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PubMed1990

Rapid activation, desensitization, and resensitization of synaptic channels of crayfish muscle after glutamate pulses.

Completely desensitizing excitatory channels were activated in outside-out patches of crayfish muscle membrane by applying glutamate pulses with switching times of approximately 0.2 ms for concentration changes. Channels were almost completely activated with 10 mM glutamate. Maximum activation was reached within 0.4 ms with greater than or equal to 1 mM glutamate. Channel open probability decayed with a time constant of desensitization of 2 ms with 10 mM glutamate and more rapidly at lower glutamate concentrations. The rate of beginnings of bursts (average number of beginnings of bursts per time bin) decayed even faster but approximately in proportion to the glutamate concentration. The dose-response curve for the channel open probability and for the rate of bursts had a maximum double-logarithmic slope of 5.1 and 4.2, respectively. Channels desensitized completely without opening at very low or slowly rising glutamate concentrations. Desensitization thus originates from a closed channel state. Resensitization was tested by pairs of completely desensitizing glutamate pulses. Sensitivity to the second pulse returned rapidly at pulse intervals between 1 and 2 ms and was almost complete with an interval of 3 ms. Schemes of channel activation by up to five glutamate binding steps, with desensitization by glutamate binding from closed states, are discussed. At high agonist concentrations bursts are predominantly terminated by desensitization. Quantal currents are generated by pulses of greater than 1 mM glutamate, and their decay is determined by the duration of presence of glutamate and possibly by desensitization.

Animals↗
PubMed1996

Involvement of the glutamatergic metabotropic receptors in the regulation of glutamate uptake and extracellular excitatory amino acid levels in the striatum of chloral hydrate-anesthetized rats.

The microdialysis technique was used to assess in vivo the putative functional role of metabotropic excitatory amino acid receptors in regulating extracellular levels of the excitatory amino acids glutamate and aspartate in the striatum of chloral hydrate-anesthetized rats. Addition of the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG) (10(-3) or 4 x 10(-3) M) in the dialysis probe did not modify the basal extracellular levels of glutamate and aspartate but induced a significant dose-dependent decrease in the KCl-elicited elevation of glutamate and aspartate extracellular levels. The effect of MCPG on glutamate extracellular concentration under K+ stimulation was reduced by the simultaneous superfusion of the metabotropic glutamate receptor agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine) (L-CCG) (10(-3) M) which had no significant effect when tested alone. In contrast, L-CCG alone significantly potentiated the KCl-elicited elevation of aspartate extracellular concentrations but failed to modify the MCPG effect on this amino acid concentration. In a parallel series of experiments, high-affinity glutamate uptake was measured ex vivo 20 min after an in vivo injection of 10 pmol of MCPG in the striatum. MCPG was found unable to modify the glutamate uptake rate. In vitro, MCPG (1-1000 microM) again had no effect on glutamate transport rate. These data suggest that metabotropic excitatory amino acid receptors (1) may act to increase the extracellular levels of glutamate and aspartate under depolarizing conditions, and (2) may not have a major role in the regulation of high affinity glutamate uptake under basal conditions. In addition, it can be assumed that the control of glutamate and aspartate extracellular levels may involve different metabotropic receptors.

Amino Acids, Dicarboxylic↗
PubMed1996

Vulnerability of CA1 neurons to glutamate is developmentally regulated.

Although it is well documented that glutamate receptor subtypes are differentially expressed during central nervous system development postnatally, how glutamate affects neurons during postnatal development is unclear. We therefore examined the development of the intrinsic neuronal response to glutamate receptor activation by studying single, hippocampal CA1 neurons that had been acutely dissociated from newborn (P1-3), 1 week old (P6-8), and 3 week old (P21-25) rats. Using laser scanning confocal microscopy and the calcium dye Fluo-3, we made time-lapse studies of the effects of glutamate stimulation on free intracellular calcium ([Ca2+]i) and simultaneous changes in neuronal morphology. In P21-25 neurons, glutamate increased [Ca2+]i fluorescence, and caused marked somal swelling, blebbing, and retraction of dendrites into the soma. These major morphological changes were followed by sudden loss of intracellular fluorescence, indicative of a loss of membrane integrity and cell death. In P6-8 neurons, glutamate increased [Ca2+]i to the same extent, but this increase was not followed by either major morphological changes or loss of membrane integrity. In P1-3 neurons, glutamate increased [Ca2+]i minimally, and no morphologic changes were observed. P1-3 neurons dissociated without enzymatic digestion demonstrated glutamate responses identical to responses seen in neurons dissociated with enzymatic digestion. In the presence of MK-801 (15 microM), glutamate still increased [Ca2+]i and caused cell death in P21-25 neurons, but the latency to these effects more than tripled. This late, MK-801-resistant [Ca2+]i increase was not eliminated by DNQX or Ni2+/Cd2+, suggesting that this increase is mediated by metabotropic receptors. These findings demonstrate that (1) hippocampal neurons from newborns are intrinsically less vulnerable to glutamate toxicity than neurons from 3 weeks old animals, and (2) multiple glutamate receptor subtypes affect the magnitude of the [Ca2+]i increase in response to glutamate in the neuronal microenvironment.

Aniline Compounds↗
PubMed2003

Glutamate stimulates neurotrophin expression in cultured Müller cells.

The uptake of excess extracellular glutamate and the secretion of neurotrophins by glial cells have been suggested to protect CNS neurons from glutamate-induced toxicity. In the retina, perturbation of glutamate transport and decreased retrograde transport of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) may contribute to ganglion cell death in experimental glaucoma. Although many studies show a clear relationship between glutamate and neurotrophic factors, such relationship has not been thoroughly investigated in the retinal environment. In the following study, we determined the effects of glutamate on early passaged rat Müller cells, specifically their expression of neurotrophic factors including BDNF, nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and glial-cell line derived neurotrophic factor (GDNF); and of glutamate receptors and transporters using immunoblots or enzyme-linked immunosorbent assays. Binding of BDNF to its cognate receptor TrkB was also determined using co-immunoprecipitation studies. Cultured Müller cells grown in the presence of glutamate were also assayed for survival using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). Our study showed that while glutamate treatment did not promote cell death, it upregulated secretion of BDNF, NGF, NT-3, NT-4, and GDNF by Müller cells. While solitary bands at approximately 13-14 kDa were observed for NGF, NT-3, and NT-4; two BDNF-reactive bands were observed in immunoblots: a faster migrating band at the reported size of the BDNF monomer (approximately 13 kDa); and a more intense band at approximately 36 kDa. GDNF-reactive bands were observed at approximately 22, approximately 28, and approximately 55 kDa. Glutamate also induced significant changes in glutamate receptor and transporter proteins, as well maintained the association of BDNF to TrkB in Müller cells. The decreased N-methyl-D-aspartate receptor (NMDAR) levels and sustained activation of TrkB by BDNF could serve as protective mechanisms for Müller cell survival. Moreover, the increased secretion of neurotrophic factors and upregulation of L-glutamate/L-aspartate transporter (GLAST) expression in Müller cells may protect retinal neurons from glutamate toxicity.

Amino Acid Transport System X-AG↗
PubMed2000

Calcium channels coupled to depolarization-evoked glutamate release in the myenteric plexus of guinea-pig ileum.

Glutamate is the major excitatory neurotransmitter in the CNS. The recent characterization of glutamate as a neurotransmitter in the enteric nervous system opened a new line of investigation concerning the role of glutamate in that system. The present study aimed to further characterize the enteric glutamate release and the calcium channels coupled to it. For this study the myenteric plexus-longitudinal muscle of guinea-pig ileum was stimulated with potassium chloride or with electrical pulses. The released glutamate was detected by spectrofluorimetry. Laser scanning confocal microscopy was used for analysis of immunolabeled enteric tissue for co-localization studies of calcium channels (N- and P/Q-type) and glutamate transporters (EAAC1). Here we report the effects of known Ca(2+)-channel blockers on glutamate release evoked by KCl-depolarization or electrical stimulation in the myenteric plexus. We find that N-type Ca(2+) channels control a major portion of evoked glutamate release from this system, with a very small contribution from L-type Ca(2+) channels. Moreover, alpha(1A)-like (P-type Ca(2+) channel) and alpha(1B)-like (N-type Ca(2+ )channel) immunoreactivity co-localized with glutamate transporters in the myenteric plexus. In addition, KCl-evoked or electrically stimulated glutamate release was sensitive to omega-agatoxin IVA, in a frequency-dependent manner, suggesting that P-type channels are also coupled to the release of glutamate. We, thus, conclude that both N-type and P-type Ca(2+) channels control most of the evoked glutamate release from the enteric nervous system, as also occurs in some parts of the CNS.

Amino Acid Transport System X-AG↗
PubMed1997

The glutathione level of retinal Müller glial cells is dependent on the high-affinity sodium-dependent uptake of glutamate.

The dependence of intracellular glutathione, an important radical scavenger, on the extracellular glutamate and cystine concentration and the velocity of the high affinity sodium/glutamate transporter was studied in freshly-isolated Müller glial cells of the guinea-pig, kept in vitro for up to 11 h. To this end the relative Müller cell glutathione levels were measured using the fluorescent dye monochlorobimane, using different concentrations of glutamate and cystine in Ringer solution. In some experiments L-buthionine-[S,R]-sulfoximine, a blocker of glutathione synthesis, or L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid, inhibitors of glutamate uptake, were added. The Müller cells maintained about 80% of the normal glutathione level when maintained in Ringer solution containing 100 microM glutamate for 11 h. When under these conditions 100 microM cystine was added, the glutathione level increased to values, which were even higher than those at the beginning of the incubation period. Addition of cystine without glutamate caused a run down of the glutathione level to about 45% of the normal level, which is comparable to the run down in pure Ringer solution. Likewise, application of L-buthionine-[S,R]-sulfoximine (5 mM) lead to a strong run down of the glutathione level even in glutamate/cystine (100 microM)-containing solution. A similar suppressing effect was observed using L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid in the presence of 100 microM cystine and glutamate. We conclude that the intracellular glutamate concentration of the Müller cells is determined by the extracellular glutamate concentration and the velocity of the sodium/glutamate uptake. Consequently, cystine uptake into Müller cells, which is performed by the cystine/glutamate antiporter, is fueled by the sodium/glutamate transporter with intracellular glutamate. Both glutamate and cystine are also substrates for glutathione synthesis. The glutathione level is logically limited by the capacity of the sodium/glutamate transporter to provide glutamate intracellularly for, first, cystine uptake and, second, direct insertion into glutathione. Accordingly, the glutathione level is reduced when the sodium/glutamate transporter is blocked. Thus, a diminution of the glutathione level should be taken into consideration when the effects of sodium/glutamate uptake failure and reduced intracellular glutamate concentrations are discussed.

2-Aminoadipic Acid↗
PubMed1999

Distribution of metabotropic glutamate receptors in the parabrachial and Kölliker-Fuse nuclei of the rat.

In the present study, we analysed the distribution and cellular localization of metabotropic glutamate receptors (1alpha, 2/3, 5) in parabrachial and Kölliker-Fuse nuclei using subtype-specific antisera. Immunolabelling revealed that different nuclei express different sets of metabotropic glutamate receptors. Metabotropic glutamate receptor la immunoreactivity was found in the Kölliker-Fuse nucleus and in several parabrachial nuclei, including the waist area, lateral crescent, medial, external medial and ventral lateral nuclei. The external lateral and internal lateral parabrachial nuclei were devoid of metabotropic glutamate receptor 1alpha immunoreactivity. Metabotropic glutamate receptor 5 immunoreactivity was observed in the Kölliker-Fuse and in the medial parabrachial nuclei, while in the remaining nuclei the staining was very weak. Again, the external lateral nucleus was devoid of metabotropic glutamate receptor 5 immunoreactivity. The metabotropic glutamate receptor 2/3 antisera stained all lateral parabrachial nuclei as well as the Kölliker-Fuse nucleus, while staining in the medial parabrachial nucleus was weak. Metabotropic glutamate receptor 1alpha immunoreactivity was observed on presumed dendritic profiles, while metabotropic glutamate receptor 5 immunoreactivity was found predominantly on neuronal cell bodies. Metabotropic glutamate receptor 2/3 immunoreactivity was present as a fine, punctate immunostaining in the neuropil. Our data suggest that glutamate release in the parabrachial and Kölliker-Fuse nuclei might induce a variety of second messenger cascades, as indicated by the presence or absence of certain types of metabotropic glutamate receptors.

Animals↗
PubMed1997

Dual-enzyme assay of glutamate in single cells based on capillary electrophoresis.

A new dual-enzyme on-column reaction method combined with capillary electrophoresis has been developed for determining the glutamate content in single cells. Glutamate dehydrogenase and glutamic pyruvic transaminase were used to catalyze the glutamate reaction. Detection was based on monitoring the laser-induced fluorescence of the reaction product NADH, and the measured fluorescence intensity was related to the concentration of glutamate in each cell. Glutamate dehydrogenase catalyzed the formation of NADH, and glutamic pyruvic transaminase drives the glutamate dehydrogenase reaction by removing a reaction product and regenerating glutamate. The detection limit of glutamate is down to the 10(-8) M level, which is 1 order of magnitude lower than previously reported detection limits based on similar detection methods. The mass detection limit of a few attomoles is far superior to that of any other reports. Selectivity for glutamate is excellent over most amino acids. The glutamate content in single human erythrocytes and baby rat brain neurons were determined with this method and the results agreed well with literature values.

Adult↗
PubMed2000

A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

Modulation of human glutamate transporter activity by phorbol ester.

Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na+-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na+-dependent glutamate uptake system with a Km of 1.7 +/- 0.42 microM that is inhibited by dihydrokainate and DL-threo-beta-hydroxyaspartate (IC50 = 0.29 +/- 0.17 microM and 2.0 +/- 0.43 microM, respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC50 = 0.56 +/- 0.05 nM), but did not affect Na+-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the Vmax. 4Alpha-phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.

ATP-Binding Cassette Transporters↗
PubMed2004

Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol.

A co-ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia-derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up-regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM-induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol-GCM) and was restored when cholesterol was added to low cholesterol-GCM. This demonstrates that glia-derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.

Amino Acid Transport System X-AG↗
PubMed1999

Clonidine inhibits and phorbol acetate activates glutamate release from rat spinal synaptoneurosomes.

UNLABELLED: Glutamate is a major neural transmitter of noxious stimulation in the spinal cord. We measured glutamate release from rat spinal synaptoneurosomes by using an enzyme-linked fluorimetric assay. Glutamate was released from spinal cord synaptoneurosomes in response to the addition of 30 mM potassium chloride, 1 mM 4-aminopyridine, or 1 microM ionomycin in the presence of external calcium. There was less release of glutamate in the absence, versus the presence, of external calcium. Clonidine significantly reduced the level of glutamate released from the spinal cord synaptoneurosomes. Tetradecanoyl phorbol acetate, an activator of protein kinase C, enhanced glutamate release. Forskolin, a protein kinase A activator, had no effect on the glutamate efflux. Our data indicate that glutamate released in the spinal cord is dependent on protein kinase C but is independent of the protein kinase A pathway. They also suggest that the inhibition of glutamate release may be the underlying mechanism of antinociception by clonidine at the spinal cord level. IMPLICATIONS: We demonstrated that synaptoneurosomes from rat spinal cord could release glutamate in response to depolarization. We showed that an activator of protein kinase C increased glutamate released from spinal cord synaptoneurosomes but that clonidine decreased it. Glutamate release may be one of the mechanisms of antinociception at the spinal cord level.

4-Aminopyridine↗
PubMed1985

Characterization of glutamate uptake into synaptic vesicles.

Recent evidence indicates that L-glutamate is taken up into synaptic vesicles in an ATP-dependent manner, supporting the notion that synaptic vesicles may be involved in glutamate synaptic transmission. In this study, we further characterized the ATP-dependent vesicular uptake of glutamate. Evidence is provided that a Mg-ATPase, not Ca-ATPase, is responsible for the ATP hydrolysis coupled to the glutamate uptake. The ATP-dependent glutamate uptake was inhibited by agents known to dissipate the electrochemical proton gradient across the membrane of chromaffin granules. Hence, it is suggested that the vesicular uptake of glutamate is driven by electrochemical proton gradients generated by the Mg-ATPase. Of particular interest is the finding that the ATP-dependent glutamate uptake is markedly stimulated by chloride over a physiologically relevant, millimolar concentration range, suggesting an important role of intranerve terminal chloride in the accumulation of glutamate in synaptic vesicles. The vesicular glutamate translocator is highly specific for L-glutamate, and failed to interact with aspartate, its related agents, and most of the glutamate analogs tested. It is proposed that this vesicular translocator plays a crucial role in determining the fate of glutamate as a neurotransmitter.

Adenosine Triphosphate↗
PubMed1989

Depolarization increases chloride-dependent glutamate sequestration in synaptic membranes of rat cerebral cortex.

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.

Aconitine↗
PubMed1999

Intracellular survival pathways against glutamate receptor agonist excitotoxicity in cultured neurons. Intracellular calcium responses.

Cultured rat cerebellar granule cells are resistant to the excitotoxic effects of N-methyl-D-aspartate (NMDA) and non-NMDA receptor agonists under three conditions: 1) prior to day seven in vitro when cultured in depolarizing concentrations of potassium [25 mM]; 2) at any time in vitro when cultured in non-depolarizing concentrations of potassium 5 mM[; and 3) when neurons, cultured in depolarizing concentrations of potassium 25 mM[ for eight days in vitro, are pretreated with a subtoxic concentration of NMDA. The focus of this paper is to determine: a) whether the resistance to excitotoxicity by NMDA and non-NMDA receptor agonists is due to a decreased intracellular calcium Ca++[i response to glutamate receptor agonists in cultured rat cerebellar granule cells; or b) whether Ca++[i levels induced by the agonists are similar to those observed under excitotoxic conditions. Granule cells, matured in non-depolarizing growth medium, treated with glutamate resulted in an increase in Ca++[i followed by a plateau that remained above baseline in virtually all neurons that responded to glutamate. The response was rapid in onset (< 10 sec) and the pattern of response heterogeneous in that cells responsive to glutamate increased their Ca++[i to different extents; some cells did not respond to glutamate. Kainate also produced significant elevations in Ca++[i. The Ca++[i response to glutamate in neurons matured in depolarizing (25 mM K+) growth medium for three days was rapid, transient and heterogeneous, which reached a plateau that was elevated above baseline levels; removing the glutamate markedly reduced the Ca++[i concentration. Activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors by kainic acid produced similar changes in Ca++[i responses. At a time when cultured cerebellar granule cells become susceptible to the excitotoxic effects of glutamate acting at NMDA receptors (day in vitro (DIV) 8) in depolarizing growth medium, glutamate elicited Ca++[i responses similar to those observed at a culture time when the neurons are not susceptible to the excitotoxic effects of glutamate (DIV 3). Pretreatment of the cultured neurons with a subtoxic concentration of NMDA, which protects all neurons against the excitotoxic effects of glutamate, did not alter the maximal Ca++[i elicited by an excitotoxic concentration of glutamate.

Animals↗
PubMed1996

Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons.

1. The effects of low micromolar concentrations of glutamate on fast excitatory synaptic responses were studied in microcultures of postnatal rat hippocampal neurons using whole-cell patch clamp recordings. 2. Glutamate depressed the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor component of excitatory autaptic currents (EACs) with an EC50 of 3.8 microM. 3. Both pre- and postsynaptic effects contributed to the depression of AMPA receptor-mediated EACs. Cyclothiazide and wheatgerm agglutinin, agents which inhibit AMPA receptor desensitization, partially reversed the depression produced by glutamate, as did pertussis toxin, an agent that blocks presynaptic inhibition mediated by metabotropic glutamate receptors. 4. In neurons in which both the AMPA and N-methyl-D-aspartate (NMDA) receptor components of EACs were examined, low concentrations of glutamate depressed the NMDA component of EACs to a greater extent. The EC50 for inhibiting the NMDA component was 1.3 microM. 5. Calcium-dependent desensitization of postsynaptic NMDA receptors contributed to the depression of NMDA receptor-mediated synaptic responses. Both depolarization of postsynaptic neurons to +70 mV to decrease Ca2+ influx via NMDA channels and inclusion of high concentrations of a calcium chelator in recording pipettes decreased the depression of NMDA receptor-mediated EACs. 6. Threo-3-hydroxy-aspartate (THA), an inhibitor of glutamate transport, depressed EACs by about 10% and increased the degree of depression produced by 2.5 microM glutamate, suggesting that glutamate transport in microcultures helps to control ambient glutamate levels. 7. Because the normal extracellular concentration of glutamate is about 1 microM, these results suggest that the ambient glutamate level is an important determinant of synaptic efficacy. Relatively small changes in extracellular glutamate can alter fast excitatory synaptic transmission by both presynaptic and postsynaptic mechanisms.

Animals↗
PubMed1991

The effect of L-glutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta.

The effect of the putative amino acid transmitter, L-glutamate, on adenylate cyclase in crude membrane preparations of the rat tapeworm Hymenolepis diminuta was investigated to determine if glutamate effects the generation of the second messenger cAMP. Addition of glutamate at 10(-3) and 5.5 x 10(-9) M resulted in significant elevations in basal activity of adenylate cyclase, while concentrations in the 10(-5)-10(-7) M range caused significant depressions below basal activity. Assays with glutamate agonists and other acidic compounds showed glutamate to be the only amino acid, dicarboxylic acid, or acidic compound capable of this pattern of stimulation and inhibition. While the response of adenylate cyclase to glutamate agonists suggested that an N-methyl-D-aspartic acid (NMDA) type receptor may be present, glutamate agents acting as NMDA antagonists in vertebrate systems were agonists. Metabolic end products of glycolysis stimulated adenylate cyclase, suggesting that these, along with metabolic glutamate may regulate glycolytic enzymes. Only 10(-3) M L-glutamate significantly stimulated adenylate cyclase activity in tissue slices, and this response was restricted to those slices rich in nervous tissues. L-Glutamate eliminated the 5-hydroxytryptamine (5-HT) stimulated adenylate cyclase response suggesting that glutamate can modulate the 5-HT stimulated elevations in adenylate cyclase activity. The data support the hypothesis that L-glutamate is a neurotransmitter-modulator in the cestode.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Mercuric chloride uncouples glutamate uptake from the countertransport of hydroxyl equivalents.

The cotransport of sodium and glutamate by system X(AG)- is believed to be coupled to the countertransport of potassium and hydroxyl ion equivalents. Accordingly, the uptake of glutamate or D-aspartate in astrocytes is accompanied by an intracellular acidification. Here, we report that HgCl2 blocks the glutamate-induced acidification with an approximate 50% inhibitor concentration (IC50) of 55 nM, an order of magnitude below its IC50 for inhibition of glutamate uptake. At 100 nM HgCl2, glutamate-induced acidification was abolished, whereas glutamate uptake was unaffected. D-Aspartate-induced acidification was equally sensitive to HgCl2, indicating that HgCl2 blocked a transporter-mediated, rather than a receptor-mediated, acidification. Unaltered responses to acute acid and alkaline loads showed that HgCl2 was not acting indirectly via a change in pH regulation. We conclude that HgCl2 acted directly on the glutamate transporter to uncouple the uptake of glutamate from the export of hydroxyl equivalents. In contrast, two other sulfhydryl reagents, p-chloromercuribenzensulfonate and N-ethylmaleimide, failed to discriminate between glutamate-induced acidification and glutamate uptake. An additional effect of > or = 100 nM HgCl2, in this case shared by p-chlormercuribenzenesulfonate, was transient intracellular acidification. There is evidence that glutamate transport is regulated by intracellular pH. Mercuric mercury may disrupt the regulation of glutamate transport at lower concentrations than those that block transport.

4-Chloromercuribenzenesulfonate↗