Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

The ontogeny of the uptake systems for glutamate,GABA, and glycine in synaptic vesicles isolated from rat brain

The ontogeny of the uptake of glutamate, GABA and glycine into synaptic vesicles isolated from rat brain has been investigated. The vesicular uptake of the three amino acids increased with developmental age in parallel with synaptogenesis, indicating a functional role of uptake of the amino acids by synaptic vesicles in the nerve terminals. Uptake of the amino acids by plasma membrane particles (synaptosomes) in brain homogenate showed a somewhat different developmental profile. The uptake of glutamate increased markedly with developmental time, while the uptake of GABA showed only a slight increase. Uptake of glycine by plasma membrane particles was very low and therefore not registered. The observed developmental increase in uptake of glycine by synaptic vesicles isolated from brain, supports previous reports indicating that glycine can be taken up by vesicles from non-glycine terminals.Special issue dedicated to Dr. Morris H. Aprison.     

Adenosine triphosphate-dependent uptake of glutamate into protein I-associated synaptic vesicles

Protein I is a neuron-specific, synaptic phosphoprotein highly localized on the surface of synaptic vesicles. We have recently isolated anti-Protein I IgG by affinity chromatography and shown that these antibodies inhibit specifically the phosphorylation of Protein I (Naito, S., and Ueda, T. (1981) J. Biol. Chem. 256, 10657-10663). In an effort to characterize Protein I-associated synaptic vesicles with respect to the types of neurotransmitters, we have now developed a procedure, using the affinity-purified anti-Protein I IgG, which allows immunoprecipitation of those synaptic vesicles which contain Protein I. The isolated vesicles are largely free of contamination from other intracellular organelles and plasma membranes. We present evidence that these vesicles isolated from bovine cortex are able to accumulate L-glutamate specifically in an ATP-dependent, temperature-dependent but Na-independent manner. Thus, the structurally similar aminoacid neurotransmitters aspartate and gamma-aminobutyric acid, as well as other neurotransmitters such as dopamine, norepinephrine, serotonin, acetylcholine, and glycine, failed to show a significant ATP-dependent uptake into these vesicles. Moreover, the ATP-dependent glutamate uptake was not inhibited effectively by glutamine, aspartate, or gamma-aminobutyric acid. The ATP-dependent glutamate uptake requires ATP hydrolysis; thus there was little accumulation of glutamate in the absence of ATP or Mg2+, or when ATP was replaced by an unhydrolyzable beta, gamma-methylene ATP analog. The glutamate uptake appears to be driven at least in part by a membrane potential generated by Mg2+-ATPase, similar to that of the catecholamine and serotonin uptakes into storage granules. These observations suggest that Protein I may be involved in some aspect of the function of glutamate-containing synaptic vesicles in the brain.     

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.     

An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.     

The content of GABA and other amino acids in bovine cerebral cortex synaptic vesicles

The content of γ-amino butyric acid (GABA) and of other water soluble amino acids in bovine brain synaptic vesicles was determined by a modified automated amino acid analysis method. Following subcellular fractionation, GABA, glutamate and aspartate were distributed largely in the supernatant fractions and in the upper layer of the sucrose gradient. Only 10–20% of the total content was associated with the vesicular fraction. On the other hand, the other water soluble amino acids, such as serine, glycine and alanine, were evenly distributed between cytoplasmic and particulate fractions in a similar pattern to that observed with cytoplasmic enzyme markers. The results may indicate specific association of GABA, glutamate and aspartate with low density particles or cytoplasmic components.     

Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.     

Turnover rates of amino acid neurotransmitters in regions of rat cerebellum

The turnover rates of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, alanine, serine, and glycine were measured in five regions of rat cerebellum. Turnover rates of the putative neurotransmitters (aspartate, glutamate, and GABA) were 2-20-fold higher than those of alanine and serine, and generally consistent with the proposed neurotransmitter functions for these amino acids. However, glutamate turnover was high and similar in magnitude in the deep nuclei and granule layer, suggesting possible release, not only from parallel fibers, but from mossy fibers as well. The differential distribution of turnover rates for GABA supports its neuronal release by Purkinje, stellate, basket, and Golgi cells, whereas aspartate may be released by both climbing and mossy fibers. The distribution of glycine turnover rates is consistent with release from Golgi cells, whereas alanine may be released from granule cell parallel fibers. Turnover rates measured in two other motor areas, the striatum and motor cortex, indicated that utilization of these amino acid neurotransmitters is differentially distributed in brain motor regions. The data indicate that turnover rate measurements may be useful in identifying neurotransmitter function where content measurements alone are insufficient.     

Uptake of GABA by rat brain synaptic vesicles isolated by a new procedure.

Uptake of GABA was demonstrated in rat brain synaptic vesicles which were prepared by a new and efficient procedure. The uptake activity co-purified with the synaptic vesicles during the isolation procedure. The purity of the vesicle fraction was rigorously examined by analysis of marker enzymes and marker proteins and also by immunogold electron microscopy using antibodies against p38 (synaptophysin). Contamination by other cellular components was negligible, indicating that GABA uptake by the synaptic vesicle fraction is specific for synaptic vesicles and not due to the presence of other structure possessing GABA uptake or binding activities. GABA uptake was ATP dependent and similar to the uptake of glutamate, which was assayed for a comparison. Both uptake activities were independent of sodium. They were inhibited by the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, indicating that the energy for the uptake is provided by an electrochemical proton gradient. This gradient is generated by a proton ATPase of the vacuolar type as suggested by the effects of various ATPase inhibitors on neurotransmitter uptake and proton pumping. Competition experiments revealed that the transporters for GABA and glutamate are selective for the respective neurotransmitters.     

Models of active transport of neurotransmitters in synaptic vesicles

Models of the active transport of neurotransmitters in synaptic vesicles were constructed. The models were used to determine the resting potential at membranes of synaptic vesicles: 40mV (monoamines and acetylcholine) and -40mV (glutamate). The potential at the membrane of a synaptic vesicle was almost absent for the transport of GABA and glycine. The neurotransmitter concentration of a cell was 0.1-18mM at the concentration of neurotransmitters in a vesicle equal to 0.5M. This result is in qualitative agreement with the relevant experimental data.     

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.     

The amphicrine pancreatic cell line, AR42J, secretes GABA and amylase by separate regulated pathways.

Treatment of AR42J cells with dexamethasone leads to an enhanced formation of amylase-containing granules and facilitates their regulated secretion. Besides the exocrine properties, AR42J cells possess a specific uptake system for [3H]GABA. The stored GABA can be released upon potassium depolarisation in a Ca(2+)-dependent manner. After treatment with dexamethasone, potassium depolarisation fails to release GABA, but instead causes a Ca(2+)-dependent secretion of amylase. Since vesicles similar to small synaptic vesicles of neurons have been identified in AR42J cells, we suggest that the regulated GABA release is mediated by this vesicle type. It is tentatively speculated that other epithelial cells, which also contain small synaptic vesicles and amino acid neurotransmitters, may release them in a similar fashion.     

IPF, a vesicular uptake inhibitory protein factor, can reduce the Ca(2+)-dependent, evoked release of glutamate, GABA and serotonin

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.     

GABAergic Growth Cones: Release of Endogenous γ-Aminobutyric Acid Precedes the Expression of Synaptic Vesicle Antigens

Growth cone fractions isolated from neonatal [postnatal day 3 (P3)] rat forebrain contain GABAergic growth cones as demonstrated by immunofluorescence staining with monospecific antibodies to gamma-aminobutyric acid (GABA). HPLC analysis shows that GABAergic growth cones release this endogenous GABA when stimulated with high K+. Endogenous GABA release is Ca2(+)-independent and, in this respect, similar to that seen previously with [3H]GABA. Isolated growth cone fractions also exhibit a K(+)-stimulated, Ca2(+)-independent release of endogenous taurine. None of the other amino acids shown to be present in isolated growth cone fractions were released, including glutamate, aspartate, and glycine. A population of dissociated cerebral cortical neurones prepared from P1 rat forebrain were GABA-immunoreactive after 1 day in culture. The cell body, neurites, and growth cones of these neurones were all stained with GABA antibodies. At this time in culture, neurones did not stain with either of two antibodies to synaptic vesicle antigens, i.e., p65 and synaptophysin. Growth cones isolated from P3 rat forebrain were also not immunoreactive with these antibodies. After about 8 days in culture, when neurones had established extensive networks of long, varicose axons and elaborately branched dendrites, many neurones and their neurites were immunoreactive for GABA antibodies. At this time in culture, p65 and synaptophysin antibodies did stain neuronal cell bodies and particularly their varicose axons. Dendrites were not stained with synaptic vesicle antibodies. These results suggest that GABAergic neurones synthesize GABA during neurite outgrowth and that GABA is present in, and can be released from, the growth cones of these neurones. The presence of GABA in GABAergic growth cones is not associated with synaptic vesicles, which explains the Ca2+ independency of both endogenous and [3H]GABA release from these growth cones.     

Synaptic Neurotransmitter-Gated Receptors

Since the discovery of the major excitatory and inhibitory neurotransmitters and their receptors in the brain, many have deliberated over their likely structures and how these may relate to function. This was initially satisfied by the determination of the first amino acid sequences of the Cys-loop receptors that recognized acetylcholine, serotonin, GABA, and glycine, followed later by similar determinations for the glutamate receptors, comprising non-NMDA and NMDA subtypes. The last decade has seen a rapid advance resulting in the first structures of Cys-loop receptors, related bacterial and molluscan homologs, and glutamate receptors, determined down to atomic resolution. This now provides a basis for determining not just the complete structures of these important receptor classes, but also for understanding how various domains and residues interact during agonist binding, receptor activation, and channel opening, including allosteric modulation. This article reviews our current understanding of these mechanisms for the Cys-loop and glutamate receptor families.To understand how neurons communicate with each other requires a fundamental understanding of neurotransmitter receptor structure and function. Neurotransmitter-gated ion channels, also known as ionotropic receptors, are responsible for fast synaptic transmission. They decode chemical signals into electrical responses, thereby transmitting information from one neuron to another. Their suitability for this important task relies on their ability to respond very rapidly to the transient release of neurotransmitter to affect cell excitability.In the central nervous system (CNS), fast synaptic transmission results in two main effects: neuronal excitation and inhibition. For excitation, the principal neurotransmitter involved is glutamate, which interacts with ionotropic (integral ion channel) and metabotropic (second-messenger signaling) receptors. The ionotropic glutamate receptors are permeable to cations, which directly cause excitation. Acetylcholine and serotonin can also activate specific cation-selective ionotropic receptors to affect neuronal excitation. For controlling cell excitability, inhibition is important, and this is mediated by the neurotransmitters GABA and glycine, causing an increased flux of anions. GABA predominates as the major inhibitory transmitter throughout the CNS, whereas glycine is of greater importance in the spinal cord and brainstem. They both activate specific receptors—for GABA, there are ionotropic and metabotropic receptors, whereas for glycine, only ionotropic receptors are known to date.Together with acetylcholine- and serotonin-gated channels, GABA and glycine ionotropic receptors form the superfamily of Cys-loop receptors, which differs in many aspects from the superfamily of ionotropic glutamate receptors. Over the last two decades, our knowledge of the structure and function of ionotropic receptors has grown rapidly. In this article, we summarize our current understanding of the molecular operation of these receptors and how we can now begin to interpret the role of receptor structure in agonist binding, channel activation, and allosteric modulation of Cys-loop and glutamate receptor families. Further details on the regulation and trafficking of neurotransmitter receptors in synaptic structure and plasticity can be found in accompanying articles.     

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.     

Depolarization-Induced Release of Amino Acids From the Vestibular Nuclear Complex

There is evidence from immunohistochemistry, quantitative microchemistry, and pharmacology for several amino acids as neurotransmitters in the vestibular nuclear complex (VNC), including glutamate, γ-aminobutyrate (GABA), and glycine. However, evidence from measurements of release has been limited. The purpose of this study was to measure depolarization-stimulated calcium-dependent release of amino acids from the VNC in brain slices. Coronal slices containing predominantly the VNC were prepared from rats and perfused with artificial cerebrospinal fluid (ACSF) in an interface chamber. Fluid was collected from the chamber just downstream from the VNC using a microsiphon. Depolarization was induced by 50 mM potassium in either control calcium and magnesium concentrations or reduced calcium and elevated magnesium. Amino acid concentrations in effluent fluid were measured by high performance liquid chromatography. Glutamate release increased fivefold during depolarization in control calcium concentration and twofold in low calcium/high magnesium. These same ratios were 6 and 1.5 for GABA, 2 and 1.3 for glycine, and 2 and 1.5 for aspartate. Differences between release in control and low calcium/high magnesium ACSF were statistically significant for glutamate, GABA, and glycine. Glutamine release decreased during and after depolarization, and taurine release slowly increased. No evidence for calcium-dependent release was found for serine, glutamine, alanine, threonine, arginine, taurine, or tyrosine. Our results support glutamate and GABA as major neurotransmitters in the VNC. They also support glycine as a neurotransmitter and some function for taurine.     

Membrane glycine transport proteins

Structurally, the simplest amino acid is glycine, and it has a number of important yet distinct functions in the body. This review focuses on the different transport systems and the associated carrier proteins for glycine that are responsible for its movement across biological membranes. Transport proteins in the class GLYT appear to be the most specific for glycine. However, the B0+ system also carries significant amounts of glycine. Other amino acid transport systems capable of carrying small amounts of glycine are ASC, asc and system L. In addition, an ATP-dependent transport process exists that takes up glycine into synaptic vesicles at nerve endings. This is known as the vesicular inhibitory amino acid transporter since, in addition to glycine, it can transport possibly two other inhibitory neurotransmitters.     

Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina

Abstract: The effects of "ischaemia" (glucose-free Krebs-bicarbonate medium gassed with N₂/CO₂ on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20-fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia-induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 m M ) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia-induced release do not involve an initial release of K or an influx of calcium.     

Taurine,amino acid transmitters,and related molecules in the retina of the Australian lungfish Neoceratodus forsteri: a light-microscopic immunocytochemical and electron-microscopic study

The morphology of the retina of the Australian lungfish Neoceratodus forsteri was investigated by means of light- and electron microscopy, whilst immunocytochemical studies were performed to determine the cellular distributions of the major amino acid neurotransmitters and other amino acids. The distributions of glycine and GABA were similar to those previously described for teleost, amphibian and mammalian retinae. Labelling was abundant in amacrine cells, whilst GABA was also present in one layer of horizontal cells and some bipolar cells. Taurine was present in both rods and cones, but, unlike the mammalian or avian retina, was absent from other cellular structures, including glial elements. Unexpectedly, the photoreceptor terminals lacked an apparent content of the excitatory amino acid transmitter glutamate. The glutamate that was present in the rods and cones occupied a crescentic arc corresponding to the location of glycogen-rich paraboloids. Asparagine was also present in rods, albeit in the modified mitochondria that formed the elipsoids of the rod inner segments. Arginine, the precursor for formation of nitric oxide, was present in glial cells, and in the paraboloids of both rods and cones.