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The increased concentration of glutamate in synaptic vesicles, mediated by the vesicular glutamate transporter (VGLUT), is an initial vital step in glutamate synaptic transmission. Evidence indicates that aberrant overexpression of VGLUT is involved in certain pathophysiologies of the central nervous system. VGLUT is subject to inhibition by various types of agents. The most potent VGLUT-specific inhibitor currently known is Trypan Blue, which is highly charged, hence membrane-impermeable. We have sought a potent, VGLUT-specific agent amenable to easy modification to a membrane-permeable analog. We provide evidence that Brilliant Yellow exhibits potent, VGLUT-specific inhibition, with a Ki value of 12 nM. Based upon structure-activity relationship studies and molecular modeling, we have defined the potent inhibitory pharmacophore of VGLUT Brilliant Yellow. This study provides new insight into development of a membrane-permeable agent to lead to specific blockade, with high potency, of accumulation of glutamate into synaptic vesicles in neurons.

Glutamate is an excitatory neurotransmitter widely used in the vertebrate central nervous systems. The synaptic transmission process is characterized by three steps: (1) presynaptic vesicular transmitter uptake, (2) presynaptic release, and (3) postsynaptic receptor activation. Presynaptic vesicular glutamate uptake plays an initial pivotal role in glutamate transmission by concentrating glutamate in the vesicular lumen prior to its release. This active glutamate transport harnesses energy derived from ATP hydrolysis, and intra- or extravesicular chloride, and is highly specific to glutamate. The uptake system consists of a vesicular glutamate transporter (VGLUT) and v-type proton-pump ATPase, which generates an electrochemical proton gradient, the driving force of the transport. The major source of ATP is likely to be supplied by glycolytic vesicle-bound enzymes, glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, rather than by mitochondrial ATP synthase. The VGLUT substrate glutamate is proposed to be synthesized by vesicle-bound aspartate amino transferase from α-ketoglutarate, not directly from glutamine. VGLUT has three isoforms, and gaged by their distributions they perform different physiological functions. The mechanism and regulation of vesicular glutamate uptake are discussed. The pharmacology of vesicular glutamate uptake is a developing field of inquiry.

Glutamate uptake into synaptic vesicles in nerve terminals is a pivotal step in glutamate synaptic transmission. Glutamate is the major excitatory neurotransmitter and, as such, the vesicular glutamate transporter (VGLUT) responsible for this uptake is involved in a variety of nervous system functions and various types of pathophysiology. As yet, no VGLUT-specific, membrane-permeable agents have been developed to affect neuronal function in intact neurons, although two potent VGLUTspecific inhibitors are known. These compounds contain diazo and highly charged sulfonic acid groups, rendering them membrane-impermeable and potentially cytotoxic. In an effort to eliminate these undesirable properties, we have developed two novel agents, Brilliant Yellow analogs 1 and 2, which are free of these two groups. We show here that these agents retain highly VGLUT-selective inhibitory activity, despite their reduction in potency, and exhibit no significant cellular toxicity. Potential use of this molecular modification is discussed.

Glutamate accumulation into synaptic vesicles is a pivotal step in glutamate transmission. This process is achieved by a vesicular glutamate transporter (VGLUT) coupled to v-type proton ATPase. Normal synaptic transmission, in particular during intensive neuronal firing, would demand rapid transmitter re-filling of emptied synaptic vesicles. We have previously shown that isolated synaptic vesicles are capable of synthesizing glutamate from α-ketoglutarate (not from glutamine) by vesicle-bound aspartate aminotransferase for immediate uptake, in addition to ATP required for uptake by vesicle-bound glycolytic enzymes. This suggests that local synthesis of these substances, essential for glutamate transmission, could occur at the synaptic vesicle. Here we provide evidence that synaptosomes (pinched-off nerve terminals) also accumulate α-ketoglutarate-derived glutamate into synaptic vesicles within, at the expense of ATP generated through glycolysis. Glutamine-derived glutamate is also accumulated into synaptic vesicles in synaptosomes. The underlying mechanism is discussed. It is suggested that local synthesis of both glutamate and ATP at the presynaptic synaptic vesicle would represent an efficient mechanism for swift glutamate loading into synaptic vesicles, supporting maintenance of normal synaptic transmission.

Glycolytic ATP synthesis by synaptic vesicles provides an efficient mechanism for fueling vesicular loading of the neurotransmitter glutamate. This is achieved in part by vesicle-bound pyruvate kinase. However, we have found that vesicular glutamate uptake, in the presence of the pyruvate kinase substrates ADP and phosphoenolpyruvate (PEP), substantially exceeds that caused by exogenous ATP. We propose that this much enhanced uptake is in part due to extra ATP produced via a mechanism involving a novel enzyme, PEP-dependent ADP synthase. We discuss implications for this enzyme in energy homeostasis and pathophysiology, as well as in efficient synaptic glutamate transmission.

Synaptic vesicle loading of glutamate is a pivotal step in glutamate synaptic transmission. The molecular machinery responsible for this step is comprised of v-type proton-pump ATPase and a vesicular glutamate transporter. Recent evidence indicates that synaptic vesicles are endowed with glycolytic ATP-synthesizing enzymes, providing energy for immediate use by vesicle-bound proton-pump ATPase. In this study, we provide evidence that synaptic vesicles are also capable of synthesizing the vesicular glutamate transporter substrate glutamate, from α-ketoglutarate and L-aspartate (as the amino group donor); glutamate thus produced is taken up into vesicles. We also report a finding thatα-ketoglutarate-derived glutamate uptake into synaptic vesicles and aspartate aminotransferase are inhibited by 2,3-pyrazinedicarboxylate. Evidence is given that this is a selective inhibitor for aspartate aminotransferase. These observations provide insight into understanding the nerve endings’ mechanism for high efficiency in glutamate transmission. Finding this inhibitor may have implications for further experimentation on the role of α-ketoglutarate-derived glutamate in glutamate transmission.

Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [(3)H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. "ATP-trapping" experiments using hexokinase and glucose suggest that ATP produced by vesicle-associated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.

Glutamate is an excitatory neurotransmitter widely used in the vertebrate central nervous systems. The synaptic transmission process is characterized by three steps: (1) presynaptic vesicular transmitter uptake, (2) presynaptic release, and (3) postsynaptic receptor activation. Presynaptic vesicular glutamate uptake plays an initial pivotal role in glutamate transmission by concentrating glutamate in the vesicular lumen prior to its release. This active glutamate transport harnesses energy derived from ATP hydrolysis, and intra- or extravesicular chloride, and is highly specific to glutamate. The uptake system consists of a vesicular glutamate transporter (VGLUT) and v-type proton-pump ATPase, which generates an electrochemical proton gradient, the driving force of the transport. The major source of ATP is likely to be supplied by glycolytic vesicle-bound enzymes, glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglycerate kinase, rather than by mitochondrial ATP synthase. The VGLUT substrate glutamate is proposed to be synthesized by vesicle-bound aspartate amino transferase from α-ketoglutarate, not directly from glutamine. VGLUT has three isoforms, and gaged by their distributions they perform different physiological functions. The mechanism and regulation of vesicular glutamate uptake are discussed. The pharmacology of vesicular glutamate uptake is a developing field of inquiry.

Noncyclic fluorine-substituted and cyclic analogs of glutamic acid were tested for their ability to inhibit glutamate uptake in isolated bovine presynaptic vesicles, in order to assess the specific structural requirements of the glutamate translocation system in the vesicle membrane. Cyclic analogs that permitted close interaction between the positive and negative charges of the glutamate molecule were effective inhibitors; maximum inhibitory potency was observed with L-trans-1-aminocyclopentane-1,3-dicarboxylic acid (L-t-ACPD), while D-t-ACPD was less active. Analogs with a larger or smaller ring (as in trans-1-aminocyclohexane-1,3-dicarboxylic acid or trans-1-aminocyclobutane-1,3-dicarboxylic acid) were also inhibitory, but somewhat less so. trans-ACPD was also taken up by the vesicles with a time course and ATP dependence similar to uptake of glutamate, and this uptake was inhibited by glutamate. The K(m) value for t-ACPD uptake was similar to its K(i) for inhibition of glutamate uptake, while its rate of uptake was lower than that of glutamate. Fluorine-substituted noncyclic analogs with substitutions at the 4-carbon were less effective than glutamic acid itself, although 4,4-difluoroglutamic acid was equal in activity to the unsubstituted compound. Inhibition by these derivatives appeared to be competitive in nature, and they probably were also transported by the vesicle uptake system.

Our aim was to review the processes of glutamate release from both biochemical and neurophysiological points of view. A large body of evidence now indicates that glutamate is specifically accumulated into synaptic vesicles, which provides strong support for the concept that glutamate is released from synaptic vesicles and is the major excitatory neurotransmitter. Evidence suggests the notion that synaptic vesicles, in order to sustain the neurotransmitter pool of glutamate, are endowed with an efficient mechanism for vesicular filling of glutamate. Glutamate-loaded vesicles undergo removal of Synapsin I by CaM kinase II-mediated phosphorylation, transforming to the release-ready pool. Vesicle docking to and fusion with the presynaptic plasma membrane are thought to be mediated by the SNARE complex. The Ca(2+)-dependent step in exocytosis is proposed to be mediated by synaptotagmin.

Synaptic vesicular accumulation of glutamate is a vital initial step in glutamate transmission. We have previously shown that Rose Bengal, a polyhalogenated fluorescein analog, is a potent inhibitor of glutamate uptake into synaptic vesicles. Here, we report the structural features of Rose Bengal required for this inhibition. Various Rose Bengal-related compounds, with systematic structural variations, were tested. Results indicate that the four iodo groups and the phenyl group attached to the xanthene moiety are critical for potent inhibitory activity. Replacement of these groups with two iodo groups and an alkyl group, respectively, results in substantial reduction in potency. Of further interest in creating high potency is the critical nature of the oxygen atom which links the two benzene rings of xanthene. Thus, the phenyl group and multiple iodo groups, as well as the bridging oxygen of xanthene, are crucial elements of Rose Bengal required for its potent inhibitory action.

Glucose metabolism is of vital importance in normal brain function. Evidence indicates that glycolysis, in addition to production of ATP, plays an important role in maintaining normal synaptic function. In an effort to understand the potential involvement of a glycolytic intermediate(s) in synaptic function, we have prepared [3-32P]1,3-bisphosphoglycerate and [32P]3-phosphoglycerate and sought their interaction with a specific nerve-ending protein. We have found that a 29-kDa protein is the major component labeled with either [3-32P]1,3-bisphosphoglycerate or [32P]3-phosphoglycerate. The protein was identified as monophosphoglycerate mutase (PGAM). This labeling was remarkably high in the brain and synaptosomal cytosol fraction, consistent with the importance of glycolysis in synaptic function. Of interest, fructose-2,6-bisphosphate (Fru-2,6-P2) inhibited PGAM phosphorylation and enzyme activity. Moreover, Fru-2,6-P2 potently stimulated release of [32P]phosphate from the 32P-labeled PGAM (EC50 = 1 microM), suggesting that apparent reduction of PGAM phosphorylation and enzyme activity by Fru-2,6-P2 may be due to stimulation of dephosphorylation of PGAM. The significance of these findings is discussed.

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, [(3)H]glutamate uptake into isolated synaptic vesicles. It also decreased vesicular [(3)H]glutamate content in the nerve ending preparation synaptosome; this decrease was reflected in reduction of depolarization-induced [(3)H]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.

Synaptic vesicles in the nerve terminal play a pivotal role in neurotransmission. Neurotransmitter accumulation into synaptic vesicles is catalyzed by distinct vesicular transporters, harnessing an electrochemical proton gradient generated by V-type proton-pump ATPase. However, little is known about regulation of the transmitter pool size, particularly in regard to amino acid neurotransmitters. We previously provided evidence for the existence of a potent endogenous inhibitory protein factor (IPF), which causes reduction of glutamate and GABA accumulation into isolated, purified synaptic vesicles. In this study we demonstrate that IPF is concentrated most in the synaptosomal cytosol fraction and that, when introduced into the synaptosome, it leads to a decrease in calcium-dependent exocytotic (but not calcium-independent) release of glutamate in a concentration-dependent manner. In contrast, alpha-fodrin (non-erythroid spectrin), which is structurally related to IPF and thought to serve as the precursor for IPF, is devoid of such inhibitory activity. Intrasynaptosomal IPF also caused reduction in exocytotic release of GABA and the monoamine neurotransmitter serotonin. Whether IPF affects vesicular storage of multiple neurotransmitters in vivo would depend upon the localization of IPF. These results raise the possibility that IPF may modulate synaptic transmission by acting as a quantal size regulator of one or more neurotransmitters.

Synaptic vesicle loading of glutamate is a pivotal step in glutamate synaptic transmission. The molecular machinery responsible for this step is comprised of v-type proton-pump ATPase and a vesicular glutamate transporter. Recent evidence indicates that synaptic vesicles are endowed with glycolytic ATP-synthesizing enzymes, providing energy for immediate use by vesicle-bound proton-pump ATPase. In this study, we provide evidence that synaptic vesicles are also capable of synthesizing the vesicular glutamate transporter substrate glutamate, from α-ketoglutarate and l-aspartate (as the amino group donor); glutamate thus produced is taken up into vesicles. We also report a finding that α-ketoglutarate-derived glutamate uptake into synaptic vesicles and aspartate aminotransferase are inhibited by 2,3-pyrazinedicarboxylate. Evidence is given that this is a selective inhibitor for aspartate aminotransferase. These observations provide insight into understanding the nerve endings' mechanism for high efficiency in glutamate transmission. Finding this inhibitor may have implications for further experimentation on the role of α-ketoglutarate-derived glutamate in glutamate transmission.

A variety of naturally occurring amino acids, their isomers, and synthetic analogs were tested for their ability to inhibit uptake of [3H]glutamate into presynaptic vesicles from bovine cerebral cortex. Strongest inhibition (Ki1mM) was observed for trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and erythro-4-methyl-L-glutamic acid (MGlu), while 4-methylene-L-glutamic acid (MeGlu) was only moderately inhibitory (Ki = approximately 3mM), indicating that the synaptic vesicle glutamate translocator has higher affinity for trans-ACPD and MGlu than for glutamate. A few other amino acids, e.g., 4-hydroxyglutamic acid, S-carboxyethyl cysteine, and 5-fluorotryptophan, were slightly inhibitory; all L- and DL-isomers of protein amino acids and longer chain acidic amino acids were without measurable inhibition. Potassium tetrathionate and S-sulfocysteine exhibited strong to moderate noncompetitive or irreversible inhibition. Inhibition by t-ACPD, MGlu, or MeGlu was competitive with glutamic acid. Each of these competitive inhibitors was also taken up by the vesicle preparation in an ATP-dependent manner, as indicated by their being recovered unchanged from filtered vesicles. Similar results were obtained with reconstituted vesicles, while glutamate uptake by partially purified rat synaptosomes was inhibited only by MGlu. These results indicate that the glutamate translocator of presynaptic vesicles has stringent structural requirements distinct from those of the plasma membrane translocator and the metabotropic type of postsynaptic glutamate receptor. They further suggest possible structural requirements of pharmacologically significant compounds that can substitute for glutamic acid in the presynaptic side of glutamatergic synapses, thus serving to moderate or control glutamate excitation and associated excitotoxic effects in these neurons.

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into bovine synaptic vesicles in a manner that is ATP dependent and requires a vesicular electrochemical proton gradient. We studied the electrical and chemical elements of this driving force and evaluated the effects of chloride on transport. Increasing concentrations of Cl- were found to increase the steady-state ATP-dependent vesicular pH gradient (delta pH) and were found to concomitantly decrease the vesicular membrane potential (delta psi). Low millimolar chloride concentrations, which cause 3-6-fold stimulation of vesicular glutamate uptake, caused small but measurable increases in delta pH and decreases in delta psi, when compared to control vesicles in the absence of chloride. Nigericin in potassium buffers was used to alter the relative proportions of delta pH and delta psi. Compared to controls, at all chloride concentrations tested, nigericin virtually abolished delta pH and increased the vesicle interior positive delta psi. Concomitantly, nigericin increased ATP-dependent glutamate uptake in 0-1 mM chloride but decreased glutamate uptake in 4 mM (45%), 20 mM (80%), and 140 mM (75%) Cl- (where delta pH in the absence of nigericin was large). These findings suggest that either delta psi, delta pH, or a combination can drive glutamate uptake, but to different degrees. In the presence of 4 mM Cl-, where uptake is optimal, both delta psi and delta pH contribute to the driving force for uptake. When the extravesicular pH was increased from 7.4 to 8.0, more Cl- was required to stimulate vesicular glutamate uptake. In the absence of Cl-, as extravesicular pH was lowered to 6.8, uptake was over 3-fold greater than it was at pH 7.4. As extravesicular pH was reduced from 8.0 toward 6.8, less Cl- was required for maximal stimulation. Decreasing the extravesicular pH from 8.0 to 6.8 in the absence of Cl- significantly increased glutamate uptake activity, even though proton-pumping ATPase activity actually decreased about 45% under identical conditions. In the absence of chloride, nigericin increased glutamate uptake at all the pH values tested except pH 8.0. Glutamate uptake at pH 6.8 in the presence of nigericin was over 6-fold greater than uptake at pH 7.4 in the absence of nigericin. We conclude from these experiments that optimal ATP-dependent glutamate uptake requires a large delta psi and a small delta pH.(ABSTRACT TRUNCATED AT 400 WORDS)

The impaired Na+-K+-ATPase activity in peripheral nerve from diabetic rats is prevented by dietary myo-inositol (MI) supplementation in vivo and corrected by protein kinase C (PKC) agonists in vitro, suggesting that PKC may mediate the effects of nerve MI depletion on Na+-K+-ATPase activity. However, little is known about the effect of diabetes on PKC activity or peptide in rat peripheral nerve. Therefore, the effect of streptozocin-induced diabetes and dietary MI supplementation on the activity and distribution of PKC in rat sciatic nerve homogenates and cytosolic and particulate fractions was explored with histone phosphorylation assay and Western-blot analysis. PKC activity but not peptide was selectively decreased in the cytosolic fraction by streptozocin-induced diabetes, and this abnormality was partially corrected by dietary MI supplementation. These results suggest that altered MI metabolism may affect nerve PKC specific activity, and this alteration may play a role in reduced Na+-K+-ATPase activity and blunted regenerative response in diabetic nerve.

We have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, we considered the possibility that 3-phosphoglycerate may act as a coupling factor, and we searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [gamma-32P]ATP and appropriate test substances and were subjected to NaDodSO4/PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of a 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion.

We have investigated factors which may affect accumulated glutamate levels in synaptic vesicles and glutamate efflux. Agents which dissipate the electrochemical proton gradient resulted in a rapid reduction of steady-state vesicular glutamate levels, which was prevented by N-ethylmaleimide. Glutamate efflux was found to occur even in the presence of an electrochemical proton gradient, but was effectively inhibited by N-ethylmaleimide. These results suggest that accumulated glutamate levels in synaptic vesicles are not maintained unless glutamate is taken up continuously by an active transport mechanism, and they could provide an explanation for the lack of convincing evidence for the enrichment of endogenous glutamate in isolated synaptic vesicles.

Immunoreactivity to synapsin I, a neuronal phosphoprotein, was localized in free-floating tissue sections prepared from lingual tissue of rats. Many nerve fibers within the tissue exhibited clear immunoreactivity including motor endplates on striated muscle, autonomic fibers innervating blood vessels or glands, and sensory fibers innervating muscles or the lingual epithelium including taste buds. Numerous immunoreactive fibers occurred within each taste bud, with fewer, fine fibers being dispersed in the epithelium between taste buds. The majority of the intragemmal immunoreactive fibers extended throughout the taste buds most of the distance outward from the basal lamina toward the surface of the epithelium. Fine, perigemmal fibers reached nearly to the epithelial surface. Ultrastructural analysis of the immunoreactive sensory fibers revealed that synapsin I-immunoreactivity occurred diffusely throughout the cytoplasm, and heavily in association with microvesicles. The synaptic vesicles at the taste receptor cell-to-afferent fiber synapse were, however, not immunoreactive for synapsin I, although these vesicles fall into the size class shown to be immunoreactive in other systems. This absence of synapsin I may be a common property of vesicles in axonless short receptor cells.

Glucose metabolism is of vital importance in normal brain function. Evidence indicates that glycolysis, in addition to production of ATP, plays an important role in maintaining normal synaptic function. In an effort to understand the potential involvement of a glycolytic intermediate(s) in synaptic function, we have prepared [3-32P]1,3-bisphosphoglycerate and [32P]3-phosphoglycerate and sought their interaction with a specific nerve-ending protein. We have found that a 29-kDa protein is the major component labeled with either [3-32P]1,3-bisphosphoglycerate or [32P]3-phosphoglycerate. The protein was identified as monophosphoglycerate mutase (PGAM). This labeling was remarkably high in the brain and synaptosomal cytosol fraction, consistent with the importance of glycolysis in synaptic function. Of interest, fructose-2,6-bisphosphate (Fru-2,6-P2) inhibited PGAM phosphorylation and enzyme activity. Moreover, Fru-2,6-P2 potently stimulated release of [32P]phosphate from the 32P-labeled PGAM (EC50 = 1 microM), suggesting that apparent reduction of PGAM phosphorylation and enzyme activity by Fru-2,6-P2 may be due to stimulation of dephosphorylation of PGAM. The significance of these findings is discussed.

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, [(3)H]glutamate uptake into isolated synaptic vesicles. It also decreased vesicular [(3)H]glutamate content in the nerve ending preparation synaptosome; this decrease was reflected in reduction of depolarization-induced [(3)H]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.

Certain forms of seizure involve excessive glutamate transmission. We have recently identified a protein, referred to as the inhibitory protein factor (IPF), which potently inhibits glutamate uptake into isolated synaptic vesicles. In an effort to understand the mechanism underlying excessive glutamate transmission associated with seizure, we have analyzed IPF content in various brain regions of the spontaneously epileptic rat, SER (tm/tm, zi/zi), the absence-seizure tremor rat, TM (tm/tm), and the seizure-free control rats zitter ZI (zi/zi) and Wistar tremor control, each at 13 weeks of age. IPF content was found to be markedly reduced in the hippocampus, but not in the other brain regions, of SER, compared to the control and TM rats. TM rats also exhibited reduced IPF content compared to seizure-free controls. These changes appear developmentally regulated; no such alteration was observed in 8-week-old rats, which rarely show seizure. These observations indicate that an aberrant decrease in IPF is associated with certain forms of seizure; this decrease could lead to an abnormal increase in the amount of exocytotically released glutamate through its excessive accumulation in synaptic vesicles.

Glutamate accumulation into synaptic vesicles is a vital step in glutamate synaptic transmission. In this study, we have explored the possibility that vesicular glutamate storage may be subject to some regulation. Synaptosomes were depolarized and subjected to [3H] glutamate under non-depolarizing conditions, and vesicular [3H] glutamate content was determined by a filter-based assay. We present evidence here that prolonged depolarization of synaptosomes leads to an increase in vesicular glutamate content. Induction of this enhanced state is time- and temperature-dependent. The enhanced state has two components, one readily reversible and the other long-lasting. The up-regulation of glutamate storage capacity could lead to an increase in quantal size and play a role in modulation of glutamate transmission efficiency.

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [(3)H]L-glutamate (Glu) content within the synaptosome (a pinched-off nerve ending preparation) leads to a change in the amount of exocytotically released [(3)H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane-permeant inhibitor (K(i) = 19 nM) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H(+)-pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [(3)H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [(3)H]Glu released in a depolarization- (induced by 4-aminopyridine) and Ca(2+)-dependent manner. In contrast, fluorescein, the halogen-free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [(3)H]Glu uptake, failed to change the amount of exocytotically released [(3)H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.

Glutamate plays an important metabolic role in virtually every vertebrate cell. In particular, glutamate is the most common excitatory neurotransmitter in the vertebrate central nervous system. As such, the mechanism by which glutamate is diverted from its normal metabolic activities toward its role as a neurotransmitter has, in recent years, been systematically investigated. In glutamatergic nerve endings, synaptic vesicles accumulate and store a proportion of the cellular glutamate pool and, in response to appropriate signals, release glutamate into the synaptic cleft by exocytosis. Glutamate accumulation is accomplished by virtue of a glutamate uptake system present in the synaptic vesicle membrane. The uptake system consists of a transport protein, remarkably specific for glutamate, and a vacuolar-type H+-ATPase, which provides the coupling between ATP hydrolysis and glutamate transport. The precise manner in which the glutamate transporter and H+-ATPase operate is currently the subject of debate. Recent data relevant to this debate are reviewed in this article. Additionally, pharmacological agents thought to specifically interact with the vesicular glutamate transporter are discussed. Finally, a newly discovered, endogenous inhibitor of vesicular uptake, inhibitory protein factor (IPF), is discussed with some speculations as to its potential role as a presynaptic modulator of neurotransmission.

Publisher Summary This chapter reviews the solubilization and reconstitution of the vesicular glutamate transport system and describes an extension of these techniques, resulting in a partial purification of the vesicular glutamate transport system and a partial separation of the glutamate transporter and proton-pump ATPase. As these techniques require a minimum of 10–20 mg synaptic vesicle protein, the chapter discusses the synaptic vesicle purification protocol for large-scale preparation of vesicles. The synaptic vesicle glutamate transport system performs a key function in neurotransmission by loading the synaptic vesicle with glutamate. Given its vital role in glutamate neurotransmission, directing glutamate to the neurotransmitter pathway away from the metabolic pathway, it potentially represents an important site of regulation of central nervous system pathogenesis. Synaptic vesicle glutamate transport requires a proton ATPase of the vacuolar type and chloride. These function to provide the appropriate intravesicular pH and electrochemical gradients that are necessary to drive glutamate transport. It is not known whether chloride stimulated, ATP-dependent glutamate transport is accomplished by several proteins, each functioning independently, or if more than one function is performed by one protein.

The ATP-dependent glutamate uptake system of synaptic vesicles was investigated in epileptic (EL) mice to determine whether glutamate uptake activity correlates with seizure susceptibility or development. Given the focal seizure onset, glutamate uptake activity was measured in four separate brain regions: cerebrum (minus hippocampus), hippocampus, cerebellum, and brain stem. The EL values were compared to those of age-matched controls; DDY and ABP/LeJ (ABP) mice. The glutamate uptake specific activity for EL cerebrum was significantly higher than that for the control mice (approx. 400 days old), but was not elevated prior to seizure onset (46 days old). No difference in glutamate uptake was observed between the strains in the other brain regions. We conclude that increased synaptic vesicle glutamate uptake is brain-region specific (cerebrum) and is associated with the development or maintenance, rather than the initial cause, of seizures in the EL model of epilepsy.

Glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, is transported into and stored in synaptic vesicles. We have purified to apparent homogeneity a protein from brain cytosol that inhibits glutamate and γ-aminobutyric acid uptake into synaptic vesicles and have termed this protein “inhibitory protein factor” (IPF). IPF refers to three distinct proteins with relative molecular weights of 138,000 (IPF α), 135,000 (IPF β), and 132,000 (IPF γ), respectively. Gel filtration and sedimentation data suggest that all three proteins share an elongated structure, identical Stokes radius (60 Å), and identical sedimentation coefficient (4.3 S). Using these values and a partial specific volume of 0.716 ml/g, we determined the native molecular weight for IPF α to be 103,000. Partial sequence analysis shows that IPF α is derived from α fodrin, a protein implicated in several diverse cellular activities. IPF α inhibits ATP-dependent glutamate uptake into purified synaptic vesicles with an IC 50 of ≈26 nM, while showing no ability to inhibit ATP-independent uptake at concentrations up to 100 nM. Moreover, IPF α inhibited neither norepinephrine uptake into chromaffin vesicles nor Na + -dependent glutamate uptake into synaptosomes. However, IPF α inhibited uptake of γ-aminobutyric acid into synaptic vesicles derived from spinal cord, suggesting that inhibition may not be limited to glutamatergic systems. We propose that IPF could be a novel component of a presynaptic regulatory system. Such a system might modulate neurotransmitter accumulation into synaptic vesicles and thus regulate the overall efficacy of neurotransmission.

We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate [( 1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [gamma-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP72 preparation.

and to require a heat-labile cytosolic macromolecule factor for maximum activity. Maximal release occurred at 5/zM free Ca 2+ and within 5 min. Of the other divalent cations tested, only barium stimulated release of vesicular glutamate. The release was inhibited by N-ethylmaleimide. results are characteristic of exocytotic release of monoamines and peptides observed in endocrine systems, and constitute direct evidence for the notion that calcium-dependent release of glutamate originates from the vesicular pool. Amassed evidence indicates that glutamate serves as a major excitatory neurotransmitter in the vertebrate central nervous system (for review, see ref. 4). Despite the increased recognition of the neurotransmitter role of glutamate, the mechanism by which glutamate is released from the nerve terminal remains in large part to be elucidated. Substantial evidence now has been accumulated that glutamate is specifically taken up into highly purified isolated synaptic vesicles in an ATP-dependent manner [6, 8, 12, 15-18, 22], suggesting that glutamate could be accumulated and stored in synaptic vesicles in vivo. This is in accord with immunocytochemical evidence provided by Storm-Mathisen et al. [21] suggesting that glutamate is concentrated in certain synaptic vesicles distinct from ~,-aminobutyric acid (GABA)-containing vesicles. Recent evidence has indicated that glutamate released in a calcium-dependent manner originates from a non-cytoplasmic pool [20]. These lines of evidence argue for the involvement of synaptic vesicles in synaptic release of glutamate. In this study, we have investigated the calcium-dependent release of glutamate from the synaptic vesicles in a more direct manner, using a permeabilized synaptosome preparation. Evidence is presented that glutamate is indeed released directly from the synaptic vesicle in a calcium-dependent manner, in the presence of a cytosolic factor.

We have demonstrated previously that L-glutamate is taken up into isolated synaptic vesicles in an ATP-dependent manner, supporting the neurotransmitter role of this acidic amino acid. We now report that a nerve terminal cytosolic factor inhibits the ATP-dependent vesicular uptake of glutamate in a dose-dependent manner. This factor appears to be a protein with a molecular weight greater than 100,000, as estimated by size exclusion chromatography, and is precipitated by ammonium sulfate (40% saturation). The inhibitory factor is inactivated by heating to 100 degrees C. Proteolytic digestion of the ammonium sulfate fraction by trypsin or chymotrypsin did not reduce, but rather increased slightly, the inhibition of glutamate uptake. Unlike the native factor, the digest retained inhibitory activity after heating, suggesting that proteolytic digestion may generate active fragments. The inhibition of ATP-dependent vesicular glutamate uptake is not species-specific, as the factor obtained from both rat and bovine brains produced an equal degree of inhibition of glutamate uptake into vesicles of each species. These observations raise the possibility that vesicular uptake of glutamate may be regulated by an endogenous factor in vivo.

Protein I is a synaptic protein which serves as an endogenous substrate for cyclic AMP- and calcium-dependent protein kinases. Antibodies raised against purified Protein I have been isolated from rabbit antiserum by affinity chromatography on Protein I-conjugated agarose column. The purified antibodies were identified as immunoglobulin G by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Ouchterlony double immunodiffusion precipitation test. The purified antibodies not only inhibited the phosphorylation of purified Protein I by exogenous cyclic AMP-dependent protein kinase, but also inhibited specifically the phosphorylation of Protein I by endogenous cyclic AMP-dependent protein kinase in a synaptic vesicle fraction, synaptic junctional complex fraction, synaptic membrane fraction, and crude homogenate of cerebrum. The purified anti-Protein I antibodies also inhibited with a similar potency calcium-dependent phosphorylation of Protein I without affecting the phosphorylation of other proteins. The Fab(t) fragment of anti-Protein I immunoglobulin G, which was produced by tryptic digestion, retained the ability to inhibit specifically the phosphorylation of Protein I. This substrate-directed, specific inhibitor of the phosphorylation of Protein I may provide a unique probe for investigating the function of Protein I phosphorylation.

Protein I, a naturally occurring substrate for cyclic AMP-dependent and calcium-dependent protein kinases, previously has been found only in mammalian brain, where it has been demonstrated to be located in neurons. Various tissues and organs outside the brain have now been examined for the possible occurrence of protein I, by using both an immunohistochemical approach and a chemical procedure involving radioimmunolabeling of polyacrylamide gels. Protein I has been found in the inner plexiform layer of the retina, in the posterior pituitary, and in the autonomic nervous system. In tissue composed predominantly of cells other than nerve cells, immunoreactivity was present only where innervation was present. Protein I appeared to be localized in some, but not all, nerve terminals and synapses.

The ATP-dependent uptake of l-glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the pep-tide-containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested; the IC5o was de-termined to be 22 μM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate trans-locator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.

The purified synapse-specific phosphoprotein Protein I was previously shown to be degraded by a bacterial collagenase, through a series of intermediates, to a collagenase-resistant fragment of molecular weight about 48,000 containing a phosphorylated serine residue. In this study, a purified synaptic membrane fraction containing Protein I was treated with Cl. his-tolyticum collagenase; membrane-bound and membrane-free proteins were then phosphorylated using [γ-32P]ATP and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. It was observed that Protein I bound to the synaptic membrane was susceptible to the collagenase and degraded to fragments of molecular weights about 68,000, 62,000, and 48,000; the 68,000 fragment remained bound to the membrane whereas the 62,000 and 48,000 fragments were dissociated from the membrane. These observations suggest that the peptide moiety of mol. wt. 6000, present in the 68,000 fragment but absent from the 62,000 fragment, may play a crucial role in anchoring Protein I to the synaptic membrane.

Although gamma-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study we have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. We present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity (Km in the millimolar range) similar to vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.

An endogenous substrate for adenosine 3':5'-monophosphate-dependent protein kinase has been solubilized, and purified about 5,000-fold to apparent homogeneity, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes. This endogenous substrate, referred to as Protein I, is apparently specific to nervous tissue, and is composed of two types of polypeptides, present in a proportion of 1 (Protein Ia, 86,000 daltons) to 2 (Protein Ib, 80,000 daltons). In the presence of cAMP-dependent Protein I kinase purified from the same membrane fractions, Proteins Ia and Ib incorporated 0.83 and 0.81 mol of phosphate into serine/mol of peptide, respectively. Proteins Ia and Ib have similar amino acid compositions and have isoelectric points of 10.3 and 10.2, respectively. Both types of polypeptide have a relatively high content of glycine and proline, and both are degraded to a peptide of 48,000 daltons by highly purified collagenase, suggesting that Proteins Ia and Ib contain some sequences similar to those observed in collagen. The sedimentation coefficient of Protein Ia and Protein Ib was determined to be 2.9 S. The data suggest that both Protein Ia and Protein Ib have an elongated shape.

The ontogeny of two endogenous substrates for cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase; EC 2.7.1.37) has been studied in rat and guinea pig cerebrum. These endogenous substrates, referred to as proteins Ia and Ib, have been shown in other studies to be specific to nervous tissue and to be enriched in the synaptic membrane and synaptic vesicle fractions of adult brain. In the present study, proteins Ia and Ib were shown to increase markedly during the time of major synaptogenesis in rat cerebrum and guinea pig cerebrum, in which the morphological development of synapses is predominantly postnatal and prenatal, respectively. Similar results were obtained either by measuring endogenous phosphorylation of proteins Ia and Ib in the synaptic membrane fraction or by measuring phosphorylation of extracted proteins Ia and Ib with added protein kinase.

We have previously provided evidence for ATP-dependent glutamate uptake into synaptic vesicles, and, based upon the unique properties of the vesicular uptake system, we have proposed that the vesicular glutamate translocator plays a crucial role in selecting glutamate for neurotransmission. In this study, we have solubilized the vesicular glutamate uptake system, proposed to consist of at least a glutamate translocator and a proton pump Mg-ATPase, from rat brain synaptic vesicles, and reconstituted the functional ATP-dependent glutamate uptake system into liposomes. The glutamate uptake in the reconstituted system is dependent upon ATP, markedly potentiated by low millimolar concentrations of chloride and inhibited by agents known to dissipate electrochemical proton gradients. Moreover, it exhibited low affinity for glutamate (Km = 2 m M ), yet high specificity for glutamate; thus, it did not recognize aspartate and other agents known to interact with glutamate receptors. These properties are indistinguishable from those observed in intact synaptic vesicles. The solubilized functional components of the glutamate uptake system, alone or as a complex, have been estimated to have a Stokes radius in the range of 69 to 84 A. The reconstitution experiments described here provide a functional assay for the solubilized vesicular glutamate uptake system and represent an initial step towards the purification of the glutamate translocator.