From the Mental Health Research Institute,
Departments of ¶ Pharmacology and
Psychiatry,
University of Michigan Medical School, Ann
Arbor, Michigan 48109-0669
Received for publication, November 14, 2002
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ABSTRACT |
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Glucose is the major source of brain energy and
is essential for maintaining normal brain and neuronal function.
Hypoglycemia causes impaired synaptic transmission. This occurs even
before significant reduction in global cellular ATP concentration, and relationships among glycolysis, ATP supply, and synaptic transmission are not well understood. We demonstrate that the glycolytic enzymes glyceraldehyde phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (3-PGK) are enriched in synaptic vesicles, forming a functional complex, and that synaptic vesicles are capable of accumulating the
excitatory neurotransmitter glutamate by harnessing ATP produced by
vesicle-bound GAPDH/3-PGK at the expense of their substrates. The GAPDH
inhibitor iodoacetate suppressed GAPDH/3-PGK-dependent, but
not exogenous ATP-dependent,
[3H]glutamate uptake into isolated synaptic
vesicles. It also decreased vesicular [3H]glutamate
content in the nerve ending preparation synaptosome; this decrease was
reflected in reduction of depolarization-induced [3H]glutamate release. In contrast, oligomycin, a
mitochondrial ATP synthase inhibitor, had minimal effect on any of
these parameters. ADP at concentrations above 0.1 mM
inhibited vesicular glutamate and dissipated membrane potential. This
suggests that the coupled GAPDH/3-PGK system, which converts ADP to
ATP, ensures maximal glutamate accumulation into presynaptic vesicles.
Together, these observations provide insight into the essential nature
of glycolysis in sustaining normal synaptic transmission.
Glycolysis plays a vital role in maintaining normal brain
function. Glucose is known to serve as the major substrate for cerebral energy under normal conditions (1). Recent evidence suggests a direct
correlation between glucose utilization and cognitive function (2).
Reduction of glucose levels results in pathophysiological states and
abnormal electrophysiological activity; however, this occurs long
before significant alteration in tissue ATP levels is detected (3-7).
Substitution of pyruvate for glucose does not support normal evoked
neuronal activity, although tissue ATP level returns to normal (8-10).
Abnormal synaptic transmission caused by hypoglycemia occurs in part if
not entirely by a presynaptic mechanism (7, 11, 12). Fleck et
al. (7) have shown that substantial reduction of extracellular
glucose results in a decrease in stimulus-evoked Glu release,
with no changes in ATP levels. These studies together suggest that
glycolysis or glycolytic intermediate(s) are necessary for normal
synaptic transmission independent of global cellular ATP levels.
In an attempt to reveal the underlying mechanism of
hypoglycemia-induced aberrant synaptic transmission, we previously
explored the possibility that glycolytic intermediates could modify
proteins localized in the nerve ending (13, 14). 3-Phosphoglycerate (3-PG)1 was demonstrated to
stimulate phosphorylation of 155- and 72-kDa proteins. The latter was
identified as glucose-1,6-bisphosphate synthetase, and
1,3-bisphosphoglycerate (1,3-BPG) was found to serve as the direct
substrate for phosphorylation of this enzyme, by donating 1-phosphate.
Both of these phosphorylated proteins are enriched in the synaptosomal
(nerve ending preparation) as well as cell body soluble fractions, but
the significance of these modifications in synaptic transmission
remains unclear.
In this paper, we show that the glycolytic intermediate 1,3-BPG forms
an acyl-enzyme intermediate with vesicle-bound glyceraldehyde phosphate
dehydrogenase (GAPDH), that vesicle-bound GAPDH exists in a complex
with 3-phosphoglycerate kinase (3-PGK), and that activation of
vesicle-associated GAPDH and 3-PGK is sufficient to support vesicular
uptake of Glu. Glutamate is now recognized as the major excitatory
neurotransmitter responsible for triggering neuronal firing, in the
vertebrate central nervous system (15-23). As such, proper Glu
synaptic transmission is not only essential for basic neuronal
communication but also is involved in learning and memory formation
(19, 20). Glutamate accumulation into synaptic vesicles in the nerve
terminal is an initial crucial step in Glu transmission (18, 22-26).
This process requires ATP to generate an electrochemical gradient,
which is the driving force for Glu uptake into synaptic vesicles
(27-34).
We present evidence that glycolytically produced ATP, in
particular that produced by GAPDH and 3-PGK, but not
mitochondria-derived ATP, is harnessed for accumulation of Glu into
synaptic vesicles in synaptosomes; Glu transported into synaptic
vesicles in this manner is released upon depolarization. These findings
could provide an explanation for hypoglycemia-induced aberrant synaptic
transmission and insight into the essential nature of glycolysis
in normal synaptic transmission.
Materials--
[ Preparation of Subcellular Fractions--
Synaptic vesicles were
prepared from bovine cerebrum through the discontinuous sucrose
gradient procedure as described previously (35). The subcellular
fractions of bovine cerebrum were prepared as described previously
(36). Synaptosomes were prepared from cerebra of male Sprague-Dawley
rats (150-200 g) and purified through the Percoll gradient
centrifugation step, as described by Dunkley et al. (37).
Protein concentration was determined by the method of Bradford (38)
with a Coomassie protein assay reagent kit (Pierce) with bovine serum
albumin as standard protein.
Synthesis of
[3-32P]1,3-BPG--
[32P]Dihydroxyacetone
phosphate (DHAP) was prepared by phosphorylation of dihydroxyacetone by
glycerol kinase with [
Radioactive compounds were analyzed by high pressure liquid
chromatography on a Whatman Partisil 10 SAX WCS column (4.6 × 250 mm), comparing their retention times with those of nonradioactive authentic standards monitored at 214 nm. The column was equilibrated with 0.4 M sodium phosphate buffer (pH 3.2), and glycolytic
intermediates and nucleotides were eluted isocratically, as described
previously (14). Retention times for glycolytic intermediates were 4.1 min for DHAP and GAP, 5.3 min for Pi, 5.9 min for
2-phosphoglycerate and 3-PG, 6.0 min for ADP, 7.4 min for
phosphoenolpyruvate, 15.4 min for 1,3-BPG, and 30.0 min for ATP.
Protein Labeling with [3-32P]1,3-BPG--
The
synaptic vesicle fraction (30 µg of protein) was preincubated at
37 °C for 30 s in 27 µl of 5 mM Tris-maleate (pH
7.4). The reaction was initiated by the addition of 3 µl of
[3-32P]1,3-BPG (240 Ci/mmol) to a final concentration of
140 nM and allowed to continue for 10 s. For
electrophoretic protein separation under neutral pH conditions, the
reaction was terminated by the addition of 10 µl of buffer containing
4% SDS, 30% sucrose, 40 mM Tris-HCl (pH 7.0), 4 mM EDTA, 160 mM 2-mercaptoethanol, and 50 µg/ml bromphenol blue; aliquots (25 µl) were subjected to
polyacrylamide gel (1% SDS and 5.6% acrylamide) without stacking gel
according to the method of Fairbanks et al. (39). For
electrophoretic protein separation under alkaline pH conditions
(standard SDS-PAGE), the reaction was terminated by the addition of 10 µl of SDS sample buffer containing 4% SDS, 40% glycerol, 0.2 M Tris-HCl (pH 6.8), 20% 2-mercaptoethanol, and 50 µg/ml
bromphenol blue; aliquots (25 µl) were subjected to polyacrylamide
gel (0.1% SDS and 12% acrylamide) according to the method of Laemmli
(40), except for omission of sample boiling. Autoradiography was
carried out as described previously (41) and analyzed using an image
analyzer (Bio-Rad Gel Doc 2000).
Immunoprecipitation--
Antibodies (10 µg) were absorbed onto
immobilized protein G (0.1 ml as 50% slurry) and chemically
cross-linked, using the Seize X mammalian immunoprecipitation kit
(Pierce). The synaptic vesicle fraction (50 µg of protein) was
stirred in 0.2 ml of buffer containing 0.32 M sucrose, 4 mM Tris-maleate (pH 7.4), and 0.2 M NaCl for 5 min at 4 °C and centrifuged at 200,000 gmax
for 1 h at 4 °C. When 32P-labeled protein was
detected, the synaptic vesicle fraction was incubated at 37 °C for
10 s with the same buffer containing 140 nM (240 Ci/mmol) [3-32P]1,3-BPG. The supernatant (180 µl) was
subjected to immunoprecipitation with immobilized antibody, according
to the manufacturer's protocol. Aliquots (20 µl) were subjected to
SDS-PAGE, except for omission of sample boiling, according to Fairbanks
et al. (39) or Laemmli (40), followed by Western blot
analysis or autoradiography, as appropriate.
Western Blot Analysis--
For analysis of subcellular
fractions, 30 µg of protein were subjected to standard SDS-PAGE (40).
In NaCl solubilization experiments, the synaptic vesicle fraction (50 µg of protein) was stirred in 0.2 ml of buffer containing 0.32 M sucrose, 4 mM Tris-maleate (pH 7.4), and
various concentrations of NaCl for 5 min at 4 °C and then
centrifuged at 200,000 × gmax for 1 h. The pellet was dissolved in 30 µl of SDS sample buffer. The protein in the supernatant (180 µl) was precipitated with 15%
trichloroacetic acid and dissolved in 30 µl of SDS sample buffer.
Aliquots (25 µl) of the 200,000 × gmax pellet and supernatant fractions
were subjected to standard SDS-PAGE.
For analysis of GAPDH and 3-PGK binding to synaptic vesicles, synaptic
vesicles (1 mg of protein) were washed twice by 20 ml of buffer
containing 0.32 M sucrose, 4 mM Tris-maleate
(pH 7.4), and 0.8 M NaCl; bound NaCl was then removed by
washing twice with 20 ml of the same buffer without NaCl. The washed
synaptic vesicles (40 µg of protein) were incubated at 37 °C for
10 min in 0.1 ml of buffer in the absence or presence of 2 µg of
purified GAPDH or 3-PGK. The synaptic vesicles were pelleted by
centrifugation at 200,000 × gmax for 1 h at 4 °C and
washed twice with the buffer. The pellet was suspended in 80 µl of
SDS sample buffer, and an aliquot (25 µl) was subjected to standard
SDS-PAGE.
Proteins were electrotransferred onto the Immobilon-P polyvinylidene
difluoride membrane (Millipore Corp.) using a semidry transfer
apparatus (Bio-Rad Trans-Blot SD). The membrane was treated with 5%
nonfat dry milk in a solution containing 50 mM Tris-HCl (pH
7.4), 0.5 M NaCl, and 0.1% Tween 20 (TBS-T) for 1 h
and then incubated for 2 h at room temperature with anti-GAPDH
monoclonal antibody at a 1:50 dilution or anti-3-PGK polyclonal
antibodies at 1:500 dilution, followed by incubation with alkaline
phosphatase-conjugated goat anti-mouse IgG or anti-rabbit IgG,
respectively, at room temperature for 1.5 h. Unbound antibodies
were washed out with TBS-T. 5-Bromo-4-chloro-3-indolyl phosphate and
nitro blue tetrazolium (Bio-Rad) were used as substrates for color
development and analyzed using an image analyzer (Bio-Rad Gel Doc 2000).
Glu Uptake into Synaptic Vesicles--
Vesicular glutamate
uptake was measured by the filtration-based assay using Whatman GF/C
filters, as described previously (27, 28), with minor modifications. In
the standard assay, aliquots (10 µg of protein) of bovine synaptic
vesicles were incubated at 30 °C for 10 min with 100 µM [3H]Glu (a specific activity of 7.4 GBq/mmol was obtained by the addition of unlabeled Glu to
[3H]Glu) in 0.1 ml of an incubation medium (pH 7.4)
containing 20 mM Hepes-KOH, 0.25 M sucrose, 4 mM MgSO4, 4 mM KCl, and 2 mM L-aspartic acid in the absence or presence
of 2 mM ATP (pH adjusted to 7.4 by the addition of
Tris-base). Prior to incubation, preincubation without ATP and
[3H]Glu was carried out for 1 min at 30 °C. When
inhibitor effect was tested, test agents were added at the start of the
preincubation period. In some experiments, ATP was replaced with a
mixture of 2 mM GAP, 2 mM Pi, 2 mM NAD, and 0.1 mM ADP (pH adjusted to 7.4 by
the addition of Tris-base) as a source of a Glu uptake activator.
Glu Content in Synaptic Vesicles within
Synaptosomes--
[3H]Glu content in synaptic vesicles
within the synaptosome was assayed as described previously (42, 43)
with minor modifications. Synaptosomes (100 µg of protein) were
suspended in 0.1 ml of oxygenated (95% O2, 5%
CO2) Krebs-Ringer buffer containing 150 mM
NaCl, 2.4 mM KCl, 1.2 mM
Na2HPO4, 1.2 mM CaCl2,
1.2 mM MgSO4, 5 mM Hepes-Tris (pH
7.4), and 10 mM glucose and preincubated at 37 °C for 10 min in the absence or presence of iodoacetate, oligomycin (Calbiochem), and pyruvate at indicated concentrations. After 3 µCi of
[3H]Glu (42.0 Ci/mmol) were added to the medium,
synaptosomes were incubated for an additional 10 min. Aliquots (10 µl) were removed and filtered on Whatman GF/C filters to determine
the total amount of [3H]Glu taken up by the synaptosomes,
and the rest were immediately frozen on dry ice. For vesicular
[3H]Glu content determination, the frozen synaptosomes
(90 µl) were thawed by adding 1.5 ml of ice-cold hypotonic solution
containing 6 mM Tris-maleate (pH 8.1) and 2 mM
aspartate and incubated for 20 min at 0 °C. Aliquots (1 ml) were
filtered on Whatman GF/C filters, and radioactivity retained on filters
was determined in a Beckman LS 6500 scintillation spectrophotometer.
Glu Release from Synaptosomes--
Glutamate release from
synaptosomes was assayed using the superfusion technique described
previously (43), with minor modifications. Synaptosomes (200 µg of
protein) were suspended in 1.5 ml of oxygenated (95%
O2/5% CO2) artificial cerebrospinal fluid
(ACSF) containing 124 mM NaCl, 5 mM KCl, 2 mM MgSO4, 1.25 mM
NaH2PO4, 22 mM NaHCO3, and 10 mM D-glucose and preincubated with or
without 300 µM iodoacetate or 2 µM
oligomycin at 37 °C for 10 min. After 3.5 µCi of
[3H]Glu (42.0 Ci/mmol) were added to the medium,
synaptosomes were incubated for an additional 10 min. An aliquot (1.0 ml) of [3H]Glu-loaded synaptosomal suspension was layered
onto a cellulose-acetate membrane filter (pore size 0.45 µm) placed
in a superfusion chamber. The synaptosomes were superfused (0.5 ml/min)
with ACSF for 60 min before application of 50 µM
4-aminopyridine (4-AP) plus 2 mM CaCl2 to
trigger depolarization of the synaptosomal membrane. In some control
experiments, synaptosomes were superfused with ACSF containing 300 µM iodoacetate or 2 µM oligomycin for the first 20 min of the superfusion period. This period was the same as the
sum of the synaptosomal preincubation and [3H]Glu-loading
periods. These procedures were all carried out at 37 °C. Fractions
were collected every 10 s for 3 min, from 30 s prior to 4-AP
application. The amount of [3H]Glu released into each
fraction was expressed as a percentage of the total
[3H]Glu taken up into synaptosomes at the end of 10 min
of [3H]Glu loading. The total [3H]Glu
loaded into synaptosomes was calculated from the amount of
[3H]Glu in 0.1 ml of loaded synaptosomes, determined by
the same filtration method used for the vesicular Glu uptake assay.
Other Biochemical Analyses--
GAPDH activity was measured
using 20 µg of protein of synaptic vesicles in a reaction mixture (1 ml) containing 0.1 M Tris-HCl (pH 8.5), 1.7 mM
sodium arsenate, 20 mM sodium fluoride, 1 mM NAD, 1 mM GAP, and 5 mM
KH2PO4 (44). Activity was calculated from the
rate of increase in NADH formation by monitoring absorbance at 340 nm
at 25 °C.
ATP levels in the synaptic vesicle and synaptosome suspensions were
determined under the same conditions described for vesicular Glu uptake
and vesicular Glu content assays, respectively, by the
luciferin/luciferase method, using an ATP bioluminescent assay kit
(Sigma-Aldrich), according to the manufacturer's protocol.
Generation of the membrane potential across the synaptic vesicle
membrane was monitored by ATP-induced fluorescence quenching of the
membrane potential-sensitive dye oxonol V (Molecular Probes, Inc.,
Eugene, OR) using a Fluorolog III fluorospectrophotometer (Horiba Jobin
Yvon Co., Ltd., Tokyo, Japan) as described previously (32, 43).
Vesicular ATPase activity was assayed by determining free inorganic
phosphate liberated upon incubation of synaptic vesicles (30 µg of
protein) with ATP, according to the method of Lanzetta et
al. (45), with minor modifications as described previously
(43).
A 37-kDa Protein Enriched in Synaptic Vesicles Is Labeled with
[3-32P]1,3-BPG and Identified as GAPDH--
Purified
synaptic vesicles were allowed to react with
[3-32P]1,3-BPG for 10 s and subjected to SDS-PAGE at
either neutral (Fig. 1A) or
alkaline (Fig. 1B) pH (39, 40). Electrophoresis at neutral
pH revealed incorporation of a radioactive moiety of
[3-32P]1,3-BPG into vesicular proteins of
Mr = 37,000 and 29,000. In contrast,
electrophoresis at alkaline pH revealed only the 29-kDa protein, which
appears to retain more of the 32P label than when
electrophoresis was conducted at neutral pH. These results indicate
that both the 29- and 37-kDa vesicular proteins can incorporate a
32P-containing moiety; however, the stability of the
labeled proteins differs depending on pH. Incorporation of the
radioactive moiety into the 37-kDa protein was stimulated by
Mg2+ but not affected by 3-PG. Labeling of the 29-kDa
protein was inhibited by Mg2+ and completely blocked by
3-PG. The linkage of the 32P-containing moiety to the
37-kDa protein was labile in the presence of 0.2 M
hydroxylamine (data not shown). These observations suggested that the
37- and 29-kDa proteins might be GAPDH and PGM, respectively. This was confirmed by immunoprecipitation with antibodies directed against these proteins (Fig. 1C). Thus, labeling of the
37-kDa GAPDH with [3-32P]1,3-BPG probably occurs by
formation of a thioester bond between a cysteine residue and the
3-phosphoglyceroyl moiety of 1,3-BPG; thioester bonds are known to be
labile at alkaline pH. Labeling of the 29-kDa PGM probably represents
phosphorylation of a histidine residue that is labile at either neutral
or acidic pH.
In order to determine the subcellular distribution of GAPDH and PGM, we
examined the amount of [3-32P]1,3-BPG radioactivity
incorporated into these two proteins present in synaptosomal cytosol,
perikaryal cytosol, microsome, synaptic vesicle, and plasma membrane
fractions (Fig. 1D). For each subcellular fraction, an
equivalent amount of protein was incubated with
[3-32P]1,3-BPG. Labeled GAPDH was found in all
subcellular fractions but was enriched in the synaptic vesicle
fraction. In contrast, PGM was most enriched in the synaptosomal
cytosol. Subcellular fractions were also subjected to Western blotting
with anti-GAPDH antibody. GAPDH was found in the greatest concentration
in the synaptic vesicle fraction (Fig. 1E), in accord with
the results obtained with the labeling method. Analysis of subcellular
fractions indicates that GAPDH not only occurs in the cytosol but also
can bind to various types of membranes. To determine the nature of the
interaction of GAPDH with synaptic vesicles, synaptic vesicles were
treated with various concentrations of NaCl (Fig. 1F). GAPDH was dissociated from synaptic vesicles with increasing salt
concentrations. At 0.8 M NaCl, only about 10% of GAPDH was
found to remain associated with synaptic vesicle membranes. At
physiologic ionic strength, GAPDH is associated with synaptic vesicle
membranes, although it does also occur in the cytosol fractions.
3-PGK Is Associated with Synaptic Vesicles via Interaction with
GAPDH--
In the glycolytic pathway, GAPDH is known to exist in a
complex with 3-PGK (46). The activities of GAPDH and 3-PGK are
energetically coupled, utilizing GAP, NAD, Pi, and ADP, to
yield 3-PG, ATP, NADH, and H+ (46, 47). Because GAPDH is
enriched in synaptic vesicles, we considered the possibility that 3-PGK
may also preferentially localize to synaptic vesicle membranes. Western
blots were conducted on subcellular fractions using anti-3-PGK antibody
(Fig. 2A). Like GAPDH, 3-PGK
was found enriched in the synaptic vesicle fraction. Immunoreactive
3-PGK was also dissociated from synaptic vesicles by increasing
concentrations of NaCl in a manner similar to that observed for GAPDH
(Fig. 2B). To determine whether GAPDH and 3-PGK exist in a
complex on synaptic vesicles, we performed experiments to determine
whether GAPDH and 3-PGK could be co-immunoprecipitated. Synaptic
vesicle salt extracts were incubated with anti-3-PGK antibody, and the
resulting immunoprecipitates were subjected to Western blotting with
anti-GAPDH antibody (Fig. 2C). Immunoreactive GAPDH was
detected in the anti-3-PGK antibody immunoprecipitates, indicating that
GAPDH and 3-PGK must exist in a tight complex. A second
co-immunoprecipitation experiment was carried out, which showed that
3-PGK can be co-precipitated with anti-GAPDH antibody (Fig.
2D). In order to determine whether GAPDH and 3-PGK directly bind to the synaptic vesicles, synaptic vesicles were first washed with
0.8 M NaCl and then incubated with purified GAPDH and/or 3-PGK. The vesicles were pelleted by centrifugation, and the amount of
vesicle-bound GAPDH or 3-PGK was determined by immunoblotting (Fig.
2E). GAPDH was found capable of binding to the synaptic vesicle with or without the addition of 3-PGK. In contrast, binding of
3-PGK to synaptic vesicles was greatly increased when GAPDH was
included with 3-PGK. These results suggest that GAPDH directly binds to
synaptic vesicles and that 3-PGK binds to vesicle-bound GAPDH.
Synaptic Vesicles Are Capable of Accumulating Glu via Activation of
Endogenous GAPDH/3-PGK--
Synaptic vesicles bearing both
GAPDH and 3-PGK should be able to synthesize ATP when GAPDH substrates
and ADP are supplied. GAPDH and 3-PGK activity could regenerate ATP for
vesicular uptake of Glu. Synaptic vesicles were incubated for various
periods with [3H]Glu in the presence or absence of 2 mM GAP, NAD, and Pi and 0.1 mM ADP.
Vesicular Glu uptake was dependent on the presence of the GAPDH/3-PGK
substrate mixture throughout the incubation period(s)
tested and was approximately linear up to 6 min under these conditions
(Fig. 3A). When any one of the
components was omitted from the incubation mixture, vesicular Glu
uptake was not supported (Fig. 3B). [3H]Glu
uptake resulting from ATP generated by vesicular GAPDH and 3-PGK was
comparable with that observed with 2 mM ATP and not increased by the addition of exogenous 3-PGK. ADP supported uptake of
[3H]Glu in a concentration-dependent manner
but was inhibitory at concentrations above 0.1 mM (Fig.
3C). These results indicate that vesicular Glu uptake can
occur by harnessing the activity of vesicle-bound GAPDH and 3-PGK.
ATP Synthesized by Vesicle-bound GAPDH/3-PGK Is More
Effective than Exogenous ATP in Glu Uptake--
Experiments shown in
Fig. 3 suggested that GAP-mediated vesicular Glu uptake probably occurs
as a result of ATP synthesis. To directly demonstrate this, synaptic
vesicles were incubated with various concentrations of GAP in the
presence of 2 mM NAD, Pi, and 0.1 mM ADP, and the amount of ATP produced, as well as Glu
uptake, was determined (Fig. 4,
A and B). ATP was synthesized in a
GAP-concentration-dependent manner in parallel to vesicular Glu uptake. Under these conditions, 12 µM ATP were
sufficient to give rise to maximal vesicular Glu uptake. This
concentration is substantially lower than the apparent
Km value for ATP (0.8-1.2 mM),
determined using exogenous ATP under standard assay conditions for
vesicular Glu uptake (28) (see Fig. 9B). These observations
raised the possibility that ATP produced by vesicle-bound GAPDH/3-PGK
might be more effective than exogenous ATP in supporting vesicular Glu
uptake. To further explore this possibility, exogenous
ATP-dependent vesicular Glu uptake was compared with
GAP-supported Glu uptake. Synaptic vesicles were incubated with various
concentrations of exogenous ATP, and the amount of ATP, as well as Glu
uptake, after 10 min of incubation was determined (Fig. 4, C
and D). At least 80-100 µM exogenous ATP were
needed to support the same maximal Glu uptake observed for
GAP-supported uptake. This ATP concentration is much higher than that
observed with the endogenous ATP-generating system. In Fig.
4E, the amount of Glu taken up into synaptic vesicles was
plotted as a function of the ATP concentration determined after a
10-min incubation. The amount of Glu accumulated per ATP level after 10 min of incubation is greater when GAPDH/3-PGK substrates are utilized.
These results suggest that the ATP generated by vesicle-bound
GAPDH/3-PGK is more effective than exogenous ATP in supporting
vesicular Glu uptake.
GAP-dependent Vesicular Glu Uptake Is Inhibited by
Iodoacetate--
In an effort to provide further evidence that
GAP-dependent Glu uptake involves GAPDH, we have determined
the effect of the GAPDH-selective inhibitor iodoacetate on vesicular
Glu uptake in the presence of GAP, NAD, Pi, and ADP. As
shown in Fig. 5A, iodoacetate
inhibited vesicular GAP-dependent Glu uptake in a concentration-dependent manner. When ATP was substituted
for the GAPDH/3-PGK substrates, iodoacetate exhibited little inhibition in the concentration range tested. Iodoacetate also inhibited GAPDH
activity and impaired the ability of synaptic vesicles to regenerate
ATP from ADP in a concentration-dependent manner. These results indicate that iodoacetate prevents, via inhibition of GAPDH,
ATP generation necessary for vesicular Glu uptake.
Characterization of the GAPDH/3-PGK
Activation-dependent Vesicular Glu Uptake
System--
The ATP-dependent vesicular Glu uptake system
is known to be markedly stimulated by low millimolar chloride (28, 34,
48-50) and harnesses an electrochemical proton gradient as the driving force (28, 29-34). In contrast to
Na+-dependent plasma membrane Glu uptake,
vesicular Glu uptake is insensitive to aspartate (28, 31, 34, 48, 50)
but potently inhibited by the membrane-permeant polyhalogenated
fluorescein Rose Bengal (43). t-ACPD also inhibits vesicular
Glu uptake by serving as a substrate for the vesicular Glu transporter
(51).
We have studied the characteristics of GAPDH/3-PGK activation-induced
vesicular Glu uptake with respect to the properties of the
ATP-dependent vesicular Glu uptake system. When
vesicle-bound GAPDH and 3-PGK were activated in the presence of their
substrates and various concentrations of chloride, maximal Glu uptake
occurred at 4 mM chloride; at higher chloride
concentration, Glu uptake was attenuated (Fig.
6A). This dependence on
chloride is indistinguishable from that observed for exogenous
ATP-induced vesicular Glu uptake (Fig. 6B). Moreover,
GAPDH/3-PGK activation-induced vesicular Glu uptake was insensitive to
Asp but affected by t-ACPD and Rose Bengal as well as by the
proton ionophore FCCP, in a manner similar to that observed with
exogenous ATP-dependent Glu uptake (Fig. 6, C
and D). These results indicate that the Glu transporter
responsible for GAPDH/3-PGK activation-induced vesicular Glu uptake is
the same as that responsible for ATP-dependent vesicular
Glu uptake.
GAPDH Substrates Enhance Vesicular Glu Uptake in the Presence of a
Low Concentration of ATP--
Since ATP generated by vesicle-bound
GAPDH/3-PGK appeared more effective than exogenous ATP in supporting
vesicular Glu uptake, it was of interest to see whether GAPDH
substrates are effective in augmenting vesicular Glu uptake in the
presence of limiting concentrations of ATP. Fig.
7 shows the effect of GAPDH substrates on
vesicular Glu uptake in the presence of ATP. The addition of a mixture
of GAP, NAD, and Pi, which alone exhibited no Glu uptake, elevated Glu uptake in the presence of a low concentration of exogenous
ATP (Fig. 7A). This suggests that ADP generated by ATPase is
recycled to ATP by 3-PGK, when vesicle-bound GAPDH is activated, and
that the regenerated ATP is utilized to give rise to additional Glu
uptake. At a much higher ATP concentration, this augmentation was much
smaller, however (Fig. 7B). This could be partly attributed to saturation of proton pump ATPase with ATP. These results are compatible with the notion that GAPDH/3-PGK-mediated vesicular Glu
uptake occurs through the ATP-dependent vesicular Glu
uptake system.
ADP Inhibits ATP-dependent Vesicular Glu
Uptake--
In order to understand the ADP inhibition observed earlier
(Fig. 3C), we have studied the effect of ADP on vesicular
Glu uptake supported by exogenous ATP. It was found that exogenous
ATP-dependent vesicular Glu uptake was also inhibited by
ADP, but not by AMP or adenosine, at concentrations higher than 0.1 mM (Fig. 8A). ADP
had no effect on ATPase activity at the concentrations tested up to 10 mM (Fig. 8B). Kinetic experiments indicate that
this inhibition is noncompetitive with respect to Glu or ATP (Fig. 9, A and B). Thus,
neither Km for Glu nor Km for ATP
was altered significantly; Km values for Glu in the
presence of 0, 0.5, and 2.0 mM ADP were 1.6, 1.6, and 1.5 mM, respectively, and those for ATP in the presence of 0, 0.5, and 2.0 mM ADP were 0.82, 0.76, and 0.79 mM, respectively. The noncompetitive inhibition with
respect to ATP was rather surprising but was consistent with ADP's
inability to inhibit ATPase activity (Fig. 8B).
The effect of ADP on membrane potential was examined in an effort to
gain further insight into the mechanism of ADP inhibition. Fig.
10A shows that ADP is
capable of dissipating preformed membrane potential in a
concentration-dependent manner. Disruption of membrane potential by 10 mM ADP was 47% (Fig. 10B). ADP
did not completely dissipate membrane potential at the concentrations
tested. The ability of ADP to reduce membrane potential would not
entirely account for its inhibitory effect on Glu uptake.
ATP Produced by Glycolysis Is Predominantly Utilized for Vesicular
Accumulation of Glu in the Nerve Ending--
The data indicating that
ATP produced by vesicular GAPDH and 3-PGK can support vesicular uptake
of Glu prompted us to postulate that Glu accumulation into synaptic
vesicles in the nerve terminal may be supported by glycolytically
produced ATP. In order to test this hypothesis, we compared the effects
of the GAPDH inhibitor iodoacetate and the mitochondrial ATP synthesis
inhibitor oligomycin on vesicular accumulation of Glu in purified
synaptosomes. When GAPDH was inhibited by iodoacetate, or mitochondrial
ATP synthesis was inhibited by oligomycin, ATP levels were
reduced in a concentration-dependent manner (Fig.
11, A and B). The
reduction in synaptosomal ATP caused by 500 µM
iodoacetate and 2 µM oligomycin was 67 and 64%,
respectively. Both reagents decreased synaptosomal
[3H]Glu uptake to a similar extent (Fig. 11, C
and D). Inhibition of synaptosomal [3H]Glu
uptake by 500 µM iodoacetate and 2 µM
oligomycin was 33 and 37%, respectively. In contrast, iodoacetate was
more effective than oligomycin in reducing vesicular
[3H]Glu content (Fig. 11, E and F).
When vesicular content was corrected for synaptosomal uptake (Fig. 11,
G and H), the inhibition of vesicular uptake
within the synaptosomes by 500 µM iodoacetate was 78%, whereas the inhibition by 2 µM oligomycin was only 16%.
Although the reduction of [3H]Glu content by iodoacetate
is probably due to inhibition of GAPDH, the possibility is not entirely
ruled out that iodoacetate could inhibit vesicular Glu uptake within
the synaptosomes by a mechanism not involving GAPDH inhibition.
However, iodoacetate did not inhibit [3H]Glu uptake by
purified synaptic vesicles in the presence of ATP (Fig.
5A).
Inhibition of GAPDH by iodoacetate would lead to reduction of pyruvate,
the precursor for the tricarboxylic acid cycle substrate acetyl-CoA and
thereby would decrease ATP production in mitochondria. To better
understand the mechanism of vesicular Glu accumulation, we further
investigated the effect of pyruvate on vesicular Glu uptake in
synaptosomes in the absence or presence of iodoacetate or oligomycin.
Pyruvate led to an increase in ATP levels when added to synaptosomes
incubated without glucose or to iodoacetate-treated synaptosomes
incubated with glucose (Fig.
12A). Increased levels of
ATP resulting from incubation with pyruvate were not observed in the
presence of oligomycin. These results are consistent with the concept
that pyruvate-induced ATP production occurs in mitochondria. However,
pyruvate failed not only to enhance vesicular Glu accumulation in the
absence of exogenous glucose but also to overcome the reduction of
vesicular Glu content caused by iodoacetate (Fig. 12B).
Oligomycin blocked pyruvate-induced increases in ATP levels (Fig.
12A) but had no significant effect on vesicular Glu content
corrected for synaptosomal uptake (Fig. 12B). In the
presence of glucose, pyruvate failed to elevate ATP levels or vesicular
Glu content, either in the absence or presence of oligomycin. These
results indicate that ATP produced by glycolysis is predominantly
utilized, whereas mitochondria-generated ATP is insufficient to support
vesicular uptake of Glu in nerve endings. These observations support
the notion that synaptic vesicle-bound GAPDH and 3-PGK play an
important role by providing ATP necessary for Glu uptake into synaptic
vesicles in the nerve terminal.
Glu Accumulated into Vesicles, at the Expense of Glycolytically
Produced ATP, Is Released upon Depolarization--
In order to
determine whether the Glu accumulated into synaptic vesicles by
glycolysis-generated ATP is released upon nerve ending stimulation, we
examined depolarization-induced release in synaptosomes. Synaptosomes
treated with either iodoacetate or oligomycin, prior to (and during)
loading with [3H]Glu, were depolarized by the potassium
channel blocker 4-AP. As shown in Fig.
13, iodoacetate treatment led to
substantial reduction in the amount of [3H]Glu released.
In contrast, oligomycin pretreatment had a much smaller effect. The
reduction in depolarization-induced Glu release is similar to the
reduction of vesicular Glu content observed with each inhibition (Fig.
11, G and H). When synaptosomes were treated with
iodoacetate or oligomycin after loading with [3H]Glu,
release was affected only to a small extent. The magnitude of this
reduction was similar to that observed when synaptosomes were treated
with oligomycin before loading with [3H]Glu.
The large differential of [3H]Glu release observed
between iodoacetate- and oligomycin-treated synaptosomes indicates that [3H]Glu accumulated in synaptic vesicles utilizing ATP
synthesized via glycolysis can be released by depolarization. Thus, the
effect of GAPDH inhibition by iodoacetate on presynaptic vesicular Glu accumulation is reflected in a decrease of Glu released by exocytosis.
We have presented evidence that GAPDH is modified by the high
energy glycolytic intermediate 1,3-BPG via covalent attachment of the
3-phosphoglyceroyl moiety. The radioactive moiety was removed by
treatment with neutral hydroxylamine. This suggests that this modification involves thioester formation between the
3-phosphoglyceroyl group and a GAPDH cysteine residue, most likely
representing the acylated enzyme intermediate (52, 53).
GAPDH was found to be bound to purified synaptic vesicles. This
observation is in agreement with the findings by Schlaefer et
al. (54) and Rogalski-Wilk and Cohen (55). Schlaefer et al. have postulated that vesicle-associated GAPDH might be
functionally coupled to ATP uptake into synaptic vesicles (54). We have
provided evidence that synaptic vesicle-bound GAPDH, coupled with
3-PGK, can produce ATP sufficient for Glu uptake by the vesicular Glu uptake system. In the presence of the glycolytic intermediate GAP,
GAPDH produces 1,3-BPG, which is then converted to 3-PG and ATP by the
GAPDH-bound 3-PGK. This conversion is coupled, since GAPDH and 3-PGK
exist in a complex (Fig. 2). Bernhard and co-workers (46, 47) have
demonstrated direct transfer of 1,3-BPG from GAPDH to 3-PGK in muscle
tissue. Inhibition of vesicle-bound GAPDH activity by iodoacetate led
to a reduction of ATP synthesis as well as to an attenuation of Glu
uptake into synaptic vesicles (Fig. 5). This was also observed in
synaptosomes (Figs. 11 and 12). Iodoacetate decreased the amount of
exocytotically released Glu (Fig. 13). Thus, it is likely that, in
synaptosomes, the ATP generated from the glycolytic intermediates
provides the energy required for the majority of the Glu taken up into
synaptic vesicles. Oligomycin inhibition of mitochondrial ATP synthesis
had little effect on vesicular Glu accumulation in synaptosomes (Figs.
11 and 12). Moreover, substitution of pyruvate for glucose, which increased ATP levels in synaptosomes, failed to elevate vesicular Glu
content (Fig. 12). These observations indicate that mitochondrially produced ATP contributes minimally to Glu transport into synaptic vesicles in synaptosomes, suggesting the critical importance of glycolytically produced ATP in vesicular Glu accumulation in the presynaptic nerve terminal.
In cardiac myocytes, there is evidence that glycolysis-derived ATP is
preferentially used to prevent ATP-sensitive K+ channels
from opening, thereby maintaining cellular membrane potential (56). In
the skeletal muscle, evidence suggests that fast twitch fibers of the
white vastus utilize ATP produced by glycolysis, whereas the slow
twitch fibers of the soleus largely use ATP synthesized by
mitochondria. The former has a high glycolytic capacity and a low
oxidative capacity, in contrast to the latter, which is opposite (57).
GAPDH activity is 3-4 times higher in the white quadriceps than in the
soleus (58). GAPDH is a component associated with the skeletal muscle
junctional "foot," which links the transverse tubule and terminal
cisternae, forming the triad, the site of excitation-contraction
coupling (59, 60). Heilmeyer et al. (60) have demonstrated
that the junctional triad is capable of producing ATP from GAP, NAD,
Pi, and ADP, indicating that localized glycolytic ATP
synthesis can occur at the site where an energy supply is immediately
needed. These observations are consistent with the concept that rapid,
energy-consuming cellular processes rely on glycolytically produced ATP.
ATP generated at the surface of the synaptic vesicle may play an
essential role in ensuring prompt Glu refilling of synaptic vesicles in
neurons. Mitochondria alone may not entirely be able to meet the ATP
requirement for maintaining normal neurotransmission, an extremely
rapid cellular process. Synaptic vesicles release their contents and
then must be rapidly refilled in order to continuously support normal
neurotransmission (61). Energy demand for vesicular uptake would be
dynamic and reflect nerve activity. The synaptic vesicle's function of
refilling Glu would be more efficiently served if ATP were generated
"locally" by vesicle-bound GAPDH/3-PGK. In isolated synaptic
vesicles, GAPDH/3-PGK-generated ATP appears more effective than
exogenous ATP in supporting vesicular Glu uptake (Fig. 4). In neurons,
mitochondria located in the nerve ending may not be close enough to
synaptic vesicles to provide sufficient ATP in a timely fashion for
sustaining adequate Glu accumulation. Moreover, ATP synthesis in
mitochondria occurs after ATP formation during glycolysis. Thus, the
synaptic vesicle-bound GAPDH-3-PGK system would have an advantage over
mitochondrial ATP synthase in its prompt response to energy demand for
refilling vesicles with the transmitter Glu.
In conclusion, we propose that the ATP produced by vesicle-bound GAPDH
and 3-PGK plays a crucial role in swiftly refilling vesicles with the
major excitatory neurotransmitter Glu (and possibly other
neurotransmitters) in the presynaptic nerve terminal. According to our
hypothesis depicted in Fig. 14, GAP
produced via glucose metabolism is converted to 1,3-BPG at the expense
of NAD and Pi by synaptic vesicle-bound GAPDH; 1,3-BPG
transfers its high energy phosphate to ADP, catalyzed by GAPDH-bound
3-PGK, forming ATP at the surface of vesicles. This ATP is utilized by
vesicular H+-pump ATPase to generate an electrochemical
proton gradient across the vesicle membrane, providing the driving
force for Glu transport into synaptic vesicles. ADP produced by ATPase
would be rapidly recycled to ATP by the GAPDH-3PGK system in the
presence of glycolysis, eliminating its potential inhibitory effect on
Glu uptake. Acute depletion of glucose could lead to a reduction of ATP
produced via glycolysis within the microenvironment of the presynaptic nerve terminal. This would diminish Glu transport into the vesicle, resulting in a decreased amount of Glu released, thereby attenuating Glu-mediated synaptic transmission. Moreover, in the prolonged absence
of glycolysis, excess ADP would be accumulated, impairing the
ATP-dependent vesicular uptake system, further contributing to reduction in vesicular Glu content and release. The mechanism outlined above could offer insight into our molecular understanding of
hypoglycemia-induced, abnormal electrophysiological activity and
resulting clinical symptoms as well as into the essential requirement
for glucose metabolism in normal synaptic transmission.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (6,000 Ci/mmol) was
obtained from PerkinElmer Life Sciences.
L-[G-3H]Glutamic acid (42.0 Ci/mmol) was
purchased from Amersham Biosciences. The affinity-purified polyclonal
antibodies (rabbit IgG) specific to recombinant monophosphoglycerate
mutase (PGM) type B was kindly provided by Oriental Yeast Co., Ltd.
(Tokyo, Japan). Anti-GAPDH monoclonal antibody (mouse IgG) and
anti-3-PGK polyclonal antibody (rabbit IgG) were purchased from U.S.
Biological (Swampscott, MA) and Accurate Chemical & Science Co.
(Westbury, NY), respectively. Glycolytic enzymes and all other
chemicals were purchased from Sigma-Aldrich unless mentioned elsewhere.
-32P]ATP in a mixture containing
5 mM Tris-HCl (pH 7.0), 40 µM
MgSO4, 10 mM dihydroxyacetone, 20 µM [
-32P]ATP (6,000 Ci/mmol), and 0.5 units of glycerol kinase (Bacillus stearothermophilus). The
reaction was performed in a volume of 40 µl at 37 °C for 20 min.
[3-32P]1,3-BPG was prepared from [32P]DHAP
by conversion to [32P]GAP with triose-phosphate
isomerase, followed by a GAPDH reaction in the presence of the lactate
dehydrogenase-coupled NAD-regenerating system. The reaction mixture
(400 µl) contained 12.5 mM triethanolamine (pH 8.0), 0.2 mM EDTA, 2 mM NAD, 2 mM sodium
pyruvate, 2 mM KH2PO4, 50 µM DHAP, 3.2 units of GAPDH (rabbit muscle), 20 units of
triose-phosphate isomerase (rabbit muscle), 10 units of lactate
dehydrogenase (rabbit muscle), and 40 µl of the
[32P]DHAP reaction mixture. The entire mixture was
incubated at 25 °C for 5 min, filtered to remove the enzymes by an
Amicon Centricon-10 concentrator at 4 °C, and put onto
DEAE-cellulose (Whatman DE32, 1.2 × 2.2 cm) previously
equilibrated with 10 mM glycylglycine, pH 7.4. Elution was
carried out with stepwise increases in the NaCl concentration: 50, 75, 100, 125, 150, and 200 mM. DHAP, GAP, and inorganic
phosphate (Pi) were eluted with 75 mM NaCl in
the same buffer. 3-PG and 1,3-BPG were eluted with 125 and 150 mM NaCl, respectively, in the same buffer. All the
compounds thus prepared were stored at
80 °C until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GAPDH is labeled with
[3-32P]1,3-BPG and enriched in synaptic vesicles.
Synaptic vesicles were incubated at 37 °C for 10 s with
[3-32P]1,3-BPG in the absence or presence of 4 mM MgSO4 (Mg2+) or 50 µM 3-PG and subjected to SDS-PAGE under neutral pH
conditions, according to the method of Fairbanks (A) or
under alkaline pH conditions (standard SDS-PAGE) according to the
method of Laemmli (B), followed by autoradiography.
C, the 0.2 M NaCl extract of the synaptic
vesicle fraction was incubated at 37 °C for 10 s with
[3-32P]1,3-BPG in the presence (anti-GAPDH, mouse IgG) or
absence (anti-PGM, rabbit IgG) of 4 mM Mg2+;
immunoprecipitated with the anti-GAPDH antibody, anti-PGM antibody,
mouse IgG (control for anti-GAPDH), or rabbit IgG (control for
anti-PGM); and subjected to SDS-PAGE under neutral pH conditions,
followed by autoradiography. D, various subcellular
fractions (30 µg of protein) as indicated were incubated at 37 °C
for 10 s with 140 nM [3-32P]1,3-BPG and
subjected to SDS-PAGE under neutral pH conditions, followed by
autoradiography. E, the subcellular fractions were separated
by standard SDS-PAGE, blotted onto polyvinylidene difluoride membranes,
and detected with anti-GAPDH. F, the synaptic vesicle
fraction was treated with various concentrations of NaCl, and the salt
extracts (supernatant (Sup)) and the salt-insoluble material
(Pellet) were analyzed for GAPDH as described above. The
data shown are representative of results obtained from three separate
experiments. Syn. cytosol, synaptosomal cytosol; Per.
cytosol, perikaryal cytosol (cell body cytosol); Pl.
membrane, plasma membrane.
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Fig. 2.
Association of 3-PGK with synaptic vesicles
mediated by GAPDH. A, various subcellular fractions as
indicated were analyzed for 3-PGK by SDS-PAGE/Western blot as described
in the Fig. 1 legend, except for use of anti-3-PGK antibodies instead
of anti-GAPDH antibody. B, the synaptic vesicle fraction was
treated with various concentrations of NaCl as indicated, and the salt
extracts (supernatant (Sup)) and the salt-insoluble material
(Pellet) were analyzed for 3-PGK by SDS-PAGE/Western
blotting probed with anti-3-PGK antibodies. The 0.2 M NaCl
extract of synaptic vesicles was subjected to immunoprecipitation with
rabbit IgG or anti-3-PGK antibodies (C) and with mouse IgG
or the anti-GAPDH antibody (D); the immunoprecipitates were
analyzed for GAPDH (C) and 3-PGK (D),
respectively. E, synaptic vesicles (SV)
were washed twice with 0.8 M NaCl and incubated in
the absence or presence of GAPDH, each with or without 3-PGK at
37 °C for 10 min, followed by two additional washings with low salt
buffer. The synaptic vesicle pellets were then analyzed for GAPDH and
3-PGK. The data shown are representative of results obtained from three
separate experiments.
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Fig. 3.
Dependence of vesicular Glu uptake on the
GAPDH/3-PGK substrate. A, time course of vesicular Glu
uptake induced by activation of vesicle-bound GAPDH and 3-PGK. Glu
uptake into synaptic vesicles was measured at 30 °C for the
indicated periods in the absence ( ) or presence (
) of 2 mM GAP, 2 mM NAD, 2 mM
Pi, and 0.1 mM ADP. B, GAPDH and
3-PGK substrate requirement for vesicular Glu uptake. Glu uptake into
synaptic vesicles was measured at 30 °C after a 10-min incubation in
the absence or presence of various agents or exogenous 3-PGK (1 unit)
as indicated. C, effect of various ADP concentrations on Glu
uptake into synaptic vesicles induced by activation of vesicle-bound
GAPDH and 3-PGK. Glu uptake was determined at 30 °C after 10-min
incubation in the presence of 2 mM GAP, 2 mM
NAD, and 2 mM Pi. Values are the mean ± S.D. of four separate experiments.
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Fig. 4.
Effect of various GAP and exogenous ATP
concentrations on vesicular Glu uptake and ATP levels after
incubation. Glu uptake into synaptic vesicles (A and
C) and ATP levels in the synaptic vesicle suspension
(B and D), after 10-min incubation at 30 °C,
were measured in the presence of 2 mM NAD, 2 mM
Pi, 0.1 mM ADP, and the indicated
concentrations of GAP (A and B) or of various
concentrations of exogenous ATP (C and D).
E, Glu uptake into synaptic vesicles measured in the
presence of various concentrations of GAP, 2 mM NAD, 2 mM Pi, and 0.1 mM ADP ( ) or
various concentrations of exogenous ATP (
) was plotted as a function
of ATP level in the synaptic vesicle suspension, each determined after
a 10-min incubation at 30 °C. Values are the mean ± S.D. of
four separate experiments.
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Fig. 5.
Iodoacetate selectively inhibits GAPDH/3-PGK
activation-dependent over exogenous ATP-dependent
vesicular Glu uptake. A, effect of various iodoacetate
concentrations on GAPDH/3-PGK activation-dependent
versus exogenous ATP-dependent vesicular Glu
uptake. The synaptic vesicle fraction (10 µg of protein) was
preincubated at 30 °C for 10 min in the presence of various
concentrations of iodoacetate, followed by an additional 10-min
incubation at 30 °C after the addition of a mixture of
[3H]Glu, 2 mM GAP, 2 mM NAD, 2 mM Pi, and 0.1 mM ADP ( ) or of a
mixture of [3H]Glu and 2 mM ATP (
).
[3H]Glu accumulated into synaptic vesicles was determined
as described under "Experimental Procedures." B, effect
of various iodoacetate concentrations on vesicle-bound GAPDH activity
and on ATP produced by activation of vesicle-bound GAPDH/3-PGK. The
synaptic vesicle fraction (10 µg of protein) was incubated in the
presence of various concentrations of iodoacetate for a total period of
20 min as described for A, except for replacement of
[3H]Glu with nonradioactive Glu. After incubation, GAPDH
activity (
) and the amount of ATP produced (
) were determined, as
described under "Experimental Procedures." Values are the mean ± S.D. of four separate experiments.
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Fig. 6.
Comparison in responsiveness to chloride,
aspartate, t-ACPD, Rose Bengal, and FCCP between
GAPDH/3-PGK activation-dependent and exogenous
ATP-dependent vesicular Glu uptake. The effect of
various chloride concentrations on Glu uptake into synaptic vesicles
was determined at 30 °C after a 10-min incubation in the presence of
2 mM GAP, 2 mM NAD, 2 mM
Pi, and 0.1 mM ADP (A) or of 2 mM ATP (B). Effect of 2 mM Asp, 2 mM t-ACPD, 0.5 µM Rose Bengal
(RB), and 25 µM FCCP on Glu uptake into
synaptic vesicles was determined at 30 °C after 10-min incubation in
the presence of 2 mM GAP, 2 mM NAD, 2 mM Pi, and 0.1 mM ADP
(C) or of 2 mM ATP (D). Values are
the mean ± S.D. of four separate experiments.
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Fig. 7.
Effect of GAPDH substrates on vesicular Glu
uptake in the presence of ATP. Glu uptake into isolated synaptic
vesicles at 30 °C after 10 min incubation in the presence of 30 µM ATP (A) or 2 mM ATP
(B) was measured in the absence or presence of GAPDH
substrates (2 mM GAP, 2 mM NAD, and 2 mM Pi) as described under "Experimental
Procedures." Values are the mean ± S.D. of four separate
experiments.
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Fig. 8.
ATP-dependent vesicular Glu
uptake is inhibited by ADP, which has no effect on vesicular ATPase
activity. A, effect of various concentrations of ADP
( ), AMP (
), or adenosine (
) on Glu uptake into synaptic
vesicles at 30 °C after 10 min incubation was determined in the
presence of 2 mM ATP. B, effect of various ADP
concentrations on vesicular ATPase activity was determined as described
under "Experimental Procedures." Values are the mean ± S.D.
of four separate experiments.
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Fig. 9.
Kinetics of ADP inhibition of vesicular Glu
uptake. A, the initial rate of vesicular Glu uptake at
30 °C in the presence of 2 mM ATP was determined with
various Glu concentrations, each in the absence ( ) or presence of
0.5 mM (
) or 2 mM ADP (
), by incubating
the synaptic vesicle fraction (10 µg of protein) for 1.5 min, as
described under "Experimental Procedures." B, the
initial rate of vesicular Glu uptake with a fixed Glu concentration
(100 µM) was determined with various ATP concentrations
in the absence (
) or presence of 0.5 mM (
) or 2 mM ADP (
), as described above. Values are the mean ± S.D. of four separate experiments.
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Fig. 10.
Effect of ADP on membrane potential in
synaptic vesicles. After oxonol V (1.3 µM) was
allowed to equilibrate with synaptic vesicles as described under
"Experimental Procedures," ATP was added (2 mM as a
final concentration). Various amounts of ADP were then added, resulting
in indicated final concentrations, followed by the addition of FCCP
(8.33 µM as a final concentration). A, time
course tracing of fluorescence quenching before and after the addition
of 0.3, 1, and 2 mM ADP. The data shown are representative
of three separate experiments. B, membrane potential as a
function of various concentrations of ADP as indicated. Values are
the mean ± S.D. of three separate experiments.
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Fig. 11.
Iodoacetate reduces vesicular
[3H]Glu content in synaptosomes more effectively than
oligomycin, whereas both agents have similar effects on synaptosomal
ATP levels in the concentration ranges tested. Synaptosomes were
preincubated at 37 °C for 10 min in the presence of various
concentrations of iodoacetate (A, C,
E, and G) or oligomycin (B,
D, F, and H), followed by the addition
of [3H]Glu; ATP level (A and B),
synaptosomal [3H]Glu uptake (C and
D), and vesicular [3H]Glu content
(E and F) were determined at 37 °C after 10 min, as described under "Experimental Procedures." Vesicular
[3H]Glu content was expressed as percentage of total
synaptosomal [3H]Glu uptake (G and
H). Values are the mean ± S.D. of four separate
experiments.
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Fig. 12.
Effect of pyruvate, glucose, oligomycin, and
iodoacetate on vesicular [3H]Glu content and ATP levels
in synaptosomes. Synaptosomes were preincubated at 37 °C for 10 min in the absence or presence of 10 mM glucose, 2 µM oligomycin, 300 µM iodoacetate, or 1 mM pyruvate; ATP level (A) and vesicular
[3H]Glu content (B) were determined at
37 °C after an additional 10-min incubation with
[3H]Glu, as described in the legend to Fig. 11. Vesicular
[3H]Glu content was expressed as percentage of total
synaptosomal [3H]Glu uptake. Values are the mean ± S.D. of four separate experiments.
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Fig. 13.
Treatment of synaptosomes with iodoacetate
prior to and during the [3H]Glu-loading period reduces
[3H]Glu release. Synaptosomes were preincubated at
37 °C for 10 min in the absence ( ,
,
, and
) or presence
of 300 µM iodoacetate (IA;
) or 2 µM oligomycin (OM;
); [3H]Glu
was loaded into the synaptosome at 37 °C for 10 min. The
[3H]Glu-loaded synaptosomes were layered onto the
membrane in the superfusion chamber and superfused at 37 °C for 20 min with ACSF in the absence (
,
,
, and
) or presence of
300 µM iodoacetate (
) or 2 µM oligomycin
(
). After further superfusion with ACSF at 37 °C for 40 min,
synaptosomes were subjected to depolarization with 50 µM
4-aminopyridine (4-AP;
,
,
,
,
) or not (
);
[3H]Glu release was measured as described under
"Experimental Procedures." Values are the mean ± S.D. of six
separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 14.
Proposed role of GAPDH and 3-PGK in
vesicular Glu uptake. The glycolytic intermediate GAP is converted
to 1,3-BPG by synaptic vesicle-associated GAPDH in the presence of NAD
and Pi. 1,3-BPG is then converted to 3-PG by
GAPDH-associated 3-PGK, accompanied by phosphorylation of ADP to form
ATP. ATP thus produced is efficiently consumed by vesicular
H+-ATPase, generating an electrochemical proton gradient in
the presence of low concentrations of chloride; this provides the
driving force for Glu uptake, which is carried out by the vesicular Glu
transporter (VGLUT). ADP generated by vesicular H+-ATPase
is efficiently converted to ATP by 3-PGK. When ADP is accumulated, Glu
uptake into synaptic vesicles would be inhibited. , membrane
potential;
pH, pH gradient.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Kiyokazu Ogita (Setsunan University, Hirakata, Japan) for technical advice in the superfusion release assay, Dr. Koji Hirata for synaptic vesicle preparation, and Oriental Yeast Co., Ltd., Tokyo, for the kind gift of affinity-purified polyclonal antibodies to recombinant PGM type B. We also thank Mary Roth for excellent assistance in preparation of the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants NS 24384, NS 36656, and NS 42200 and a grant from Taisho Pharmaceutical Co., Ltd. (Tokyo, Japan).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.
§ On leave from the Dept. of Biological Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, 467-8603 Nagoya, Japan.
** To whom correspondence should be addressed: Mental Health Research Institute at MSRB II, C570D, The University of Michigan, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0669. Tel.: 734-763-3790; Fax: 734-936-2690; E-mail: tueda@umich.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M211617200
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ABBREVIATIONS |
---|
The abbreviations used are: 3-PG, 3-phosphoglycerate; t-ACPD, trans-1-aminocyclopentane-1,3-dicarboxylic acid; 4-AP, 4-aminopyridine; 1, 3-BPG, 1,3-bisphosphoglycerate; DHAP, dihydroxyacetone phosphate; FCCP, carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; GAP, glyceraldehyde-3-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 3-PGK, 3-phosphoglycerate kinase; PGM, monophosphoglycerate mutase; ACSF, artificial cerebrospinal fluid.
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REFERENCES |
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