Glycolysis and Glutamate Accumulation into Synaptic Vesicles

ROLE OF GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE AND 3-PHOSPHOGLYCERATE KINASE*

Atsushi IkemotoDagger §, David G. BoleDagger , and Tetsufumi UedaDagger ||**

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [gamma -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.

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 [gamma -32P]ATP in a mixture containing 5 mM Tris-HCl (pH 7.0), 40 µM MgSO4, 10 mM dihydroxyacetone, 20 µM [gamma -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.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.


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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.

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.


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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.

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.


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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 (triangle ) 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.

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.


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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 (triangle ) 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.

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.


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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 (triangle ). [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.

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.


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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.

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.


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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.

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).


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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 (triangle ) 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 (open circle ) or presence of 0.5 mM (black-triangle) or 2 mM ADP (black-square), 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 (open circle ) or presence of 0.5 mM (black-triangle) or 2 mM ADP (black-square), as described above. Values are the mean ± S.D. of four separate experiments.

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.


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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.

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).


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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.

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.


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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.

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.


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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 (open circle , , , and triangle ) or presence of 300 µM iodoacetate (IA; black-square) or 2 µM oligomycin (OM; black-triangle); [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 (open circle , , black-square, and black-triangle) or presence of 300 µM iodoacetate () or 2 µM oligomycin (triangle ). After further superfusion with ACSF at 37 °C for 40 min, synaptosomes were subjected to depolarization with 50 µM 4-aminopyridine (4-AP; , black-square, black-triangle, , triangle ) or not (open circle ); [3H]Glu release was measured as described under "Experimental Procedures." Values are the mean ± S.D. of six separate experiments.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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. Delta Psi , membrane potential; Delta pH, pH gradient.


    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.

    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

    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.

    REFERENCES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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