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The Journal of Neuroscience, January 15, 1999, 19(2):562-569
Synthesis of the Antioxidant Glutathione in Neurons: Supply by
Astrocytes of CysGly as Precursor for Neuronal Glutathione
Ralf
Dringen,
Brigitte
Pfeiffer, and
Bernd
Hamprecht
Physiologisch-chemisches Institut der Universität, D-72076
Tübingen, Germany
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ABSTRACT |
Deficiency of the antioxidant glutathione in brain appears to be
connected with several diseases characterized by neuronal loss. To
study neuronal glutathione metabolism and metabolic interactions between neurons and astrocytes in this respect, neuron-rich primary cultures and transient cocultures of neurons and astroglial cells were
used. Coincubation of neurons with astroglial cells resulted within 24 hr of incubation in a neuronal glutathione content twice that of
neurons incubated in the absence of astroglial cells. In cultured
neurons, the availability of cysteine limited the cellular level of
glutathione. During a 4 hr incubation in a minimal medium lacking all
amino acids except cysteine, the amount of neuronal glutathione was
doubled. Besides cysteine, also the dipeptides CysGly and  GluCys
were able to serve as glutathione precursors and caused a
concentration-dependent increase in glutathione content. Concentrations
giving half-maximal effects were 5, 5, and 200 µM for
cysteine, CysGly, and GluCys, respectively. In the transient cocultures, the astroglia-mediated increase in neuronal glutathione was
suppressed by acivicin, an inhibitor of the astroglial ectoenzyme -glutamyl transpeptidase, which generates CysGly from glutathione. These data suggest the following metabolic interaction in glutathione metabolism of brain cells: the ectoenzyme -glutamyl transpeptidase uses as substrate the glutathione released by astrocytes to generate the dipeptide CysGly that is subsequently used by neurons as precursor for glutathione synthesis.
Key words:
astrocytes; CysGly; glutathione; metabolic coupling; neurons; neurodegeneration; oxidative stress
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INTRODUCTION |
Evidence is growing that glutathione
plays an important role in the detoxification of reactive oxygen
species in brain. Deficiencies in brain glutathione metabolism appear
to be connected with several neurodegenerative diseases (Bains and
Shaw, 1997 ). For example, a lowered glutathione content has been found
in the substantia nigra pars compacta of patients who suffered from
Parkinson's disease (Sofic et al., 1992; Sian et al., 1994). In
several animal models, it has been demonstrated that glutathione is
essential for normal brain function. In newborn rats, induction of
glutathione deficiency by application of buthionine sulfoximine (BSO),
an inhibitor of the first step in glutathione synthesis, caused
mitochondrial damage in brain (Jain et al., 1991). Furthermore,
reduction of the brain glutathione content by BSO enhanced the toxic
effects of insults, which are discussed to act by generation of
reactive oxygen species, i.e., reperfusion after ischemia (Mizui et
al., 1992) or treatment with 6-hydroxydopamine (Pileblad et al., 1989) and 1-methyl-4-phenylpyridinium (Wüllner et al., 1996).
On the cellular level, neurons are considered to contain less
glutathione than astroglial cells (for review, see Cooper, 1997). In
cell culture, values for the amount of glutathione in neurons have been
reported. However, these values vary in a range from <1 nmol/mg
protein (Raps et al., 1989) to 40 nmol/mg protein (Pileblad et al.,
1991). Recently it has been demonstrated that the amount of glutathione
in neurons and astroglial cells varies with the brain region from which
the cells have been prepared. Neurons in cultures prepared from the
cortex contain less glutathione than astroglial cultures from cortex.
In contrast, neuronal and astroglial cultures prepared from striatum or
mesencephalon contain almost identical levels of glutathione (Langeveld
et al., 1996).
So far little is known about the metabolism of glutathione in brain
neurons. As in many other cell types (Bannai and Tateishi, 1986), the
availability of cysteine appears to limit the glutathione content in
cultured neurons. In culture, it has been demonstrated that neurons are
unable to take up cystine (Sagara et al., 1993) and, therefore, rely on
the presence of cysteine as substrate for glutathione synthesis (Sagara
et al., 1993; Kranich et al., 1996). It has been suggested that
astrocytes provide the cysteine essential for neuronal glutathione
synthesis by intracellular reduction of cystine and subsequent release
of cysteine to neurons (Sagara et al., 1993). Indeed, the presence of
astroglial cells increased strongly the content of glutathione in
neurons (Bolanos et al., 1996), supporting the idea of a metabolic
interaction between astrocytes and neurons regarding glutathione metabolism.
The data presented here demonstrate that the dipeptide CysGly is an
excellent precursor of neuronal glutathione. The results suggest that
astroglial cells generate this precursor via their ectoenzyme
-glutamyl transpeptidase from the glutathione that they release.
Part of this work has been published in abstract form (Dringen et al.,
1998b).
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MATERIALS AND METHODS |
Materials. DMEM and horse serum were obtained
from Life Technologies (Eggenstein, Germany). CysGly and GluCys were
obtained from Bachem (Bubendorf, Switzerland). Fetal calf serum,
glutathione (GSH), glutathione disulfide (GSSG), glutathione
reductase from yeast (EC 1.6.4.2), insulin, and NADH were purchased
from Boehringer Mannhein (Mannheim, Germany). NADPH was from Applichem
(Darmstadt, Germany). Acivicin, bovine serum albumin, BSO, cytosine
arabinoside, 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB),
poly-D-lysine, progesterone, putrescine, transferrin, and
5-sulfosalicylic acid were obtained from Sigma (Deisenhofen, Germany).
Sodium pyruvate and sodium selenite were purchased from Fluka (Neu-Ulm,
Germany). L-Cysteine, streptomycine sulfate, penicillin G,
and Triton X-100 were from Serva (Heidelberg, Germany). All other
chemicals, of the highest purity available, were obtained from Merck
(Darmstadt, Germany). Cell culture dishes (35 mm in diameter), 6-well
dishes, cell culture inserts with Anopore membranes (25 mm in
diameter), and 96-well microtiter plates were from Nunc (Wiesbaden, Germany).
Neuron-rich primary cultures. Neuron-rich primary cultures
were prepared from the brains of embryonic Wistar rats and maintained as described (Löffler et al., 1986). Briefly, the brains of the embryonic day 16 (E16) rats were prepared and mechanically disrupted by
passing through nylon meshes with 135 and 20 µm pores. One million
viable cells were seeded into poly-D-lysine-coated plastic culture dishes (35 mm in diameter) in 2 ml of 90% DMEM and 10% horse
serum containing penicillin and streptomycin (PS; 20 U/ml penicillin G
and 20 µg/ml streptomycin sulfate). After 24 hr, the cells were
washed with 2 ml of DMEM/PS and then incubated in 2 ml of
glia-conditioned DMEM/PS (Löffler et al., 1986) containing the
five components particular to medium N2 (Bottenstein and Sato, 1979).
After 3 d in culture, the cells were treated for 24 hr with
cytosine arabinoside in a final concentration of 5 µM.
Subsequently, the cells were incubated in glia-conditioned DMEM/PS/N2.
The cultures were used at an age of 5-7 d. In this range, the specific
glutathione content did not depend on the age of the culture. The
neuron-rich primary cultures contain minor contaminations of astroglial
cells but no oligodendroglial or ependymal cells (Löffler et al.,
1986).
Transient cocultures. Astroglia-rich primary cultures were
prepared as described (Hamprecht and Löffler, 1985) by seeding 1 million viable cells in 25 mm cell culture inserts (Nunc) on an Anopore
membrane with a pore size of 20 nm and incubating in 4 ml 90% DMEM and
10% fetal calf serum/PS. The culture medium was renewed every 7 d. At a culture age between 17 and 21 d, the transient coculture
was initiated by transferring a cell culture insert with astroglial
cells into a well of a 6-well plate containing a neuron-rich primary
culture of a culture age between 5 and 7 d (Fig.
1). These neuron-rich cultures were
prepared as described above. We seeded 1.5 million viable cells
per well of a poly-D-lysine-coated 6-well dish. The
membrane of the insert allows traffic of small, diffusible substances
between the cells of the two culture types but prevents cell contacts.
The protein content of the neuronal and astroglial compartment at the
onset of the coculture was 143 ± 34 µg and 264 ± 48 µg
of protein per well or cell culture insert, respectively
(n = 15, from five cocultures).
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Figure 1.
Transient cocultures. 1,
Neuron-rich and astroglia-rich primary cultures were prepared and
maintained as described in Materials and Methods. The astroglial cells
were seeded and cultivated on Anopore membranes, permeable for low
molecular weight compounds. 2, After separate incubation
of the neuronal and astroglial cultures, the coculture was initiated by
transfer of an astroglia-containing culture insert to a well containing
cultured neurons. 3, After an appropriate incubation
period, glutathione levels were determined in the two compartments of
the coculture and in the coculture medium.
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Experimental incubation. The culture medium of neuron-rich
primary cultures (prepared on plastic dishes 35 mm in diameter) was
replaced by 2 ml of a minimal medium (MM+) containing (in mM) 44 NaHCO3, 110 NaCl, 1.8 CaCl2, 5.4 KCl, 0.8 MgSO4, 0.92 NaH2PO4, and 5 glucose adjusted with
CO2 to pH 7.4), containing the amino acids and dipeptides
indicated. The cells were incubated for up to 6 hr in the incubator
(Heraeus, Hanau, Germany) containing a humidified atmosphere of 10%
CO2 and 90% air. The incubation was terminated by washing
the cells twice with 2 ml of ice-cold PBS (10 mM potassium
phosphate buffer, pH 7.4, containing 150 mM NaCl) to
completely remove sulfhydryl-group-containing compounds that might
interfere with the glutathione assay. Subsequently, the cells were
lysed in 200 µl of 1% (w/v) sulfosalicylic acid on ice for 2 min.
The cell lysates were scraped off the dish, and, after centrifugation
(1 min, 16000 × g), the supernatant was used for
measuring the content of total glutathione (GSx) or GSSG.
The cells of the transient coculture were incubated in a total volume
of 2 ml DMEM/PS in the presence or the absence of acivicin (100 µM). All incubations were performed in the cell incubator containing a humidified atmosphere of 10% CO2 and 90%
air. The coculture was terminated by collecting the coculture medium,
washing the cells of both the astroglial and the neuronal compartment twice with 2 ml of ice-cold PBS, and lysing the cells with 200 µl of
1% (w/v) sulfosalicylic acid. The lysates were collected as described above.
Glutathione assay. GSx (amount of GSH plus 2 times amount of
GSSG) was measured in a microtiter plate assay, as described previously
(Dringen and Hamprecht, 1996). The standards were carried through
exactly the same procedure as the cell extracts. Ten microliters of the
cell lysates or GSSG standards (0-500 pmol of GSx/10 µl) were
transferred into 90 µl of water placed in wells of microtiter plates.
After addition of 100 µl reaction mixture (0.1 M sodium phosphate buffer, pH 7.5, containing 1 mM EDTA, 0.3 mM DTNB, 0.4 mM NADPH, and 1 U/ml glutathione
reductase), the increase in absorbance at 405 nm was detected in 15 sec
intervals over a range of 2.5 min using a microtiter plate reader
(Titertek Plus MS212; ISN Biomedicals, Meckenheim, Germany).
Glutathione contents were evaluated with the aid of the software
delivered with the plate reader by using a calibration curve
established with standard samples. Because the cells were washed twice
with PBS before the cells were lysed, the remaining sulfhydryl
compounds were present in an amount too low to interfere with the
detection of glutathione in the lysates.
GSSG was quantified after derivatization of reduced glutathione with
2-vinylpyridine (2VP; Griffith, 1980), as described previously (Dringen
and Hamprecht, 1996). Briefly, 130 µl of the protein-free supernatant
was mixed with 5 µl 2VP and adjusted with 0.2 M Tris to a
pH value between 5 and 7. Standard amounts of GSSG were treated the
same way. After 1 hr incubation at room temperature, 10 µl of the
2VP-treated samples or standards were assayed as described above using
a calibration curve established between 0 and 50 pmol of GSSG per well.
The extinction at 405 nm was followed over 5 min in 30 sec intervals.
Determination of cell viability. Cell viability was assessed
by determining the activity of lactate dehydrogenase (LDH) in the
incubation medium, as previously described (Dringen et al., 1998a).
Fifty microliters of the incubation medium collected from the dishes
after the incubation of the cells were added to 150 µl of 80 mM Tris/HCl buffer, pH 7.2, containing 200 mM
NaCl in a well of a microtiter plate. After addition of 200 µl
reaction mixture (80 mM Tris/HCl buffer, pH 7.2, containing
200 mM NaCl, 3.2 mM pyruvate, and 0.4 mM NADH), the decrease in the absorbance at 340 nm was
recorded for up to 10 min in 30 sec intervals at room temperature using
the microtiter plate reader. The LDH activity in the incubation buffer
was compared with the LDH activity in incubation medium after total
lysis of the cells by an incubation at room temperature (30 min) after
the addition of Triton X-100 to 1% (w/v) final concentration. Zero
percent viability corresponds to 100% LDH activity in the buffer.
Determination of protein content. The content of glutathione
per dish of a neuron-rich or astroglia-rich primary culture was normalized to the protein content of this dish. For this purpose, the
precipitates of the sulfosalicylic acid precipitation were dried and
resuspended in 0.5 M NaOH. An aliquot part of the solution was analyzed for its protein content according to the method described by Lowry et al. (1951). Bovine serum albumin was used as a standard.
Statistical analysis. Because the absolute values for GSx
content of the neuron cultures as well as the degree of increase of
glutathione content after application of cysteine or a cysteine substitute varied strongly between independently prepared cultures, an
inclusion of data obtained from different cultures for one statistical
analysis was not always appropriate. However, within one culture the
GSx values obtained from replica plates were almost identical (SD
<7%). Therefore, unless stated otherwise, the data presented on the
glutathione content of neuron-rich primary cultures represent
triplicates ± SD obtained from replica plates of one culture. All
results shown were reproduced with comparable results on at least one
independently prepared culture.
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RESULTS |
Glutathione content in neuron cultures
The total glutathione content (GSx) of neuron-rich primary
cultures varied among independently prepared cultures. In 27 individual experiments performed in triplicates of replica plates on 15 independently prepared cultures, GSx values were obtained between the
extreme values 13.7 ± 0.3 and 44.2 ± 0.9 nmol of GSx/mg
protein with an average amount of 23.7 ± 6.0 nmol of GSx/mg
protein. However, the glutathione content in replica plates of one
culture was almost constant with an average SD <7% (27 experiments).
The amount of GSx found represented almost exclusively GSH. GSSG was
encountered only in small amounts in the range of the detection limit
of the method used for its determination. The GSSG content accounted for maximal 2.5% of the total glutathione content (Table
1).
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Table 1.
Content of glutathione in cell lysates and cell viability
before and after incubation of neurons in the presence of cysteine,
CysGly, or GluCys
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Glutathione content in transient neuron-astroglia cocultures
To study metabolic interactions between brain neurons and
astrocytes in the synthesis of the antioxidant glutathione, a transient coculture system was established (Fig. 1). After the incubation periods
indicated, the neuronal and astroglial compartments were separated from
each other (Fig. 1), and glutathione content was determined in the
cells of both compartments as well as in the incubation medium. After
onset of the incubation, the glutathione content of neurons incubated
in the absence of astroglial cells remained almost constant (Fig.
2A). In contrast, after
application of astroglial cells, the glutathione content in the
neuronal compartment increased (Fig. 2A) and reached,
after 24 hr of incubation, twice the initial level of glutathione or
165% of the glutathione amount of cells incubated in the absence of
astroglial cells (Figs. 2A, 7). In contrast to the
glutathione content in the neuronal compartment of the coculture, that
in the astroglial compartment remained almost unchanged during the
coculture with neurons (Fig. 2B). In the medium of
the coculture, glutathione was hardly detectable during the first 10 hr
of the incubation. However, after 24 hr of incubation, the glutathione
concentration in the medium exceeded 2 µM (Fig.
2C, Table 2).
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Figure 2.
Glutathione content in astroglia-neuron
coculture. The neurons were incubated in DMEM/PS in the absence
(open circles) or the presence (closed
circles) of cell culture inserts containing astroglial cells.
The glutathione content was determined at the time points indicated in
the neuronal (A, closed circles) and
astroglial (B) compartment of the coculture, in
neuron-rich cultures incubated in the absence of astroglial cells
(A, open circles), and in the incubation
medium of the coculture (C). At the onset of the
incubation, the primary cultures of neurons and astroglial cells were
6- and 21-d-old, respectively. The data presented are triplicates ± SD obtained in one experiment. Using independent cultures, the
experiment was repeated twice with comparable results.
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Table 2.
Glutathione content of the neuronal compartment and the
incubation medium of transient cocultures in the presence (100 µM) or the absence (0 µM) of acivcin
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Glutathione content in neurons after application of glutamine,
cysteine, and glycine
To identify glutathione precursors, the availability of which
limit the synthesis of glutathione in neurons, neuron-rich primary cultures were incubated for 4 hr in a minimal medium containing glucose
(5 mM) (MM+) and the amino acids indicated (Fig.
3). The high viability of the cells was
not altered by this incubation (data not shown). In the absence of
amino acids or in the presence of glutamine, glycine, or glutamine plus
glycine, no increase in neuronal glutathione content was found in
respect to the amount in untreated cells (Fig. 3). In contrast,
application of cysteine in the absence or the presence of glycine
and/or glutamine caused a significant increase in the amount of
neuronal glutathione, at least doubling the content in comparison to
that of untreated cells (Fig. 3).
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Figure 3.
Glutathione content in a 6-d-old neuron-rich
primary culture after incubation for 4 hr in MM+ containing the amino
acids indicated in concentrations of 1 mM. Before the
incubation, the cells contained 23.3 ± 3.3 nmol of GSx/mg of
protein. The data presented are triplicates ± SD. The experiment
was repeated on an independent culture with comparable results.
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Dipeptides as precursors for neuronal glutathione synthesis
To study the ability of neurons to use the cysteine-containing
dipeptides CysGly and GluCys as precursors for glutathione synthesis, the glutathione content of neurons was measured 4 hr after
incubation of the cells with the dipeptides. Both dipeptides were able
to substitute for cysteine in doubling glutathione content in neurons
within 4 hr (Fig. 4, Table 1). After
incubation of the cells with cysteine or one of the two dipeptides, the
increased glutathione content represented almost exclusively GSH; only
minor amounts of GSSG were detectable (Table 1). Under these conditions the viability of the cells was practically unchanged (Table 1).
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Figure 4.
Effect of BSO on the utilization of CysGly and
GluCys for glutathione synthesis in 6-d-old neuron-rich primary
cultures. The cells were incubated for 4 hr in MM+ containing Gln + Cys + Gly, Gln + CysGly, or Gly + GluCys in concentrations of 1 mM each in the absence (open bars) or the
presence (hatched bars) (0.5 mM) of BSO.
Before the incubation, the cells contained 28.0 ± 0.7 nmol of
GSx/mg of protein. The data presented are triplicates ± SD. The
experiment was repeated with comparable results using an independent
culture.
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The first enzyme in glutathione synthesis is the -glutamylcysteine
synthetase. To investigate its involvement in the utilization of the
dipeptides as precursors for glutathione synthesis in neurons, BSO, an
inhibitor of this enzyme (Griffith and Meister, 1979), was applied to
neurons together with the dipeptides. The presence of BSO not only
completely prevented the increase in neuronal GSx content during
incubation with cysteine or one of the dipeptides, but also caused,
within 4 hr of incubation compared with the initial content, a decrease
of at least 30% in the neuronal content of glutathione (Fig. 4). This
finding for neuronal glutathione synthesis from GluCys as precursor
contrasts with the situation described for kidney (Anderson and
Meister, 1983), brain (Pileblad and Magnusson, 1992), and for cultured
astroglial cells (Dringen et al., 1997b), in which GluCys can bypass
the -glutamylcysteine synthetase reaction.
The glutathione content of neurons in culture was rapidly enhanced
after application of dipeptides (Fig. 5).
After 1 hr of incubation, neurons incubated with GluCys contained
glutathione levels higher than those found in neurons fed with cysteine
or CysGly. Maximal glutathione contents were observed after ~4 hr of
incubation if cysteine, CysGly, or GluCys were fed in concentrations of 1 mM (Fig. 5). By making the concentrations of cysteine
plus glycine, CysGly, or GluCys limiting for glutathione synthesis, the dependency of the cellular glutathione content on the
concentrations of these substrates was determined. Exogenous cysteine
plus glycine, or CysGly or GluCys were used for neuronal glutathione
synthesis with half-maximal effects at respective concentrations of
about 5, 5, and 200 µM (Fig.
6). Maximal glutathione levels were
attained at concentrations of ~30 µM cysteine plus
glycine or CysGly, whereas GluCys had to be present in a
concentration of 1 mM to raise neuronal glutathione to a
maximal level (Fig. 6). In contrast to the presence of dipeptides,
cysteine in concentrations higher than 100 µM led to a
submaximal glutathione content in neurons (Fig. 6). This reduction in
maximal glutathione content of neurons incubated with cysteine at a
concentration of 1 mM (78.2 ± 8.7%) rather than with
100 µM (100%) was reproducible and significant (p < 0.001; n = 12 from four
experiments) but was not accompanied by a decrease in the cell
viability (data not shown).
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Figure 5.
Time course of the glutathione content in 5-d-old
neuron-rich primary cultures during incubation with amino acids and/or
dipeptides. The cells were incubated for the time periods indicated in
MM+ containing glutamine and either cysteine plus glycine (open
circles) or CysGly (closed circles), or glycine
plus GluCys (closed triangles). The amino acids or
dipeptides were applied in concentrations of 1 mM. The data
presented are triplicates ± SD. Using an independent culture, the
experiment was repeated with comparable results.
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Figure 6.
Glutathione content in a 6-d-old neuron-rich
primary culture after incubation with amino acids and/or dipeptides.
The cells were incubated for 4 hr in MM+ containing glutamine (1 mM) and either cysteine plus glycine (open
circles) or CysGly (closed circles) in the
concentrations indicated or in MM+ containing glycine (1 mM) plus GluCys (closed triangles) of
varied concentration. Before the incubation the cellular glutathione
content was 34.0 ± 1.5 nmol of GSx/mg of protein. The data
represent triplicates ± SD. The experiment was repeated with
comparable results using an independently prepared culture.
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Effect of acivicin on the GSx content in cocultures
Because CysGly is the product of a reaction catalyzed by GT, it
is hypothesized that this dipeptide is generated from the GSH released
by astroglial cells. Thus, CysGly would be produced in equimolar
concentration to the GSH used as substrate, and, subsequently, could
become substrate for neuronal glutathione synthesis (see Fig. 8). To
test for an involvement of GT in the astroglia-induced increase in
the neuronal glutathione level in the cocultures, acivicin was applied,
an inhibitor of the ectoenzyme GT (Stole et al., 1994; Dringen et
al., 1997a). Presence of acivicin in a concentration of 100 µM during an incubation for 24 hr did not significantly
alter the glutathione content in neurons compared with cells incubated
in the absence of the inhibitor (Fig. 7). However, acivicin completely prevented the astroglia-induced increase in the glutathione content of the neuronal compartment of the coculture
(Fig. 7). No statistically significant difference
(p > 0.1) was observed between the glutathione
content of neurons cultivated in the absence of astroglial cells and
neurons cultivated in the presence of astroglial cells plus acivicin
(Fig. 7). In addition, during incubation of the cocultures with
acivicin, the amount of glutathione in the medium after 24 hr of
incubation was 234 ± 67% (n = 12;
p < 0.001) of that found in the medium of cocultures
incubated in the absence of acivicin. In all four experiments
performed, acivicin raised the level of glutathione in the coculture
medium more than it lowered that level in the cocultured neurons (Table
2).
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Figure 7.
Effect of acivicin and astroglial cells on the
glutathione content in neuron-rich primary cultures. Neurons were
incubated for 24 hr in DMEM/PS in the presence or the absence of
acivicin (100 µM) and/or a cell culture insert containing
an astroglia-rich primary culture. The GSx values obtained in four or
five experiments, each performed in triplicates on independent
cultures, were compared with the GSx content of neurons before onset of
the incubation. Compared to the glutathione content of untreated
neurons and neurons incubated without astroglial cells, with acivicin,
or with astroglial cells plus acivicin, the GSx content of neurons
after incubation with astroglial cells in the absence of acivicin is
highly significantly different (p < 0.00001) as analyzed by Student's t test.
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DISCUSSION |
The glutathione content of neuronal cultures reported in the
literature varies strongly from one report to another (see introductory remarks). These differences might be attributed to differences in the
preparation techniques, to species differences, to the use of different
brain areas for the preparation of the cultures (Langeveld et al.,
1996), or to different culture conditions. Variations in the
composition of the glia-conditioned medium (Löffler et al., 1986)
that was used in the present study for the maintenance of the
neuron-rich cultures might explain the alterations in the glutathione
content of different batches of neurons. The cysteine precursor
containing medium conditioned by glial cells was collected from several
astroglial cultures and was stored for different time periods before
application to the neuron cultures. Therefore, the concentration of
unstable glia-derived compounds might differ from batch to batch of the
medium. That could be the case for compounds containing sulfhydryl
groups and for growth factors, which have been reported to influence
neuronal glutathione metabolism (Spina et al., 1992; Pan and
Perez-Polo, 1993; Cheng and Mattson, 1995; Mattson et al., 1995).
A glutathione content well above the highest value reported for
untreated neurons in culture (40 nmol of GSx/mg of protein; Pileblad et
al., 1991) was reached, if cysteine, CysGly, or GluCys was
administered to cultured neurons. Because only marginal amounts of GSSG
were found in the cultured neurons, these cells appeared not to suffer
from oxidative stress during cultivation. This finding is in accord
with previous data showing that GSSG accounts for <1% of the
glutathione in whole brain (Cooper et al., 1980; Rehncrona et al.,
1980).
In contrast to cysteine, the availability of glutamine or glycine did
not limit neuronal glutathione synthesis. Even in a minimal medium,
only cysteine or a cysteine precursor had to be present to double
neuronal glutathione levels during 4 hr of incubation, indicating that
glutamate/glutamine and glycine were present in the cells in
concentrations sufficient for glutathione synthesis. Besides cysteine,
also the dipeptides CysGly and GluCys served as precursors for
neuronal glutathione. Like cysteine, CysGly was efficiently used by
neurons as glutathione precursor in micromolar concentrations. In
contrast, GluCys had to be applied in at least one order of
magnitude higher concentration to obtain maximal glutathione levels.
The concentrations of cysteine, CysGly, and GluCys providing
half-maximal effects on glutathione synthesis are lower in neurons than
in astroglial cells (Dringen et al., 1997b), indicating that neurons
are more efficient in utilizing these compounds as glutathione
precursors than astroglial cells.
The inhibition by the -glutamylcysteine synthetase inhibitor BSO of
the utilization by neurons of CysGly and GluCys for glutathione
synthesis indicates that both dipeptides must be hydrolyzed before
their constituent amino acids serve as substrates for glutathione synthesis. For GluCys, an extracellular cleavage has to be strongly considered, unless one wants to invoke a futile cycle of intracellular synthesis and degradation of GluCys in neurons. The decrease in
glutathione levels of BSO-exposed neurons in spite of the presence of
sufficient glutathione precursors demonstrates a rapid turnover of
neuronal glutathione.
The level of cysteine is low in extracellular fluids, because this
amino acid is rapidly oxidized by oxygen (Ishii and Bannai, 1985).
Therefore, the question arises as to how neurons, cells known to be
unable to use cystine, acquire the cysteine essential for glutathione
synthesis. Astrocytes could provide a cysteine precursor for neuronal
glutathione synthesis. At least in culture, the presence of astroglial
cells has been reported to maintain (Sagara et al., 1993) or even
increase glutathione levels in cultured neurons (Bolanos et al., 1996).
These findings were confirmed in the present study. The glutathione
level in the neuronal compartment was doubled within 24 hr in the
presence of astroglial cells, indicating that (1) in the absence of
astroglial cells at least one precursor for glutathione is limiting
neuronal glutathione synthesis, and (2) that a metabolite was provided
from the astroglial cells to the neurons able to bypass this
limitation. Cysteine has been reported to be released from astroglial
cells in cystine-containing culture medium (Sagara et al., 1993).
However, this release has to take place against the sodium gradient,
which enables astroglial cells to efficiently take up cysteine (Sagara
et al., 1993). The appearance of cysteine in the culture medium of
astroglial cells can also be explained as a consequence of the release
of glutathione from astroglial cells (Yudkoff et al., 1990; Sagara et
al., 1996; Dringen et al., 1997b), because GSH liberates cysteine from
cystine by forming mixed disulfides (Deneke et al., 1995). Therefore, it can be doubted that the cysteine found in the culture medium of
astroglial cells was indeed released from astroglial cells.
Recently it has been reported that the GSH released from astroglial
cells is partially used as substrate of the astroglial ectoenzyme GT
(Dringen et al., 1997a). One product of the GT reaction is the
dipeptide CysGly (Meister et al., 1981; Huseby, 1988), which is used by
neurons in micromolar concentrations as precursor for glutathione. This
finding suggests the involvement of astroglial GT in the generation
of the cysteine precursor provided by astroglial cells for neuronal
glutathione synthesis (Fig. 8). To prove
this hypothesis, the GT inhibitor acivicin was applied to the
incubation medium of neuron-astroglia cocultures. The presence of
acivicin totally prevented the astroglia-induced effect on the
glutathione content in the neuronal compartment of the coculture.
Although the specificity of acivicin for inhibiting GT has not been
fully established, this finding indicates that GT is involved in the
supply of a glutathione precursor from astroglial cells to neurons. The
finding that neurons incubated for 24 hr with acivicin contain a
glutathione level identical to neurons cultured in the absence of
acivicin demonstrates that acivicin, in contrast to BSO, did not alter
glutathione turnover in neurons by inhibition of glutathione synthesis
but rather inhibited GT in the astroglial compartment of the
coculture. The amount of glutathione in the media preserved by the
inhibition of GT is at least as high as the increase in the
glutathione content in the neuronal compartment in the absence of
acivicin. Therefore, in the coculture the CysGly most likely generated
by GT would allow for the increase of glutathione in the neuronal
compartment of the coculture. Steady-state concentrations of CysGly in
the coculture medium will be at best in the low micromolar range, which
will make it difficult to identify this dipeptide in the coculture
medium. Such a task might even be more difficult, because the
involvement of an ectopeptidase in the utilization of the dipeptide has
to be envisaged as an alternative to the uptake of CysGly into neurons
(Fig. 8).
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Figure 8.
Scheme of the proposed metabolic interaction
between neurons and astrocytes regarding glutathione synthesis. The GSH
released from astrocytes is substrate for the astroglial ectoenzyme
GT. CysGly, one product of the GT reaction, serves as a precursor
of neuronal glutathione. It is not known whether the hydrolysis of
CysGly occurs in the extracellular space by a neuronal ectopeptidase or
after uptake of the dipeptide in the neurons. In addition, glutamine is
released from astrocytes and used by neurons as precursor for glutamate
necessary for glutathione synthesis. X represents an
acceptor of the -glutamyl moiety transferred from glutathione by
GT, the most likely candidates being glutamine and
H2O.
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Besides the dipeptide CysGly, the second product of the GT reaction
is the -glutamyl derivative of the second substrate. Excellent
substrates of this enzyme are the amino acids glutamine, methionine,
and cystine (Tate and Meister, 1974; Thompson and Meister, 1975). In
addition, also H2O can serve as an acceptor of the
-glutamyl moiety (Binkley and Nakamura, 1948; Meister et al., 1981).
Further studies are required to show which products besides CysGly are
generated by astroglial GT and what the functions and the metabolism
of these compounds might be in the nervous system.
Besides the supply of a cysteine precursor, other compounds might also
be provided to neurons by astroglial cells during coculture. For the
increase in glutathione content in the neuronal compartment of the
coculture, a contribution of glial-derived growth factors has to be
considered, because an influence of neurotrophic factors on neuronal
glutathione metabolism has been reported (see above). However, it is
somewhat unlikely that such factors released from the astroglial
compartment would accumulate within a few hours in the coculture medium
to a concentration sufficient to strongly modulate the expression
levels of enzymes involved in glutathione synthesis. Although such an
effect cannot be ruled out, the availability of a cysteine precursor
predominantly limits neuronal glutathione synthesis. Therefore, the
role of growth factors in the astroglia-induced increase in neuronal
glutathione might be at best a supporting one.
With the release of glutamine by astroglial cells (Robinson et al.,
1998) and the extracellular generation of CysGly from glutathione,
astroglial cells provide precursors for all three constituting amino
acids of glutathione to neurons. For normal brain function, it might be
essential that it is not the substrates for the enzymes involved in
glutathione synthesis that are directly delivered to neurons, because
glutamate, cysteine, and glycine might harm neurons (Olney et al.,
1972; Choi, 1988; Kemp and Leeson, 1993; Puka-Sundvall et al., 1995).
In contrast, no toxic effects have been reported for glutamine and CysGly.
In conclusion, the data presented here suggest that CysGly generated by
GT from the glutathione released from astrocytes serves as precursor
for neuronal glutathione synthesis (Fig. 8). This metabolic interaction
between astrocytes and neurons provides several new targets of
interference that might be envisaged for therapeutical purposes.
Substances able to increase either glutathione release, activity of
GT, or utilization of CysGly by neurons could elevate glutathione
concentrations in brain neurons. As a consequence, those neurons would
be protected better against oxidative stress.
| |
FOOTNOTES |
Received Aug. 10, 1998; revised Oct. 26, 1998; accepted Oct. 27, 1998.
We thank Dr. Heinrich Wiesinger for critically reading this manuscript.
Correspondence should be addressed to Dr. Ralf Dringen,
Physiologisch-chemisches Institut der Universität,
Hoppe-Seyler-Strasse 4, D-72076 Tübingen, Germany.
| |
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Abstract
Introduction
