Aspartate aminotransferase isotope exchange reactions:
implications for glutamate/glutamine shuttle hypothesis
George A.
Kimmich,
James A.
Roussie, and
Joan
Randles
Department of Biochemistry and Biophysics, School of
Medicine and Dentistry, University of Rochester, Rochester, New
York 14642
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ABSTRACT |
Aspartate aminotransferase
(AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate
(Asp) to a keto acid acceptor
oxaloacetate (OA) or
-ketoglutarate
(KG), respectively. Data presented here show that AAT catalyzes two
partial reactions resulting in isotope exchange between
3H-labeled Glu or 3H-labeled Asp and the
cognate keto acid in the absence of the keto acid acceptor required for
the net reaction. Tritiated keto acid product was detected by release
of 3H2O from C-3 during base-induced
enolization. Tritium released directly from C-2 (or C-3) by the enzyme
was also evaluated and is a small fraction of that released because of
exchange to the keto acid pool. Exchange is dependent on AAT
concentration, time-dependent, proportional to the amino-to-keto acid
ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor.
Enzymatic conversion of [3H]KG to Glu by glutamic
dehydrogenase (GDH) or of [3H]OA to malate by malic
dehydrogenase (MDH) "protects" the label from release by base,
showing that base-induced isotope release is from keto acid rather than
a result of release during the exchange process. AAT isotope exchange
is discussed in the context of the glutamate/glutamine shuttle
hypothesis for astrocyte/neuron carbon cycling.
glutamate/oxaloacetate transaminase; transaminase
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INTRODUCTION |
MORE THAN
60% of all central nervous system (CNS) synapses are glutamatergic
(22), indicative of the predominant role that glutamate
(Glu) must play in the numerous neurotransmitter-mediated events
required for brain function. Nevertheless, Glu becomes a serious
neurotoxin if extracellular levels similar to those achieved during
neuronal activity are sustained for even short time intervals (5,
11, 20, 23, 25, 28, 33, 41). Ordinarily, neurotoxicity is
avoided because of the activity of sodium-coupled transport systems in
cells surrounding glutamatergic synapses. Glu uptake by these systems
restores the low extracellular level (~1 µM) characteristic of
quiescent neurons.
Both neurons and glial cells have capability for Glu reuptake (1,
7, 18, 27, 29). However, when antisense DNA is used in
"knockdown" experiments to block expression of specific transporter
isoforms, the most severe pathophysiology occurs when either of the two
primarily astrocytic isoforms (GLT1 or GLAST) is eliminated
(28). Gene "knockout" experiments confirm this conclusion. GLT1 knockouts exhibit lethal spontaneous seizure activity
(34), and mice depleted of GLAST survive but show reduced motor coordination (38). These experiments imply that Glu
reuptake by astrocytes is particularly important for avoiding
neurotoxicity. However, a problem is created for glutamatergic neurons
even if astrocytes take up only a portion of the synaptic Glu. Neurons are believed to be incapable of catalyzing net Glu synthesis from the
two- and three-carbon precursors provided by long-chain fatty acid and
glucose metabolism, respectively, because they do not express pyruvate
carboxylase, the anaplerotic enzyme required for net formation of TCA
cycle intermediates (17, 31, 43). Instead,
metabolic intermediates with four or five carbon atoms are thought to
be necessary for returning carbon from astrocytes to neurons to allow
continuing resynthesis of the neuronal Glu required for sustaining
neurotransmission. Carbon must be returned that is at least equal in
amount to that represented by astrocytic Glu capture.
Of the various intermediates receiving attention, glutamine (Gln) is
often viewed as one of the most likely candidates for carbon return
from astrocyte to neuron. Astrocytes have Gln synthetase that can
produce Gln from Glu in a single step (26). Moreover, neurons have capability for sodium-coupled Gln uptake and can resynthesize Glu in a single step catalyzed by phosphate-activated glutaminase (15). There are no known neuronal Gln
receptors, so extracellular return of carbon as Gln would not induce
inappropriate neural activity. The proposed neuron-astrocyte-neuron
carbon cycle is commonly called the Glu/Gln shuttle hypothesis
(3, 30, 35, 37).
Questions regarding the shuttle hypothesis have been raised on the
basis that astrocytes may metabolize some of the Glu they "capture"
for energy production via the TCA cycle. To the extent that this
occurs, neurons that release Glu will face a carbon deficit unless
four- or five-carbon intermediates in some form(s) other than Gln are
also returned. Most of the proposed limitations to the shuttle concept
are based on observations showing that astrocytes take up isotopically
labeled Glu rapidly and produce a wide variety of labeled TCA cycle and
other metabolic intermediates (8, 9, 14, 32, 33, 39, 40,
42). These labeled intermediates are considered alternative
candidates to Gln for shuttling carbon back to glutamatergic neurons.
The higher the rate of formation of a particular intermediate and the
larger its pool size, the more credence it is awarded as an alternative carbon shuttle participant.
The work presented here shows that aspartate aminotransferase (AAT; EC
2.6.1.1) catalyzes a rapid exchange of isotope between Glu and its
cognate keto acid
-ketoglutarate (KG), in the absence of
oxaloacetate (OA) or other keto acid acceptors for the amino group. The
enzyme also catalyzes aspartate (Asp)/OA isotope exchange. The exchange
reactions allow rapid incorporation of isotope from Glu into KG or from
Asp into OA. In the whole cell context, label exchanged into either
keto acid pool will then be distributed to other TCA cycle
intermediates even though no net consumption of Glu or Asp occurs in
the process. Astrocytes are known to express AAT in high activity
(10, 19), so it is likely that similar aminotransferase-catalyzed isotope exchange events occur in intact cells. To the extent they do, rapid labeling of TCA cycle intermediates from labeled Glu exaggerates the calculated rate of net KG production and thus the need for alternatives to Gln for replenishing the neuronal
Glu pool.
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METHODS |
Glu/KG exchange.
Enzyme-catalyzed partial reactions were characterized by determining
whether AAT can transfer isotope between tritiated amino acid and its
cognate keto acid in the absence of the substrate keto acid required
for the full net reaction. Glu/KG exchange was studied at room
temperature (20-25°C) by incubating 0.5 U AAT/ml with 50 µM
[2,3-3H]Glu [0.5-1 × 105
disintegrations per minute (dpm)/ml] and 10-200 µM of unlabeled KG in a medium containing (in mM) 100 KCl, 10 NaCl, and 10 Tris · PO4 buffer (pH 7.0). Control experiments in
which AAT was omitted were run in parallel. The total incubation volume
was 0.1-1.0 ml depending on the number of samples to be taken. One
unit of AAT is defined as the amount required to convert 1 µmol/min
of KG to Glu at pH 7.5 and 37°C with Glu and OA present at saturating concentrations. Incubation with AAT was allowed for intervals from 5 to
120 min. Aliquots (100 µl) were taken from the incubation mixture at
the times indicated, and enzyme activity was terminated by adding each
sample to 100 µl of either 0.1 M NaOH or 1 mM aminooxyacetate (AOA),
an AAT inhibitor (16). Enzyme-catalyzed transfer
of the amino group from [2,3-3H]Glu to unlabeled KG
produces KG labeled with tritium at C-3 and unlabeled Glu. Each
3H-labeled Glu molecule undergoing Glu/KG exchange
liberates tritium at C-2 to the incubation medium as
3H2O. The amount of C-3 tritium present at C-3
of the keto acid was measured by adding NaOH to force tautomerization
of keto acid to the enol form
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Each keto-enol transition for a given molecule releases
half of the C-3 tritium as 3H2O. Label loss
after net conversion of [2,3-3H]Glu to
[3-3H]KG proves that all keto acid C-3 3H is
lost as 3H2O when NaOH is added. The amount of
labeled keto acid was determined by the amount of "volatile"
isotope produced by NaOH treatment and lost during subsequent sample evaporation.
After enzymatic activity was terminated, 100-µl samples were
evaporated to dryness in a fume hood. The dry residue was dissolved in
200 µl of water, and tritium remaining in an aliquot was determined by liquid scintillation spectrometry (Beckman LS-200; 50% counting efficiency). Tritiated water in each sample at the time enzyme activity
was stopped was calculated as the difference (in dpm) in evaporated
samples compared with samples incubated without enzyme.
Although commercial preparations of tritiated Glu are produced as
[2,3-3H]Glu, C-2 tritium is usually a low percentage of
total label because of losses during storage by exchange with
H+ in the solvent. The manufacturer characterized the
preparation used here as having 8% of the total label at C-2.
Evaporation of the initial tritiated stock resulted in loss of ~8%
of the total dpm, suggesting that all of the original C-2 label may
indeed have been lost, leaving tritium exclusively at C-3. To evaluate this possibility, the fraction of C-2 tritium was determined by cycling
[2,3-3H]Glu through KG several times by the combined
action of AAT and glutamic dehydrogenase (GDH), as shown by the
following reaction sequence
The amount of Glu taken to KG and reconverted to Glu is
dependent on the amount of OA available and was determined by
spectrophotometric monitoring of the amount of NADH oxidized. The
extent of cycling is dependent on the OA-to-Glu ratio provided that the
NADH concentration exceeds that of OA. For the conditions indicated
above, the amount of NADH oxidized shows that 3.2 times the
initial amount of Glu is taken through a Glu
KG
Glu cycle before OA
is expended as the system progresses toward equilibrium. KG remains at
a low level throughout the incubation because the GDH equilibrium
constant (Keq = 6.9 × 105
at pH 7) highly favors Glu formation (4). The only label
lost after evaporation of samples taken from these experiments is
released either from C-2 of the tritiated Glu sample or from the small amount of KG present after the GDH reaction is completed. GDH converts
93% of KG back to Glu under these conditions, as determined by
spectroscopic measurements of NADH oxidation with 50 µM KG standards.
Data shown in RESULTS indicate that little or no C-2 tritium is present in the labeled Glu and that volatile tritium originates solely from C-3 of KG.
The fraction of total label present in KG at isotopic equilibrium is
equal to the mole fraction of KG. This value was used to calculate the
theoretical limit to expected NaOH-induced label loss shown in Figs. 2,
3, 5, 6, and 7.
For samples stopped with AOA, the amount of
3H2O provides a measure of label released
directly by the enzyme during Glu/KG exchange. Under these conditions,
Glu/KG exchange results in loss of C-2 Glu tritium but C-3 tritium is
retained in KG. In samples stopped with NaOH, the high pH of the
treated sample forces KG to the enol form. Any KG labeled as a result
of Glu/KG exchange loses its C-3 tritium as
3H2O during keto-enol tautomerization. The
difference in the amount of label lost during evaporation of samples
stopped with NaOH compared with that lost in samples stopped with AOA
provides a measure of total tritium conveyed to the KG pool as a result
of Glu/KG exchange. In some cases, KG in the sample was converted to
Glu by adding 300 µM NADH, 1 mM NH
, and 0.1 U/ml of
GDH before the addition of NaOH. Tritium is not released from C-3 of
Glu by NaOH. All experiments were performed a minimum of three times
with similar results in each trial. Data shown in Fig. 2 are means ± SE; all other data are mean values of duplicate samples taken for
each set of conditions. The range for each set of duplicates is
typically ~3% (9 of 10 sample pairs), or smaller than the symbol
size used in the figures.
In some experiments Glu was converted to KG on a net basis. In these
cases, AAT was incubated with (in µM) 50 Glu, 230 OA, and 300 NADH.
After different time intervals, 1 U/ml of malic dehydrogenase (MDH) was added. The amount of OA remaining at
the time of MDH addition was determined from the decrease in
optical density at 340 nm due to NADH oxidation as OA is
converted to malate (Mal). This approach provides a
means of determining the fraction of [3H]Glu converted to
labeled KG as a function of time and allows a comparison to the
fraction of label that can then be released from KG by adding NaOH.
Asp/OA exchange.
Isotope exchange associated with this partial reaction was evaluated by
procedures fundamentally similar to those described in Glu/KG
exchange. In this case, AAT was incubated at room
temperature with 50 µM [2,3-3H]Asp (0.5-1.0 × 105 dpm/ml) and 10-200 µM of unlabeled OA. KG and
Glu were absent. Again, reactions were stopped with NaOH to determine
the amount of label released during the exchange process and by
enolization of the [3H]OA product. The amount of tritium
at C-2 of the tritiated Asp was evaluated by net conversion of Asp to
Mal by incubating (in µM) 50 [2,3-3H]Asp, 250 KG, and
300 NADH with 0.5 U/ml AAT and 1 U/ml MDH. Parallel experiments in
which NADH oxidation was monitored in a spectrophotometer allowed
determination of the total amount of Asp converted to OA under these conditions.
Materials.
[2,3-3H]Glu and [2,3-3H]Asp were purchased
from New England Nuclear. AAT, GDH, and MDH were purchased from
Sigma-Aldrich (St. Louis, MO) and stored at 4°C until used. All other
chemical reagents were purchased from Sigma-Aldrich at the highest
purity available.
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RESULTS |
Aminotransferases catalyze transfer of an amino group from an
amino acid donor to a keto acid acceptor via a pyridoxal cofactor covalently bound to the enzyme (21, 24). A pyridoximine
intermediate formed during the reaction sequence from the original
amino group remains bound to the enzyme. Formation of the intermediate
results in conversion of amino acid to its cognate keto acid, the first product of the complete net reaction. The reaction mechanism is "ping-pong" in that the cognate keto acid is released from the enzyme before binding of the second substrate (also a keto acid) required for the full reaction sequence. After binding the substrate keto acid, the amino group is transferred from the enzyme-pyridoximine intermediate to produce a new amino acid as the second product of the
reaction. This transfer results in concomitant restoration of the
pyridoxal form of the free enzyme. Mechanisms of this type are
designated as ping-pong because the enzyme shifts back and forth
between two different catalytic forms during the full reaction sequence. The net reaction and a schematic representation of
the ping-pong mechanism for the full sequence catalyzed by AAT are
where Enz is enzyme. Ping-pong reaction mechanisms always involve
"partial" reactions related to the formation and subsequent expenditure of an enzyme intermediate. If the free energy change for a
partial reaction is moderate in magnitude, so that unidirectional forward and reverse rates are similar and sufficiently rapid, significant bidirectional fluxes will occur between one substrate and
its product in the absence of the second substrate and product. No net
interconversion of substrate and product occurs except that due to
formation of the tiny amount of pyridoximine-enzyme intermediate
necessary to support the steady-state partial reaction sequence. Two
partial reactions can be predicted for the AAT reaction mechanism
one
between Glu and KG and another between Asp and OA. Occurrence of either
partial reaction results in loss of a hydrogen atom from C-2 of the
amino acid to the aqueous medium, as shown schematically for the Glu/KG
partial reaction (absence of OA and Asp)
and the Asp/OA partial reaction (absence of KG and Glu)
If partial reactions indeed occur for AAT, they may be detected by
the formation of 3H2O from an amino acid
substrate tritiated at C-2. As shown above, if the amino acid is
tritiated at both C-2 and C-3, the keto acid product retains the C-3
tritium. Because amino acid C-2 tritium is usually limited in amount
because of spontaneous losses during storage, the degree to which
either partial reaction has occurred is better determined by the amount
of tritium in the keto acid pool as described in METHODS.
The degree to which AAT catalyzes a Glu/KG partial reaction was
evaluated by incubating the enzyme with 50 µM
[2,3-3H]Glu and either 50 or 200 µM of unlabeled KG. As
shown in Fig. 1, the amount of tritium
retained after sample evaporation decreases as a function of time and
in a manner that is dependent on the amount of KG present. Much more
label is released when samples are treated with NaOH instead of AOA,
showing that most of the volatile tritium was released by enolization
of the KG pool rather than by the enzyme during the exchange process.

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Fig. 1.
Aspartate aminotransferase (AAT)-catalyzed isotope
exchange between 3H-labeled glutamate (Glu) and unlabeled
-ketoglutarate (KG). Loss of tritium as a function of time after
incubation of 0.5 U/ml AAT at room temperature (23°C) with 50 µM
[2,3-3H]Glu and 50 or 200 µM unlabeled KG is shown.
Enzyme activity was stopped at the indicated time by adding 1 M NaOH in
3 trials and with aminooxyacetate (AOA), an aminotransferase inhibitor,
in the remaining trial as indicated. AOA was added at the start of 1 experiment to inhibit AAT, and NaOH was added at the indicated times as
a means of detecting any base-induced loss of tritium from
[3H]Glu not due to enzyme-catalyzed isotope exchange into
KG. The data for each time course are representative of 3 experiments
with similar results in each case. Each data point shown here and in
Figs. 3-7 is the mean value for duplicate samples taken from the
same experiment. Range for the duplicates typically is <3%, or
smaller than the symbol size used. dpm, Disintegrations per minute.
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Figure 2 shows the amount of tritium
remaining after a 60-min incubation interval when 50 µM
[3H]Glu was incubated with six different concentrations
of unlabeled KG. Total tritium loss is markedly dependent on the size
of the KG pool, as suggested by the results shown in Fig. 1. The
maximal tritium loss expected for each KG concentration is also shown in Fig. 2. These values were calculated from the amount of label expected in the KG pool if isotopic equilibrium had been achieved between Glu and KG. After a 60-min incubation interval, the tritium loss observed for each KG concentration was ~80% of the calculated maximal value.

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Fig. 2.
Base-induced tritium loss as a function of concentration
of unlabeled KG after a 60-min incubation to allow Glu/KG isotope
exchange. The incubation mixture included 50 µM
[2,3-3H]Glu and 0.5 U/ml AAT. Enzyme activity was stopped
by addition of 1 M NaOH before evaporation of samples. Each data point
is the mean ± SE for 3 separate experiments. The dashed line
shows the theoretical limit of the expected tritium loss, assuming that
NaOH liberates all isotopic label in the KG pool and that isotopic
equilibrium was achieved between Glu and KG.
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In experiments similar to those summarized in Figs. 1 and 2, NADH and
NH
were included in the incubation mixture. After 60 min of incubation to permit isotope exchange to the KG pool, GDH was
added to some of the samples and incubation continued for another 40 min to allow conversion of any labeled KG to Glu. Samples taken when
only AAT is present lose 70% of the total label after NaOH treatment
and evaporation. After GDH treatment, nearly all of the tritium is
"protected" by conversion of KG to Glu and was retained after NaOH
and evaporation as shown in Fig. 3. When
both AAT and GDH were present at the start of the experiment, only 7%
of the total label was lost after the 100-min incubation interval, the
same amount as when GDH was added after 60 min.

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Fig. 3.
Isotope exchange between 50 µM
[3H]Glu and 200 µM unlabeled KG followed by net
conversion of [3H]KG to [3H]Glu by 1 U/ml
glutamic dehydrogenase (GDH). GDH ( ) protected
virtually 100% of the tritium otherwise lost after NaOH treatment and
evaporation when only AAT was present ( ). When AAT and
GDH were both present from the start of the experiment
( ), 7% of the total tritium was still lost, indicating
that a small pool of KG may not have been converted to Glu and was
still available for supporting isotope exchange.
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Figure 4 shows results obtained when
tritiated Glu was converted to KG on a net basis by incubating AAT with
50 µM Glu and 230 µM OA. The amount of OA remaining after various
incubation intervals, determined from the optical density change after
MDH was added to each sample, showed that 41.5 µM OA was utilized after an 80-min incubation, indicating that an equal amount of Glu
(83%) was converted to KG during that time. The percentage of label
lost after NaOH treatment after various time intervals was determined
in parallel experiments under identical conditions but with
[2,3-3H]Glu present. The percentage of label lost during
evaporation is approximately equal to the percentage of Glu converted
to KG in each sample, as shown.

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Fig. 4.
Time dependence and % net conversion of
[3H]Glu to [3H]KG during incubation of 0.5 U/ml AAT with 50 µM [2,3-3H]Glu and 250 µM
oxaloacetate (OA; left axis). The % of tritium remaining in
nonvolatile form after NaOH treatment is shown on right
axis. The amount of Glu converted to KG is equal to the amount of
OA consumed in the net AAT reaction, i.e., the difference between
initial OA and residual OA at the time the sample was taken. Residual
OA was determined from the change in optical density after 1 U/ml malic
dehydrogenase (MDH) was added to the reaction mixture. The reaction
mixture included 300 µM NADH.
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In another approach, Glu was converted to KG and then cycled back to
Glu by incubating 50 µM [2,3-3H]Glu with AAT and GDH in
the presence of 200 µM OA, 300 µM NADH, and 1 mM
NH
as described in METHODS. The decrease
in NADH showed that 3.2 times the initial amount of Glu was cycled from
Glu to KG and then back to Glu before OA became limiting. Despite the
high degree of Glu turnover with both enzymes present, only 5% of the
total tritium was lost after treatment of the final incubation mixture
with NaOH and subsequent evaporation (Fig.
5). This loss probably is C-3 tritium
released from the small amount of KG not cycled back to Glu rather than from C-2 of the [3H]Glu substrate (see
DISCUSSION). When only AAT was present to allow net KG
formation, ~70% of the total tritium was lost.

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Fig. 5.
Net conversion of tritiated Glu to tritiated KG followed
by GDH-catalyzed reconversion of tritiated KG back to Glu. Tritium
remaining in nonvolatile form after evaporation of NaOH-treated samples
is shown as a function of time during incubation of 0.5 U/ml AAT with
50 µM [2,3-3H]Glu, 200 µM OA, 300 µM NADH, and 1 mM
NH . When only AAT was present during the 2-h
incubation interval, 65% of the isotope was converted to
3H2O after NaOH treatment and lost during
subsequent evaporation. When GDH was added after 60% of the isotope
had been converted to a potentially volatile form, but before NaOH was
added to stop enzymatic activity, the label was progressively
"protected" from later release by NaOH. When AAT and GDH were both
present at the outset, only 4% of the label was released by NaOH
treatment. GOT, glutamate/oxaloacetate transaminase.
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Experiments similar to those described above show that AAT also
catalyzes isotope exchange between Asp and OA. When AAT was incubated
with 50 µM [2,3-3H]Asp and variable amounts of
unlabeled OA, the amount of NaOH-induced label loss was a function of
the Asp-to-OA ratio, as shown in Fig. 6.
If MDH was added after allowing 60 min of isotope exchange with 200 µM OA, much of the label otherwise released by NaOH was protected,
because of conversion of [3H]OA to [3H]Mal.
However, 24% of the total label was still lost during evaporation, as
shown. In some cases, AAT was incubated with 50 µM
[3H]Asp, 250 µM KG, and MDH so that
[3H]OA produced from Asp by AAT was converted immediately
to Mal. Under these circumstances, ~25% of the total label also is
lost after treatment with NaOH and evaporation, as shown in Fig.
7. Net conversion of Asp to OA and the
Asp/OA exchange reaction are blocked by AOA.

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Fig. 6.
AAT-catalyzed isotope exchange between 50 µM
[3H]Asp and unlabeled OA as a function of OA
concentration. In one case, MDH was added before NaOH treatment but
after 75% of the Asp tritium was shifted to OA where it is subject to
base-induced release. Two-thirds of the potentially releasable label
was protected from release during subsequent NaOH treatment. Lack of
protection for the other one-third indicates that ~24% of the total
tritium is at C-2 of the [2,3-3H]Asp. The dotted line
shows the theoretical limit for 3H2O formation
expected if isotopic equilibrium had been achieved before NaOH
treatment, after correction for the loss of C-2 tritium. The reaction
medium contained 300 µM NADH.
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Fig. 7.
Net conversion of tritiated Asp to tritiated OA followed
by MDH-catalyzed conversion of tritiated OA to malate. Tritium
remaining in nonvolatile form after evaporation of NaOH-treated samples
is shown as a function of time during incubation of 0.5 U/ml AAT with
(in µM) 50 [2,3-3H]Asp, 250 KG, and 300 NADH
( ). MDH added after 60 min protected over half of the
tritium otherwise released by NaOH, but 24% remained releasable
( ). When AAT and MDH were present from the start
( ), 22% of the label was lost after NaOH and
evaporation. The top dashed line is the average of 9 samples taken over
90 min but with AOA present from the start of incubation
( ). The bottom dashed line is the loss expected from
the amount of OA produced from Asp, determined by spectroscopic
measurement, after correction for the calculated tritium loss from C-2.
The difference between this value and the observed label loss relates
to MDH "pulling" the AAT reaction toward completion more fully than
when only transaminase is present.
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DISCUSSION |
The data presented here show that AAT catalyzes two partial
reactions in addition to the two-substrate, two-product net reaction usually considered. Each partial reaction leads to isotope exchange between an amino acid substrate and its cognate keto acid, under conditions where net interconversion of the two reactants cannot occur.
Release of label in volatile form (3H2O) after
NaOH-induced enolization of keto acid is a rapid and sensitive method
for detecting each partial reaction. When the full net reaction is
allowed, the percentage of [3H]Glu converted to KG proves
to be the same as the percentage of total tritium released after
evaporation of NaOH-treated samples (Fig. 4), showing that base
releases tritium from KG quantitatively. Production of volatile tritium
requires AAT, is time dependent, and is inhibited by AOA, an
aminotransferase inhibitor (Fig. 1). Nearly all of the tritium released
in Fig. 1 is dependent on NaOH addition, showing that little releasable
tritium came from C-2 of Glu. This was confirmed by experiments in
which tritiated KG was converted to [3H]Glu with GDH. In
one case, labeled KG was produced by exchange with tritiated Glu (Fig.
3). In the other, [3H]Glu was cycled three or four times
on a net basis to KG and then back to Glu (Fig. 5). Only 5-7% of
the total label was lost in each case. Both experiments show that
strong base does not induce tritium release from amino acid. The
cycling experiment in particular indicates that no more than 5% of
tritium in the original [2,3-3H]Glu could have been at
C-2.
It is important to recognize that, in both experiments, a small pool of
KG remains after GDH action that may explain the small amount of label
lost after NaOH treatment. The Keq for AAT is ~1.0, and the reported Keq for Glu formation
from KG by GDH is 6.9 × 105 at pH 7.0 (4). With these values and the initial concentrations of
Glu, OA, NADH, and NH
present in the experiment shown
in Fig. 5, it can be calculated that <1% of the initial 50 µM Glu
would be present as KG if both reactions achieve equilibrium.
Spectrophotometric measurement of NADH oxidized during the 60-min
incubation interval shows that the reaction actually reaches 93% of
the calculated equilibrium value. The 5-7% label loss in the
experiments shown in Figs. 3 and 5 therefore can be explained by
base-induced loss of C-3 tritium from the small pool of labeled KG
remaining in the incubation mixture because of the lack of full
equilibrium for GDH. We conclude that virtually all the volatile
tritium produced from Glu because of Glu/KG exchange results from
base-induced enolization of [3H]KG, with consequent
release of C-3 tritium.
As shown in Figs. 2 and 6, isotope exchange is dependent on relative
pool sizes of the amino/keto acid pair. Figures 2, 3, and 5 include a
comparison of the observed fraction of tritium released during sample
evaporation and the maximum fraction predicted to be in KG. As shown,
the approach to equilibrium is dependent on the time allowed for the
exchange process and on the KG-to-Glu ratio. For the AAT concentration
used (0.5 U/ml), approximate isotopic equilibrium is established in 60 min at room temperature when Glu and KG are present at 50 and 10 µM,
respectively. As the concentration of KG is raised, more total tritium
is transferred to the keto pool but longer incubation times are
required to approach equilibrium. At 500 µM KG, isotope in the KG
pool after 1 h is ~80% of the calculated equilibrium value.
Although label in volatile form is routinely detected after NaOH
treatment to induce release of C-3 tritium, it is also possible that
some label originates from spontaneous enolization of keto acid before
addition of base. Tautomeric tritium loss is probably modest in
magnitude because of the very low percentage of enol KG expected at pH
7.0. Nevertheless, if rate constants are high for the keto-enol
transition, some C-3 label could be lost. If appreciable
tautomerization occurs at pH 7.0, then label will remain in the
incubation medium as 3H2O after conversion of
labeled KG to Glu and be lost during sample evaporation. As described
above, the 5-7% tritium loss after sample treatment with GDH
probably originates from NaOH-induced enolization of the small amount
of KG (~3.5 µM) remaining after enzyme treatment. Base-induced
tritium release from this small KG pool fully accounts for the observed
tritium loss and implies that tautomeric label release due to
spontaneous tautomerization at pH 7 is negligible.
Figures 6 and 7 show that AAT also catalyzes an Asp/OA exchange.
Exchange between 50 µM [3H]Asp and 200 µM unlabeled
OA results in release of ~75% of the total label after 60 min.
However, if MDH is then added to convert labeled OA to MAL, only
two-thirds of the volatile label present before MDH is protected (Fig.
6), in contrast to the case with Glu, where GDH added after [3H]Glu/KG exchange protects nearly all tritium from
NaOH-induced loss. The 24% label loss after MDH treatment could be due
to spontaneous tautomerization of OA occurring more readily than for
KG. Insight into this possibility is provided in Fig. 7. In this case,
net conversion of [3H]Asp to OA by incubation of AAT with
250 µM unlabeled KG resulted in loss of 59% of the total label.
Spectroscopic data showed that 60% of the 50 µM Asp was converted to
OA if only AAT was present, in good agreement with the fraction of
label lost. When MDH is then added, it "pulls" the Asp
MAL
conversion 99% to completion. After MDH is added at 60 min, the
base-induced tritium loss is still 25%. The same loss (24%) is
observed if both AAT and MDH are present from the start of the
experiment, despite the fact that the OA pool is much smaller in this
case during the entire incubation interval. The data are consistent
with a substantial percentage of total label in C-2 of
[3H]Asp (unlike the case for [3H]Glu), with
no spontaneous tautomerization of either KG or OA.
It should be noted that AAT can also catalyze a direct exchange of C-3
tritium with solvent H+ under certain circumstances.
Nuclear magnetic resonance (NMR) and proton magnetic resonance (PMR)
studies show that C-3 deuterium/hydrogen exchange occurs when AAT is
incubated in 2H2O with 100-200 mM Glu and
0.1-2 mM KG (2, 6). The exchange probably results
from polarity of an imine intermediate formed by the enzyme that
influences 3H lability at the adjacent C-3. Rearrangement
of the initial imine to an enamine is thought to be facilitated by
transfer of a C-3 hydrogen atom (as H+) to a basic group in
close proximity on the enzyme. If the substrate proton extracted by the
enzyme exchanges with a solvent deuteron, reversal of the imine-enamine
transition allows deuterium incorporation to the pyridoxal-bound carbon
skeleton that can either be converted back to Glu or appear as
[2H]KG if the keto acid is released by the enzyme. With
Glu as substrate, a small pool of KG in the medium is mandatory for
allowing NMR detection of H/2H exchange, even though the
required enzyme-bound intermediates could be expected to originate
solely from Glu. KG probably prevents rapid dissociation of KG derived
from the Glu substrate, allowing the enzyme to sustain a significant
titer of bound [3H]KG in equilibrium with the
3H-enamine intermediate required for H/2H
exchange. Exchange of C-3 tritium due to equilibria among imine-enamine enzyme intermediates is especially pronounced for alanine
aminotransferase (2, 6).
It is important to recognize that the mechanism of
aminotransferase-catalyzed H/2H exchange with solvent
described above allows H+ (or tritium) release from C-3 of
amino acid substrates as a direct result of enzyme activity
(36). However, once tritium is released by this process,
it would not be restored to the substrate by reversal of the AAT
reaction or by action of another enzyme because of 3H
dilution in the massive H+ pool represented by aqueous
solvent. Our results clearly show that conversion of KG to Glu by GDH,
or of OA to MAL by MDH, prevents formation of volatile label that
otherwise is released by NaOH. The NaOH-induced label release must
originate from the keto acid pool, rather than directly from
3H/solvent exchange at C-3 via an enzyme-bound intermediate.
As might be expected, the magnitude of C-3 H exchange with solvent is
highly dependent on enzyme concentration. Substrate/solvent H/2H exchange observed by NMR occurs at enzyme
concentrations 40- to 80-fold higher (20-40 U/ml) than those used
here and with amino acid concentrations (100-200 mM) more than
100-fold higher (2, 6). The moderate enzyme and substrate
concentrations used in our experiments prevent C-3 tritium release
directly by the enzyme from making a significant contribution, relative
to the rate of tritium exchange between the Glu and KG pools. Tritium
release from an enzyme intermediate as the basis of
3H2O formation would be detected after stopping
the enzyme activity with AOA. The tritium release as
3H2O described here does not occur if the AAT
reaction is stopped with AOA; hence it is primarily (if not entirely) a
result of release from the KG pool by strong base. Together, the data
are consistent with an active isotope exchange between substrate and cognate keto acid, with little or no isotope release from enzyme-bound intermediate(s).
Glu/Gln shuttle.
The Glu/KG partial reaction of AAT described here raises significant
questions regarding some of the challenges raised against the Glu/Gln
shuttle hypothesis. Most of those challenges call attention to the fact
that astrocytes incubated with isotopically labeled Glu produce a wide
variety of labeled TCA cycle and related metabolic intermediates. The
rapidity of formation of these products has been interpreted as
indicative of substantial net Glu metabolism by astrocytes rather than
conversion to Gln and subsequent return of carbon in that form to
glutamatergic neurons (9, 14, 32, 33, 39, 40). One study
showed that astrocytes convert uniformly labeled-[13C]Glu
to Glu and Gln labeled at carbons 1, 2, and 3 (33). This labeling pattern can be explained if
part of the uniformly labeled KG product produced by AAT makes one turn
of the TCA cycle and is then converted back to Glu either by GDH or
AAT. The observation was interpreted as a partial restoration of Glu
that had been converted to KG on a net basis, after one circuit through
the cycle. Thus actual net Glu loss was less than that otherwise
calculated from the amount of 14CO2 produced
from [1-14C]Glu, which loses all its 14C
label at KG dehydrogenase. The original interpretation of the data
concluded that some net consumption of Glu occurs, even after correction for reconversion of KG to Glu.
Hassel and Brathe (12, 13) suggested that the apparent
need for a carbon cycle is debatable because neurons can incorporate label from 14CO2 into Glu. They proposed that
malic enzyme (ME) may allow conversion of pyruvate to Mal that is then
converted to Glu via TCA cycle activity without a requirement for
pyruvate carboxylase. However, enzymes that catalyze carboxylation
events typically require biotin as cofactor, which is important to
provide sufficient affinity for the CO2 substrate. ME is
not a biotin enzyme. Our own observations indicate that ME produces
pyruvate, not Mal. At physiological concentrations of Mal and pyruvate,
>95% of the Mal present is converted to pyruvate with concomitant
NADPH production, rather than formation of Mal and NADP. This is
consistent with the conventional view that ME functions to produce
NADPH to support reductive biosynthetic events.
AAT-catalyzed isotope exchange raises another explanation for the wide
distribution of label among metabolites that is observed after
incubating astrocytes with labeled Glu. The Glu/KG partial reaction can
transfer labeled carbon from Glu to KG. All TCA cycle intermediates
produced subsequently will be labeled, but this implies no net
consumption of Glu. Moreover, isotope exchange is a two-way street.
Once isotope is distributed among TCA cycle intermediates, formation of
[1,2,3-13C]Glu from the original
UL-[13C]Glu may imply nothing more than an ongoing
exchange cycle that transfers isotope from differentially labeled KG
back to Glu that now will gain the same labeling pattern as the KG
involved in the exchange process. The initial partial reaction can
allow rapid formation of labeled metabolic intermediates from labeled
Glu with no net Glu oxidation. The second exchange event allows
production of [1,2,3-13C]Glu from
UL-[13C]Glu but would not imply net Glu synthesis. To the
extent that any isotope exchange occurs, there is less net Glu
oxidation than would otherwise be concluded from the distribution of
isotope in metabolic intermediates. Labeling patterns alone do not
provide insight into the magnitude of net Glu consumption or production.
Furthermore, astrocytes may catalyze net synthesis of Glu from KG via
GDH activity under the same conditions in which simultaneous AAT-catalyzed isotope exchange allows formation of labeled metabolites. It is important to remember that equilibrium for the reaction catalyzed
by GDH lies greatly in favor of Glu formation. At pH 7.4, the
equilibrium ratio of Glu to KG established by GDH ranges from a few
hundred- to a few thousandfold, depending on the NH
concentration and the NADH-to-NAD ratio sustained in the mitochondria. It is important to note that glucose was present in all of those studies in which [13C]Glu was used to study isotope
distribution in metabolites. Astrocytes express pyruvate carboxylase
(31, 43), the anaplerotic enzyme necessary for providing
net synthesis of KG from glucose. These cells therefore have the
capacity for net Glu production, even while exchange events cause
isotope distribution in metabolites that would suggest net Glu
metabolism. When glucose is present, the pool size of TCA cycle
intermediates and other metabolites may convey more information
regarding glucose metabolism than that of Glu. Accurate interpretation
of metabolism from isotope distribution data is particularly complex
and difficult if AAT-catalyzed partial reactions occur concomitantly.
The rate of AAT in vivo is believed to be seven to eight times faster
than the rate-limiting step for TCA cycle activity. This pronounced
rate difference allows cellular pools of AAT reactants and products to
be near thermodynamic equilibrium. Therefore, it is tempting to assume
that the net AAT reaction allows isotope introduced to the cellular Glu
pool to achieve rapid isotopic equilibrium with the KG pool. However,
the steady-state KG pool is ~20-fold larger than that for KG. Net AAT
activity is limited by OA availability such that complete turnover of
the entire steady-state OA pool delivers only a small amount of label
from Glu to KG and then on to TCA cycle intermediates. In contrast,
isotopic equilibrium due to the AAT partial reaction can label the KG
pool ~20-fold faster. Indeed, label transfer by AAT from Glu to KG is
nearly undetectable at physiological concentrations of OA compared with KG at its physiological concentration. We conclude that the Glu/KG partial reaction of AAT may be a much greater determinant of isotope incorporation from Glu to the TCA metabolite pool than the net AAT
reaction with OA. Label incorporation to these metabolites must be
interpreted with caution when assessing quantitative aspects of Glu
metabolism. Otherwise, the role played by a Glu/Gln shuttle in the CNS
carbon cycle may be underestimated.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant RO1-DK-15365-27.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
G. A. Kimmich, Dept. of Biochemistry and Biophysics, School
of Medicine and Dentistry, Univ. of Rochester, Rochester, NY 14642 (E-mail: george_kimmich{at}urmc.rochester.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 30, 2002;10.1152/ajpcell.00487.2001
Received 10 October 2001; accepted in final form 28 January 2002.
 |
REFERENCES |
1.
Amara, SG,
and
Kuhar MJ.
Neurotransmitter transporters.
Annu Rev Neurosci
16:
73-93,
1993[ISI][Medline].
2.
Babu, UM,
and
Johnston RB.
Nuclear magnetic resonance studies of D2O-substrate exchange reactions catalyzed by glutamic pyruvic and glutamic oxaloacetic transaminases.
Biochemistry
15:
5671-5678,
1976[ISI][Medline].
3.
Balazs, R,
Machiyama Y,
Hammond BJ,
Julian T,
and
Richter D.
The operation of the gamma-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro.
Biochem J
116:
445-461,
1970[ISI][Medline].
4.
Bergmeyer, HU,
Klotzsch H,
Mollering H,
Nelbock-Hochstetter M,
and
Beaucamp K.
Methods of Enzymatic Analysis. New York: Academic, 1965, p. 967-1037.
5.
Choi, DW.
Glutamate neurotoxicity and diseases of the nervous system.
Neuron
1:
623-634,
1988[ISI][Medline].
6.
Cooper, JL.
Proton magnetic resonance studies of glutamate-alanine transaminase-catalyzed deuterium exchange.
J Biol Chem
251:
1088-1096,
1976[Abstract].
7.
Danbolt, NC.
Glutamate uptake.
Prog Neurobiol
65:
1-105,
2001[ISI][Medline].
8.
Dennis, SC,
and
Clark JB.
The pathway of glutamate metabolism in rat brain mitochondria.
Biochem J
168:
521-527,
1977[ISI][Medline].
9.
Farinelli, SE,
and
Niklas WJ.
Glutamate metabolism in rat brain cortical astrocyte cultures.
J Neurochem
58:
1905-1915,
1992[ISI][Medline].
10.
Fonnum, F.
The distribution of glutamate decarboxylase and aspartate transaminase in subcellular fractions of rat and guinea-pig brain.
Biochem J
106:
401-412,
1968[ISI][Medline].
11.
Frandsen, A,
and
Schousboe A.
Development of excitatory amino acid induced cytotoxicity in cultured neurons.
Int J Dev Neurosci
8:
209-216,
1990[ISI][Medline].
12.
Hassel, B,
and
Brathe A.
Cerebral metabolism of lactate in vivo: evidence for neuronal pyruvate carboxylation.
J Cereb Blood Flow Metab
20:
327-336,
2000[ISI][Medline].
13.
Hassel, B,
and
Brathe A.
Neuronal pyruvate carboxylation supports formation of transmitter glutamate.
J Neurosci
20:
1342-1347,
2000[Abstract/Free Full Text].
14.
Hassel, B,
Sonnewald U,
and
Fonnum F.
Glial-neuronal interactions as studied by cerebral metabolism of [2-13C]-acetate and [1-13C]-glucose: an ex vivo 13C NMR spectroscopic study.
J Neurochem
64:
2773-2782,
1995[ISI][Medline].
15.
Hogstad, S,
Svenneby G,
Torgner IA,
Kvamme E,
Hertz L,
and
Schousboe A.
Glutaminase in neurons and astrocytes cultured from mouse brain; kinetic properties and effects of phosphate, glutamate, and ammonia.
Neurochem Res
13:
383-388,
1988[ISI][Medline].
16.
John, RA,
and
Charteris A.
The reaction of amino-oxyacetate with pyridoxal phosphate-dependent enzymes.
Biochem J
171:
771-779,
1978[ISI][Medline].
17.
Kaufman, E,
and
Driscoll BF.
Carbon dioxide fixation in neuronal and astroglial cells in culture.
J Neurochem
58:
258-262,
1992[ISI][Medline].
18.
Lester, HA,
Mager S,
Quick MW,
and
Corey JL.
Permeation properties of neurotransmitter transporters.
Annu Rev Pharmacol Toxicol
34:
219-249,
1994[ISI][Medline].
19.
Magee, SC,
and
Phillips AT.
Molecular properties of the multiple aspartate aminotransferases purified from rat brain.
Biochemistry
10:
3397,
1971[ISI][Medline].
20.
Meldrum, B.
Amino acids as dietary excitotoxins
a contribution to understanding neurodegenerative disease.
Trends Pharmacol Sci
11:
379-387,
1990[ISI][Medline].
21.
Metzler, ED,
Ikawa M,
and
Snell EE.
A general mechanism for vitamin B6-catalyzed reactions.
J Am Chem Soc
76:
648-652,
1954[ISI].
22.
Nicholls, DG.
CNS Neuro-Transmitters and Neuromodulators: Glutamate. New York: CRC, 1995, p. 35-52.
23.
Olney, JW.
Excitotoxic amino acids and neuropsychiatric disorders.
Annu Rev Pharmacol Toxicol
30:
47-71,
1990[ISI][Medline].
24.
Oshima, T,
and
Tamiya N.
Mechanism of transaminase action.
Biochem J
78:
116-119,
1961[ISI].
25.
Patel, AJ,
Weir MD,
Hunt A,
Tahourdin CSM,
and
Thomas DGT
Distribution of glutamine synthetase and glial fibrillary acidic protein and correlation of glutamine synthetase with glutamate decarboxylase in different regions of the rat cerebral nervous system.
Brain Res
331:
1-9,
1985[ISI][Medline].
26.
Rothman, JM,
and
Olney JW.
Glutamate and the pathology of hypoxic/ischemic brain damage.
Ann Neurol
19:
105-111,
1986[ISI][Medline].
27.
Rothman, S.
Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death.
J Neurosci
4:
1884-1891,
1984[Abstract].
28.
Rothstein, JD,
Hoberg MDH,
Pardo CA,
Bristol LA,
Jin L,
Kuncl RW,
Kanai Y,
Hediger MA,
Wang Y,
Schielke JP,
and
Welty DF.
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate.
Neuron
16:
675-686,
1996[ISI][Medline].
29.
Rothstein, JD,
Martin L,
Levey AI,
Dykes-Hobert M,
Jin L,
Wu D,
Nash N,
and
Kuncl RW.
Localization of neuronal and glial glutamate transporters.
Neuron
13:
713-725,
1994[ISI][Medline].
30.
Schousboe, A,
Westergaard N,
Waagepetersen HS,
Larsson OM,
Bakken IJ,
and
Sonnewald U.
Trafficking between glia and neurons of TCA cycle intermediates and related metabolites.
Glia
21:
99-105,
1997[ISI][Medline].
31.
Shank, RP,
Bennett GS,
Freytag SO,
and
Campbell GLM
Pyruvate carboxylase: an astrocyte specific enzyme implicated in the replenishment of amino acid neurotransmitter pools.
Brain Res
329:
364-367,
1985[ISI][Medline].
32.
Sonnewald, U,
Westergaard N,
Petersen SB,
Unsgard G,
and
Schousboe A.
Metabolism of [U-13C]-glutamate in astrocytes studied by 13C NMR spectroscopy: incorporation of more label into lactate than into glutamine demonstrates the importance of the tricarboxylic acid cycle.
J Neurochem
61:
1179-1182,
1993[ISI][Medline].
33.
Sonnewald, U,
Westergaard N,
and
Schousboe A.
Glutamate transport and metabolism in astrocytes.
Glia
21:
56-63,
1997[ISI][Medline].
34.
Tanaka, K,
Watase K,
Manabe T,
Yamada K,
Watanabe M,
Takahashi K,
Iwama H,
Nishikawa T,
Ichihara N,
Hori S,
Takimoto M,
and
Wada K.
Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1.
Science
276:
1699-1702,
1997[Abstract/Free Full Text].
35.
Van den Berg, CJ,
and
Garfinkel D.
A simulation study of brain compartments: metabolism of glutamate and related substances in mouse brain.
Biochem J
123:
211-218,
1971[ISI][Medline].
36.
Walter, U,
Luthe H,
Gerhart F,
and
Soling HD.
Hydrogen exchange at the carbon of amino acids during transamination.
Eur J Biochem
39:
395-403,
1975.
37.
Waniewski, RA,
and
Martin DL.
Exogenous glutamate is metabolized to glutamine and exported by rat primary astrocyte cultures.
J Neurochem
47:
304-313,
1986[ISI][Medline].
38.
Watase, K,
Hashimoto K,
Kano M,
Yamada K,
Watanabe M,
Inoue Y,
Okuyama S,
Sakagawa T,
Ogawa S,
Kawashima N,
Hori S,
Takimoto M,
Wada K,
and
Tanaka K.
Motor discoordination and increased susceptibility to cerebellar injury in GLAST mutant mice.
Eur J Neurosci
10:
976-988,
1998[ISI][Medline].
39.
Westergaard, N,
Sonnewald U,
and
Schousboe A.
Release of
-ketoglutarate, malate and succinate from cultured astrocytes: possible role in amino acid neurotransmitter homeostasis.
Neurosci Lett
176:
105-109,
1994[ISI][Medline].
40.
Westergaard, N,
Sonnewald U,
and
Schousboe A.
Metabolic trafficking between neurons and astrocytes: the glutamate glutamine cycle revisited.
Dev Neurosci
17:
203-211,
1995[ISI][Medline].
41.
Whetsell, WO,
and
Shapira NA.
Biology of disease
neuroexcitation, excitotoxicity and human neurological disease.
Lab Invest
68:
372-387,
1993[ISI][Medline].
42.
Yu, AC,
Schousboe A,
and
Hertz L.
Metabolic fate of 14C-labeled glutamate in astrocytes in primary cultures.
J Neurochem
39:
954-960,
1982[ISI][Medline].
43.
Yu, ACH,
Drejer J,
Hertz L,
and
Schousboe A.
Pyruvate carboxylase activity in primary cultures of astrocytes and neurons.
J Neurochem
41:
1484-1487,
1983[ISI][Medline].
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