table 1. For purposes of quantitation, the standard regression plots of peak area versus concentration were drawn by measuring series concentrations of CSSC, CSH, CSSG, GSH, and GSSG. The minimal detectable concentrations in our experiments were 0.50 μM for thiols and acidic amino acids and 0.25 μM for disulfides.

Table 1. HPLC retention times for thiols, disulfides, and acidic amino acids
ChemicalRetention time (min)
  1. The listed standard chemicals are reacted with iodoacetic acid and FDNB to produce N-dinitrophenyl derivatives, which are spectrophotometrically detected at 365 nm. The HPLC mobile phase starts with 5 min of isocratic elution at 80% A/20% B, followed by 10 min of gradient elution to 1% A/99% B, and held at 1% A/99% B for 5 min. The flow rate is 1.5 ml/min.

Glutamic acid6.29
CSSC7.78
Homocysteic acid8.48
CysGly9.32
Cysteic acid9.47
Aspartic acid9.80
CSH10.75
CSSG13.13
GSH14.23
GSSG16.02

Quantification of intracellular GSH and related compounds in astrocytes and neurons

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

The cultured cells, both astrocytes and neurons, were ruptured and lysed in 0.4 ml of distilled water per 1 × 106 cells and then subjected to three cycles of freezing (-80°C) and thawing. The solution was collected after centrifugation. Two-tenths milliliter of the sample was immediately used for HPLC measurement. The rest was used for protein quantitation.

Protein content was determined at 565 nm by the method of Bradford (1976). A standard curve, covering a range of 1-25 μg/ml, was made using bovine serum albumin. The sample concentrations were adjusted to the range of the standard curve. The mean ± SEM protein contents of the cell lysate were 351.3 ± 51.8 μg/ml for astrocytes and 95.4 ± 15.3 μg/ml for neurons. Under these conditions, the minimal detectable concentrations of thiols and acidic amino acids were 1.42 nmol/mg for astrocytes and 5.24 nmol/mg for neurons, and those of disulfides were 0.71 nmol/mg for astrocytes and 2.62 nmol/mg for neurons.

Most of the data reported here represent two independent experiments, each performed in duplicate or in triplicate culture dishes or samples.

All animal procedures were approved by the Committee on Animal Care of the University of British Columbia.

CSSG is found in CSSC-containing ACM

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

We first analyzed the components and concentrations of CSH and related compounds in ACM. MEM was added to the confluent astrocyte cultures at a concentration of 1.33 ml per 1 × 106 cells (10 ml per flask). As shown in Fig. 1, the control medium contains mainly CSSC (0 h in culture dishes). Analysis of the control medium after it has been in the incubator for 48 h shows no difference with that at 0 h. Several new peaks appeared in ACM compared with control medium. Three of them were clearly identified as CSH, CSSG, and GSH. The finding of CSSG in ACM was meaningful. The direct efflux of CSSG is unlikely because no CSSG was detected in astrocytes (see Table 4). On the other hand, extracellular CSSG can be produced when GSH reacts with CSSC nonenzymatically: GSH + CSSC [LEFT RIGHT ARROW] CSH + CSSG.

image

Figure 1. HPLC chromatograms of CSH and related compounds in control medium, ACM, and the standards. Control medium was serum-free CSSC-containing MEM. The ACM was made by adding the culture medium to the confluent astrocytes for 24 h. The peaks in the control medium and ACM were identified by referring to the standards. A: Control MEM contains mainly CSSC. B: Several new peaks besides CSSC were found in 24-h ACM. Three of them can be identified as CSH, CSSG, and GSH. C: The standards were CSSC, CSH, CSSG, GSH, and GSSG. The concentration of each chemical is 10 μM. The peak area of disulfide is about double that of thiol. AUFS, absorbance units full scale.

Download figure to PowerPoint

Table 2. Content of thiols, disulfides, and acidic amino acids [glutamic acid (Glu) and aspartic acid (Asp)] in astrocytes and neurons
 Content (nmol/mg of protein)
 GSHCSHGSSGGluAsp
  1. The confluent astrocyte cultures and neuron-enriched cultures at 7 DIV were ruptured and lysed in distilled water by freezing and thawing. Protein content was determined by the method of Bradford (1976). The samples were immediately analyzed by HPLC. Data are mean ± SEM values of five determinations. CSSC, CSSG, and CysGly were not detected. ND, not detectable.

Astrocytes59.1 ± 6.05.02 ± 0.380.73 ± 0.1233.7 ± 6.64.50 ± 0.94
Neurons50.8 ± 1.5NDND194 ± 663.0 ± 3.6

At 37°C and pH 7.4, the equilibrium constant K of the above reaction is 3.2, and the velocity constant k is 610 L/mol × min (Jocelyn, 1967). That means the reaction can rapidly proceed forward. The appearance of CSSG in ACM leads us to suggest that CSH may not be released, but instead is produced extracellularly by the reaction of GSH, which is released by astrocytes, and the stable amino acid CSSC. This suggestion is consistent with the fact that GSH, not CSH, is maintained at a high intracellular level. CysGly was not detected in the ACM.

The changes in concentration of CSH and related compounds in ACM were measured as shown in Fig. 2. CSSC content decreased with time of incubation, with a beginning concentration of 76.9 ± 3.4 μM. CSH and CSSG levels increased rapidly from 0 and then decreased when CSSC content diminished at 24 and 48 h. GSH was barely detectable at 6 h. This can be explained by the high concentration of CSSC in the medium. The theoretical value of GSH calculated from the K value (3.2) and concentrations of CSSC, CSH, and CSSG is 0.07 μM at 6 h, well below our minimal detectable level, 0.5 μM. Therefore, GSH may not be detected in this situation. The values of the equilibrium constant K calculated from Fig. 2 are 4.66 at 12 h, 4.19 at 24 h, and 4.57 at 48 h. A small amount of GSSG was found at 48 h. GSSG may be generated by two possible mechanisms: autoxidation of GSH and the thiol/disulfide exchange reaction. The latter can also occur nonenzymatically (Jocelyn, 1967): CSSG + GSH [LEFT RIGHT ARROW] CSH + GSSG.

image

Figure 2. Time course of content of CSH and related compounds in ACM. Confluent astrocytes in flasks were rinsed with Hanks' solution three times. The serum-free CSSC-containing MEM was added at a concentration of 1.33 ml per 1 × 106 cells. Samples of the ACM were taken at 0, 6, 12, 24, and 48 h and processed immediately for HPLC analysis. The calculated values of the equilibrium constant K for the equation GSH + CSSC [LEFT RIGHT ARROW] CSH + CSSG are as follows: K12h = 4.66, K24h = 4.19, and K48h = 4.57. Data are mean ± SEM (bars) values of five determinations.

Download figure to PowerPoint

GSH is released by astrocytes in CSSC-free medium

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

To obtain direct evidence for GSH release from astrocytes, we analyzed the conditioned medium from CSSC-free MEM. CSSC-free MEM (M-2289) was added to the confluent astrocyte cultures at a concentration of 0.66 ml per 1 × 106 cells (5 ml per flask). In this case, the extracellular thiol/disulfide exchange reaction between CSSC and GSH can be excluded. As shown in Table 2, CSH and CSSG were no longer found, whereas GSH was the only component detected. This result indicates that there is no direct release of CSH and CSSG by astrocytes. Astrocytes release GSH to the medium from an intracellular reservoir of GSH. Because there is no CSSC as a source for GSH synthesis, the detected GSH concentration may not reflect the potential levels the astrocytes can release. The small content of GSSG is assumed to be the autoxidation product from its reduced form. The dipeptide of CysGly was not detected in these experiments.

Table 3. Content of CSH and related compounds in ACM made from CSSC-free medium
  Content (μM)
TimeCSSCCSHCSSGGSHGSSGCysGly
  1. The confluent astrocyte cultures were rinsed with Hanks' solution three times, and the cystine-free MEM was added at a concentration of 0.66 ml per 1 × 106 cells. The samples were collected at 2, 4, 6, and 12 h and analyzed using HPLC methods. Data are mean ± SEM values of five determinations. ND, not detectable.

2 hNDNDND0.66 ± 0.11NDND
4 hNDNDND1.06 ± 0.03NDND
6 hNDNDND1.43 ± 0.09NDND
12 hNDNDND3.18 ± 0.090.32 ± 0.19ND

Analysis of CSH and related compounds in CSF and in plasma of artery and vein

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

It is of course critical to determine whether the in vitro mechanisms studied above are applicable to the in vivo situation. Plasma of the carotid artery and of the internal jugular vein, which represent blood supply and return of the brain, and CSF were studied. Our results (Table 3) showed that the components of CSH and related compounds in CSF and blood are surprisingly different. No CSSC was detected in CSF, whereas it occurs at high concentration in plasma. Vein plasma clearly has a lower level of CSSC than artery plasma. GSH showed a relatively high concentration in CSF, whereas it was not detected in plasma. CSH and CSSG were detected in both CSF and plasma, with a low level in CSF. No clear differences in the concentration of CSH and CSSG were detected in the artery and vein. GSSG was detected at a low concentration in CSF. No CysGly was detected in CSF, artery, or vein.

Table 4. Content of CSH and related compounds in CSF and in plasma of the carotid artery and internal jugular vein
  Content (μM)
 CSSCCSHCSSGGSHGSSGCysGly
  1. Three-month-old rats were used. CSF was taken from the cerebellomedullary cistern. Arterial and venous plasma was taken from the carotid artery and internal jugular vein, respectively. All the samples were centrifuged at 300 g for 2 min and immediately processed for HPLC analysis. Data are mean ± SEM values and concentration range with five to seven samples as indicated. The contents of CSH and CSSG have no difference between artery and vein. ND, not detectable.

  2. ap < 0.001, different from artery value.

CSF (n = 7)      
Mean ± SEMND1.12 ± 0.140.50 ± 0.085.87 ± 0.290.22 ± 0.12ND
Range 0.68-1.750.28-0.834.92-7.110-0.80 
Artery (n = 7)      
Mean ± SEM35.2 ± 1.24.94 ± 0.693.17 ± 0.21NDNDND
Range30.8-40.13.32-7.492.37-3.72   
Vein (n = 5)      
Mean ± SEM26.3 ± 0.9a5.42 ± 0.793.06 ± 0.35NDNDND
Range23.9-29.22.48-7.002.34-4.35   

Thiols are not found in NCM

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

Whether neurons can provide thiols by themselves was studied to determine whether astrocytes are the major releasers of thiols. Neuron-enriched cultures were prepared by using the glial-neuronal coculture method in serum-free medium as described in Materials and Methods. At 7 DIV, the astrocyte feeder layers were removed. Neuron-enriched cultures were rinsed with Hanks' solution three times. CSSC-supplemented or CSSC-free MEM (M-2289) were added at a concentration such that the medium volume/cell number ratio was the same as that of ACM. As shown in Fig. 3, no CSH or GSH was detected in CSSC-supplemented NCM. CSSC levels showed no obvious change. Low levels of CSSG were found in CSSC-supplemented NCM, suggesting that there is still a tiny amount of GSH released by neurons and the extracellular thiol/disulfide exchange reaction, but the amount of thiols is too small to be detected. We cannot exclude the possibility that residual glial contamination (<5%) of these cultures accounts for the GSH release observed.

image

Figure 3. Time course of CSSC and CSSG content in NCM. Neuron-enriched cultures were prepared with the glial-neuronal coculture method in serum-free medium. At 7 DIV, the cultures were rinsed with Hanks' solution three times. The serum-free CSSC-containing MEM was added at a concentration of 1.33 ml per 1 × 106 cells, the same as the medium volume/cell number ratio of ACM. CSH, GSH, GSSG, and CysGly were not detected. Data are mean ± SEM (bars) values of five determinations.

Download figure to PowerPoint

NCM prepared from CSSC-free MEM was also analyzed. The medium was added at a concentration of 0.66 ml per 1 × 106 cells. The samples were collected at 6 and 12 h. The thiols and disulfides, including CSH, CSSC, CSSG, GSH, GSSG, and CysGly, were not detected in five determinations.

Contents of GSH and related compounds in astrocytes and neurons

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

As the cross-membrane concentration gradient influences the kinetics of membrane transport, the intracellular contents of GSH and related compounds were investigated. Confluent astrocytes (2-6 weeks in culture) and neuron-enriched cultures (7 DIV) were analyzed. As shown in Table 4, there are high levels of GSH in astrocytes and neurons, with higher concentrations in astrocytes. Low levels of CSH and GSSG were found in astrocytes but not in neurons. The GSH/CSH ratio is 11.8:1, and the GSH/GSSG ratio is 81.5:1 in astrocytes. CSSC, CSSG, and the dipeptide CysGly were not detectable in either astrocytes or neurons. In contrast with the relatively lower levels of GSH, neurons contain much higher levels of glutamate and aspartate than astrocytes (Table 4).

Autoxidation of CSH and GSH

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

CSH and GSH are unstable under aerobic conditions. We assayed the autoxidation rates of CSH and GSH (100 μM each) in phosphate-buffered solution. As shown in Fig. 4, CSH was oxidized to CSSC, and GSH oxidized to GSSG under conditions of 37°C, pH 7.4, and 100% air. A very small amount of cysteic acid (∼ 1-2 μM) was also found as a by-product of CSH autoxidation (data not shown).

image

Figure 4. Autoxidation of CSH and GSH. CSH or GSH (100 μM) in phosphate-buffered solution was incubated under conditions of pH 7.4, 37°C, and 100% air. The samples were collected at 0, 6, 12, 24, and 48 h and analyzed by HPLC. Data are mean ± SEM (bars) values of four determinations. A: CSH is oxidized to CSSC. B: GSH is oxidized to GSSG.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES

GSH is the major cellular antioxidant, acting to protect neurons in the face of oxidative stress. CSH is an important amino acid that is a major contributor to the maintenance of intracellular GSH levels. In this study, we demonstrate the mechanism by which astrocytes provide CSH to neurons indirectly by releasing GSH. (a) In the absence of CSSC, as the results from CSSC-free ACM show (Table 2), it is GSH, not CSH, that is found in the ACM. This result indicates that GSH is the direct product released by astrocytes. Astrocytes release GSH from their intracellular reservoirs without requiring further supplies of thiol in the CSSC-free medium. Hence, the GSH concentrations measured here under these conditions may not reflect the potential levels of secretion that astrocytes may reach. (b) In the presence of CSSC, as the results from CSSC-containing ACM show (Figs. 1 and 2), CSH, CSSG, and GSH were found in the ACM. Intracellular CSSG was not detected in astrocytes, and CSH was present at much lower intracellular levels than GSH (Table 4). The high levels of CSSG and CSH in the ACM cannot be explained by direct cellular release. However, extracellular CSSG and CSH can be produced when GSH reacts with CSSC nonenzymatically: GSH + CSSC [LEFT RIGHT ARROW] CSH + CSSG. The reaction can rapidly proceed forward with an equilibrium constant K of 3.2 (Jocelyn, 1967). Our data suggest that astrocytes release GSH directly, which then reacts with the stable amino acid CSSC extracellularly to generate CSH and CSSG (Fig. 5). It should be noted that CSSG does not accumulate in ACM (Fig. 2). We found that CSSG can replace CSSC to support astrocyte survival, whereas astrocytes cannot survive in CSSC-free medium without addition of other thiols or disulfides (authors' unpublished data). It is possible that CSSG might either be taken up by astrocytes directly or further react with GSH to generate CSH and GSSG (Jocelyn, 1967).

image

Figure 5. A model shows how astrocytes may provide CSH to neurons. Our data suggest that blood mainly supplies CSSC to the brain. CSSC is taken up by astrocytes and used to synthesize GSH. Astrocytes release GSH to the extracellular fluid, where GSH reacts with CSSC to generate CSH and CSSG nonenzymatically. CSH is then taken up by neurons for GSH synthesis.

Download figure to PowerPoint

This hypothesis is supported by the results of transporter studies and provides a new understanding of the physiological significance of GSH efflux. GSH efflux has been found in fibroblasts (Bannai and Tsukeda, 1979), hepatocytes (Fariss and Reed, 1983; Aw et al., 1986), and astrocytes (Sagara et al., 1996). Kinetic studies from astrocytes suggest that GSH efflux is carrier-mediated, and the Km value is 127 nmol/mg of protein (Sagara et al., 1996). GSH transporters of astrocytes are ion-independent, and net transport depends on the concentration gradient of GSH. Because the intracellular concentration of GSH is several orders of magnitude greater than the extracellular concentration, the transport of GSH operates as an efflux process. CSH transporters, in contrast, belong to the Na+-dependent system ASC (Kilberg et al., 1981; Franchi-Gazzola et al., 1982). Recently, two members of the ASC transporter family, ASCT1 and ASCT2, have been cloned and functionally studied (Arriza et al., 1993; Shafqat et al., 1993; Utsunomiya-Tate et al., 1996). CSH, as well as some other neutral amino acids, is taken up by cells with high affinities, coupling to the cotransport of Na+. These results suggest a crossmembrane movement of GSH efflux and CSH influx. Consistent with the transporter data, we show that it is GSH, not CSH, that is released by astrocytes. CSH is generated extracellularly and then taken up by neurons for GSH synthesis.

Cooper and Kristal (1997) have previously indicated that transfer of sulfur amino acids and peptides between astrocytes and neurons occurs and that astrocytes play an important role in sulfur homeostasis in the CNS. It was found that astrocytes and neurons have completely different capacities in the utilization of CSSC and CSH. The uptake rate of radiolabeled CSSC by astrocytes is much higher than that of neurons (Sagara et al., 1993). Kranich et al. (1996) found that cultured astrocytes could make use of CSSC to synthesize GSH to their maximal degree. Cultured neurons, instead, make use of CSH, rather than CSSC. Similar to these results, our data show that CSSC was used rapidly within 48 h in astrocyte-enriched cultures (Fig. 2), whereas CSSC levels showed almost no change in neuron-enriched cultures (Fig. 3). These results indicate that astrocytes have a strong reducing ability, converting CSSC to CSH in their cell bodies. This ability may account for the enhanced synthesis and release of GSH by astrocytes. Our data show that astrocytes release substantial amounts of GSH in ACM, whereas neurons release little.

Whether γGT is involved in thiol generation is still unclear. Dringen et al. (1999) found that adding the dipeptide CysGly, the product of the γGT-catalyzed reaction, to neuron-enriched primary cultures increases the GSH content of neurons. Acivicin, a γGT inhibitor, suppresses the astrocyte-mediated increase in neuronal GSH content. These researchers suggested that the ectoenzyme γGT uses GSH released by astrocytes to generate CysGly, which is subsequently used by neurons. However, no evidence thus far shows the existence of CysGly in ACM or CSF. The localization of γGT in the CNS is crucial for the study of its function. γGT is used extensively as a marker of brain microvessels. It is abundant in the choroid plexus (Tate et al., 1973). γGT-positive cells include endothelial cells (Orte et al., 1999), pericytes (Risau et al., 1992), and the endfeet of astrocytes (Zhang et al., 1997). This type of localization is consistent with the suggested role of γGT in the transport of amino acids across the blood-brain barrier (Meister and Anderson, 1983; Cooper and Kristal, 1997). However, it does not prove that γ-GT can generate thiol in the extracellular space of the CNS. GSH released by astrocytes into the extracellular space may not reach the ectoenzyme γGT owing to the restrictive distribution of this enzyme (only around brain microvessels). A recent experiment has shown the detection of several γ-glutamyl derivatives of amino acids in the perfusates of brain slices (Li et al., 1999). This result could be explained by the existence of a large amount of exposed brain microvessels in brain slices. GSH released by astrocytes could react with the ectoenzyme γ-GT on the surface of these microvessel cells, which may not represent the situation of integral tissue. Further screening of γ-glutamyl derivatives in conditioned medium and CSF is required to clarify the involvement of γGT in thiol generation.

We are most interested in the applicability of the in vitro results to in vivo situations. By analyzing CSH, GSH, and related compounds in CSF and in plasma of the carotid artery and internal jugular vein, the following conclusions are obtained. First, CSSC is substantially transported from blood to the CNS. Both artery and vein contain high concentrations of CSSC, and there is also an obvious positive arteriovenous concentration difference, indicating an uptake by the brain. Second, blood GSH cannot be the thiol source of the brain. GSH was not detected in plasma of the carotid artery and internal jugular vein, whereas it occurs at a relatively high level in CSF. The calculated GSH concentrations from the thiol/disulfide exchange equilibrium constant K (3.2) and CSH, CSSC, and CSSG concentrations are 0.14 and 0.20 μM for artery and vein, respectively, below detectable levels. Third, the other substances considered, such as CSH, CSSG, and CysGly, are less likely to be the thiols or disulfides transported from blood to the brain. CSH and CSSG have no arteriovenous differences, suggesting no net uptake across the blood-brain barrier within the CNS. The dipeptide CysGly was not detectable in blood and CSF, nor was it found in ACM in our in vitro studies. Fourth, the thiol/disulfide exchange reaction occurs in the CNS. CSH, GSH, and CSSG were found in CSF. The calculated concentration of CSSC (0.03 μM) is below our detectable level, which may explain why CSSC was not detected in CSF.

Several discrepancies exist in the study of thiol and disulfide transport across the blood-brain barrier. Wade and Brady (1981) found that little [35S]CSSC is taken up by the brain using a carotid injection technique. By contrast, Hwang et al. (1980) showed [35S]CSSC uptake by isolated brain capillaries. Our finding of CSSC levels in plasma is not unique as Felig et al. (1973) observed a similar positive arteriojugular venous concentration difference of CSSC (9.0 ± 4.2 μM) in humans. For CSH and GSH, our data from plasma levels suggest no substantial uptake of CSH and GSH by the brain, in contrast with the observation of [35S]CSH uptake (Wade and Brady, 1981) and [35S]GSH uptake (Kannan et al., 1990). It is postulated that CSH and GSH are permeable across the blood-brain barrier and that the movement is bidirectional. GSH is also important in the γ-glutamyl cycle. However, their net transport across the blood-brain barrier contributes little to the thiol/disulfide pool of the CNS.

It should be mentioned that a sharp gradient of GSH exists in different blood vessels. It has long been known that the liver is the major organ releasing GSH into blood (Anderson et al., 1980; Fariss and Reed, 1983). The hepatic vein plasma has the highest levels of GSH observed (26 μM) (Anderson et al., 1980). We did not detect GSH in the carotid artery and internal jugular vein. The explanation for this could be that GSH released from the liver rapidly proceeds to the thiol/disulfide exchange reaction in blood.

Owing to its critical role in intracellular GSH synthesis, CSH must be maintained at a stable level. However, CSH is unstable and will be oxidized to CSSC under aerobic conditions. Likewise, GSH and other thiols will also be oxidized to the corresponding disulfides. Our experiments show that the stable levels of thiols in the brain are maintained by astrocytes. The choroid plexus might also play a role in the formation and recycling of GSH in the CSF (Anderson et al., 1989).

Extensive studies have shown that astrocytes play diverse functions in maintaining the stable internal milieu of the brain, such as participating in the blood-brain barrier (Wolburg and Risau, 1995), regulating extracellular potassium (Newman, 1995), and taking up glutamate (Martin, 1995). Astrocytes have also been shown to have neuroprotective effects against H2O2-induced oxidative stress (Langeveld et al., 1995; Desagher et al., 1996; Drukarch et al., 1997). In this study, we show that astrocytes indirectly provide CSH to neurons for GSH synthesis. Taken together, these data indicate that astrocytes play a critical role in protecting neurons from oxidative stress.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. Procedures for obtaining CSF and plasma of artery and vein
  5. HPLC assay of thiols, disulfides, and associated compounds
  6. Quantification of intracellular GSH and related compounds in astrocytes and neurons
  7. RESULTS
  8. CSSG is found in CSSC-containing ACM
  9. GSH is released by astrocytes in CSSC-free medium
  10. Analysis of CSH and related compounds in CSF and in plasma of artery and vein
  11. Thiols are not found in NCM
  12. Contents of GSH and related compounds in astrocytes and neurons
  13. Autoxidation of CSH and GSH
  14. DISCUSSION
  15. Acknowledgements
  16. REFERENCES
  • 1
    Anderson M.E., Bridges R.J., Meister A. (1980) Direct evidence for inter-organ transport of glutathione and that the non-filtration renal mechanism for glutathione utilization involves gamma-glutamyl transpeptidase.Biochem. Biophys. Res. Commun. 96 848853.
  • 2
    Anderson M.E., Underwood M., Bridges R.J., Meister A. (1989) Glutathione metabolism at the blood-cerebrospinal fluid barrier.FASEB J. 3 25272531.
  • 3
    Arriza J.L., Kavanaugh M.P., Fairman W.A., Wu Y.N., Murdoch G.H., North R.A., Amara S.G. (1993) Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family.J. Biol. Chem. 268 1532915332.
  • 4
    Aw T.Y., Ookhtens M., Ren C., Kaplowitz N. (1986) Kinetics of glutathione efflux from isolated rat hepatocytes.Am. J. Physiol. 250 G236G243.
  • 5
    Banker G.A. (1980) Trophic interactions between astroglial cells and hippocampal neurons in culture.Science 209 809810.
  • 6
    Bannai S. & Tsukeda H. (1979) The export of glutathione from human diploid cells in culture.J. Cell. Physiol. 112 265272.
  • 7
    Beutler E. (1989) Nutritional and metabolic aspects of glutathione.Annu. Rev. Nutr. 9 287302.
  • 8
    Bradford M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72 248254.
  • 9
    Cooper A.J. & Kristal B.S. (1997) Multiple roles of glutathione in the central nervous system.Biol. Chem. 378 793802.
  • 10
    Desagher S., Glowinski J., Premont J. (1996) Astrocytes protect neurons from hydrogen peroxide toxicity.J. Neurosci. 16 25532562.
  • 11
    Dringen R., Pfeiffer B., Hamprecht B. (1999) Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione.J. Neurosci. 19 562569.
  • 12
    Drukarch B., Schepens E., Jongenelen C.A.M., Stoof J.C., Langeveld C.H. (1997) Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency.Brain Res. 770 123130.
  • 13
    Fariss M.W. & Reed D.J. (1983) Measurement of glutathione and glutathione disulfide efflux from isolated rat hepatocytes, inIsolation, Characterization and Use of Hepatocytes (Harriss R. A. and Cornell N. W., eds), pp. 349355. Elsevier, New York.
  • 14
    Felig P., Wahren J., Ahlborg G. (1973) Uptake of individual amino acids by the human brain.Proc. Soc. Exp. Biol. Med. 142 230231.
  • 15
    Franchi-Gazzola R., Gazzola G.C., 'Asta V., Guidotti G.G. (1982) The transport of alanine, serine, and cysteine in cultured human fibroblasts.J. Biol. Chem. 257 95829587.
  • 16
    Hwang S.M., Weiss S., Segal S. (1980) Uptake of L-[35S]cystine by isolated rat brain capillaries. J. Neurochem. 35 417424.
  • 17
    Jocelyn P.C. (1967) The standard redox potential of cysteine-cystine from the thiol disulphide exchange reaction with glutathione and lipoic acid.Eur. J. Biochem. 2 327331.
  • 18
    Kannan R., Kuhlenkamp J.F., Jeandidier E., Trinh H., Ookhtens M., Kaplowitz N. (1990) Evidence for carrier-mediated transport of glutathione across the blood-brain barrier in the rat.J. Clin. Invest. 85 20092013.
  • 19
    Kilberg M.S., Handlogten M.E., Christensen H.N. (1981) Characteristics of system ASC for transport of neutral amino acids in the isolated rat hepatocyte.J. Biol. Chem. 256 33043312.
  • 20
    Kranich O., Hamprecht B., Dringen R. (1996) Different preferences in the utilization of amino acids for glutathione synthesis in cultured neurons and astroglial cells derived from rat brain.Neurosci. Lett. 219 211214.
  • 21
    Langeveld C.H., Jongenelen C.A.M., Schepens E., Stoof J.C., Bast A., Drukarch B. (1995) Cultured rat striatal and cortical astrocytes protect mesencephalic dopaminergic neurons against hydrogen peroxide toxicity independent of their effect on neuronal development.Neurosci. Lett. 192 1316.
  • 22
    Li X., Wallin C., Weber S.G., Sandberg M. (1999) Net efflux of cysteine, glutathione and related metabolites from rat hippocampal slices during oxygen/glucose deprivation: dependence on γ-glutamyl transpeptidase. Brain Res. 815 8188.
  • 23
    Martin D.L. (1995) The role of glia in the inactivation of neurotransmitters, inNeuroglia (Kettenmann H. and Ransom B. R., eds), pp. 732745. Oxford, New York.
  • 24
    McCaffery C.A., Raju T.R., Bennett M.R. (1984) Effects of cultured astroglia on the survival of neonatal rat retinal ganglion cells in vitro.Dev. Biol. 104 441448.
  •  
    McCarthy K.D. and De Vellis J. (1985) Preparation of separate astroglia and oligodendroglial cell cultures from rat cerebral tissue.J. Cell Biol. 85 890902.
  • 26
    Meister A. (1989) Metabolism and function of glutathione, inGlutathione, Part A (Dolphin A., Poulson R., and Avramovic O., eds), pp. 367474. John Wiley & Sons, New York.
  • 27
    Meister A. & Anderson M.E. (1983) Glutathione.Annu. Rev. Biochem. 52 711760.
  • 28
    Newman E.A. (1995) Glial cell regulation of extracellular potassium, inNeuroglia (Kettenmann H. and Ransom B. R., eds), pp. 717731. Oxford, New York.
  • 29
    Orte C., Lawrenson J.G., Finn T.M., Reid A.R., Allt G. (1999) A comparison of blood-brain barrier and blood-nerve barrier endothelial cell markers.Anat. Embryol. (Berl.) 199 509517.
  • 30
    Ratan R.R., Murphy T.H., Baraban J.M. (1994) Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione.J. Neurosci. 14 43854392.
  • 31
    Reed D.J., Babson J.R., Beatty P.W., Brodie A.E., Ellis W.W., Potter D.W. (1980) High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides.Anal. Biochem. 106 5562.
  • 32
    Risau W., Dingler A., Albrecht U., Dehouck M., Cecchelli R. (1992) Blood-brain barrier pericytes are the main source of γ-glutamyltranspeptidase activity in brain capillaries. J. Neurochem. 58 667672.
  • 33
    Sagara J., Miura K., Bannai S. (1993) Maintenance of neuronal glutathione by glial cells.J. Neurochem. 61 16721676.
  • 34
    Sagara J., Makino N., Bannai S. (1996) Glutathione efflux from cultured astrocytes.J. Neurochem. 66 18761881.
  • 35
    Shafqat S., Tamarappoo B.K., Kilberg M.S., Puranam R.S., McNamara J.O., Guadano-Ferraz A., Fremeau R.T. (1993) Cloning and expression of a novel Na+-dependent neutral amino acid transporter structurally related to mammalian Na+ glutamate contransporters.J. Biol. Chem. 268 1535115355.
  • 36
    Tate S.S., Ross L.L., Meister A. (1973) The gamma-glutamyl cycle in the choroid plexus. Its possible function in amino acid transport.Proc. Natl. Acad. Sci. USA 70 14471449.
  • 37
    Utsunomiya-Tate N., Endou H., Kanai Y. (1996) Cloning and functional characterization of a system ASC-like Na+-dependent neutral amino acid transporter. J. Biol. Chem. 271 1488314890.
  • 38
    Vernadakis A. (1988) Neuron-glia interrelations.Int. Rev. Neurobiol. 30 149224.
  • 39
    Wade L.A. & Brady H.M. (1981) Cysteine and cystine transport at the blood-brain barrier.J. Neurochem. 37 730734.
  • 40
    Wang X.F. & Cynader M.S. (1999) Effects of astrocytes on neuronal attachment and survival shown in a serum-free co-culture system.Brain Res. Brain Res. Protoc. 4 209216.
  • 41
    Wolburg H. & Risau W. (1995) Formation of the blood-brain barrier, inNeuroglia (Kettenmann H. and Ransom B. R., eds), pp. 763776. Oxford, New York.
  • 42
    Yudkoff M., Pleasure D., Cregar L., Lin Z., Nissim I., Stern J. (1990) Glutathione turnover in cultured astrocytes: studies with [15N]glutamate. J. Neurochem. 55 137145.
  • 43
    Yuzaki M., Mikoshiba K., Kagawa Y. (1993) Cerebellar astrocytes specifically support the survival of Purkinje cells in culture.Biochem. Biophys. Res. Commun. 197 123129.
  • 44
    Zhang H.F., Ong W.Y., Leong S.K., Laperche Y. (1997) Species differences in the localisation of gamma-glutamyl transpeptidase immunopositive cells at the blood-brain interface.J. Hirnforsch. 38 323330.