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PubMed1983

pH dependence of the Coxiella burnetii glutamate transport system.

The transport of glutamate, apparently a primary energy source for Coxiella burnetii, has been examined. C. burnetii is shown to possess a pH-dependent active transport system for L-glutamate with an apparent Kt of 61.1 microM and Vmax of 8.33 pmol/s per mg at pH 3.5. Both L-glutamine and L-asparagine competitively inhibited transport of glutamate, but D-glutamate, L-aspartate, L-glutamate-gamma-methyl ester, methionine sulfoximine, or alpha-ketoglutarate did not compete. This transport system is both temperature and energy dependent. Uptake of glutamate is highly sensitive to uncouplers of oxidative phosphorylation such as 2,4-dinitrophenol and carbonyl cyanide-m-chlorophenyl hydrazone that decrease the proton motive force across the cytoplasmic membrane. ATPase inhibitors such as dicyclohexylcarbodiimide or metabolic poisons such as KCN, NaF, or arsenite were much less effective as inhibitors of glutamate transport. Uptake of glutamate did not appear to be coupled to Na+ symport as in Escherichia coli since no monovalent cation requirement could be demonstrated. Instead, the Vmax of glutamate transport showed good correlation with the transmembrane pH gradient (delta pH). From these results, we propose that L-glutamate transport by C. burnetii is energized via a proton motive force.

Adenosine Triphosphate↗
PubMed1987

Involvement of glutamate in the respiratory metabolism of Bradyrhizobium japonicum bacteroids.

Bradyrhizobium japonicum bacteroids were isolated anaerobically and supplied with 14C-labeled succinate, malate, aspartate, or glutamate for periods of up to 60 min in the presence of myoglobin to control the O2 concentration. Succinate and malate were absorbed about twice as rapidly as glutamate and aspartate. Conversion of substrate to CO2 was most rapid for malate, followed by succinate, glutamate, and aspartate. When CO2 production was expressed as a proportion of total carbon taken up, malate was still the most rapidly respired substrate, with 68% of the label absorbed converted to CO2. The comparable values for succinate, glutamate, and aspartate were 37, 50, and 38%, respectively. Considering the fate of labeled substrate not respired, greater than 95% of absorbed glutamate remained as glutamate in the bacteroids. In contrast, from 39 to 66% of the absorbed succinate, malate, or aspartate was converted to glutamate. An increase in the rate of CO2 formation from labeled substrates after 20 min appeared to coincide with a maximum accumulation of label in glutamate. The results indicate the presence of a substantial glutamate pool in bacteroids and the involvement of glutamate in the respiratory metabolism of bacteroids.

Aspartic Acid↗
PubMed1992

Mechanisms of glial swelling induced by glutamate.

The mechanisms of glutamate-induced glial swelling have been studied using an in vitro model that permits detection of cell volume changes with high accuracy. The model allows for a close control of the extracellular environment to study in isolation the effect of defined extracellular alterations occurring in brain under pathophysiologic conditions. Glutamate was applied in concentrations between 50 microM and 10 mM to either C6 glioma cells or astrocytes from primary culture. Glutamate uptake was assessed by HPLC measurements of amino acids in the extracellular medium. Glutamate at all concentrations tested caused glial swelling, which, however, was moderate, with maximal average volume increases between 5.0 +/- 1.92 and 18.38 +/- 1.6% of control at 50 microM and 5 mM glutamate, respectively. Swelling was concentration dependent and correlated with glutamate uptake. After removal of all extracellular glutamate by glial uptake, cell volume spontaneously normalized. Pretreatment of the cells for 90 min with ouabain (1 mM) to abolish the extracellular/intracellular Na+ gradient, prevented glutamate-induced swelling. It is concluded that while glial cells readily accumulate glutamate from the extracellular environment to protect neurons from excitotoxic effects, swelling results from the increase of intracellular osmotic activity due to the uptake of Na+ and glutamate.

Animals↗
PubMed1993

Splanchnic bed utilization of glutamine and glutamic acid in humans.

To study the fate of enterally delivered nonessential amino acids, glutamine and glutamate, 14 healthy adults were infused in the postabsorptive state with [2-15N]glutamine and [15N]glutamate for 7 h by intravenous (iv) and nasogastric (ng) tube routes. The amount of enterally delivered tracer that was sequestered by the splanchnic bed on the first pass was 54 +/- 4 and 88 +/- 2% for the [2-15N]glutamine and [15N]glutamate tracers, respectively. Only 46 and 12% of the ng glutamine and glutamate tracers entered systemic blood, respectively. The relative amount of 15N transferred from glutamate to glutamine, the transaminating amino acids leucine, isoleucine, valine, and alanine, and to proline was significantly higher when the [15N]glutamate was infused by the ng vs. iv route. The same was also true for [2-15N]glutamine, which presumably transferred 15N after it was converted to glutamate. Thus we conclude that the splanchnic bed sequesters over one-half of the glutamine and almost all of the glutamate delivered to it in the postabsorptive state. There is production of transaminating amino acids in the splanchnic bed, and the splanchnic bed produces simultaneously both glutamine from glutamate and glutamate from glutamine.

Adult↗
PubMed2002

Characterization of vesicular glutamate transporter in pancreatic alpha - and beta -cells and its regulation by glucose.

Glutamate has been suggested to play an important role in the release of insulin and glucagon from pancreatic cells via exocytosis. Vesicular glutamate transporter is a rate-limiting step for glutamate release and is involved in the glutamate-evoked exocytosis. Two vesicular glutamate transporters (VGLUT1 and -2) have recently been cloned from the brain. In this report, we first functionally characterized vesicular glutamate transporter in cultured pancreatic alpha- and beta-cells, and then detected mRNA expression of VGLUT1 and -2 in these cells. We also investigated the effect of high or low level of glucose on vesicular glutamate transport in cultured pancreas cells. Our results suggest that both alpha- and beta-cells contain functional vesicular glutamate transporter. The transport characteristics are similar to the cloned neuronal VGLUT1 and -2 in regard to ATP dependence, substrate specificity, kinetics, and chloride dependence. VGLUT2 mRNA is expressed in both alpha- and beta-cells, whereas VGLUT1 is only expressed in beta-cells. High (12.8 mM) and low (2.8 mM) concentrations of glucose increased vesicular glutamate transport in beta- and alpha-cells, respectively. VGLUT2 mRNA was significantly increased in beta- and alpha-cells by high and low glucose concentration, respectively. This increase in VGLUT2 mRNA was suppressed by actinomycin D. We conclude that both alpha- and beta-cells possess functional vesicular glutamate transporters regulated by alteration in glucose concentration, partly via the transcriptional mechanism.

Animals↗
PubMed2001

Effect of acute exposure to ammonia on glutamate transport in glial cells isolated from the salamander retina.

A rise of brain ammonia level, as occurs in liver failure, initially increases glutamate accumulation in neurons and glial cells. We investigated the effect of acute exposure to ammonia on glutamate transporter currents in whole cell clamped glial cells from the salamander retina. Ammonia potentiated the current evoked by a saturating concentration of L-glutamate, and decreased the apparent affinity of the transporter for glutamate. The potentiation had a Michaelis-Menten dependence on ammonia concentration, with a K(m) of 1.4 mM and a maximum potentiation of 31%. Ammonia also potentiated the transporter current produced by D-aspartate. Potentiation of the glutamate transport current was seen even with glutamine synthetase inhibited, so ammonia does not act by speeding glutamine synthesis, contrary to a suggestion in the literature. The potentiation was unchanged in the absence of Cl(-) ions, showing that it is not an effect on the anion current gated by the glutamate transporter. Ammonium ions were unable to substitute for Na+ in driving glutamate transport. Although they can partially substitute for K+ at the cation counter-transport site of the transporter, their occupancy of these sites would produce a potentiation of < 1%. Ammonium, and the weak bases methylamine and trimethylamine, increased the intracellular pH by similar amounts, and intracellular alkalinization is known to increase glutamate uptake. Methylamine and trimethylamine potentiated the uptake current by the amount expected from the known pH dependence of uptake, but ammonia gave a potentiation that was larger than could be explained by the pH change, and some potentiation of uptake by ammonia was still seen when the internal pH was 8.8, at which pH further alkalinization does not increase uptake. These data suggest that ammonia speeds glutamate uptake both by increasing cytoplasmic pH and by a separate effect on the glutamate transporter. Approximately two-thirds of the speeding is due to the pH change.

ATP-Binding Cassette Transporters↗
PubMed1997

Intracellular Ca2+ release mediated by metabotropic glutamate receptor activation in the leech giant glial cell.

We have investigated the effects of glutamate and glutamate receptor ligands on the intracellular free Ca2+ concentration ([Ca2+]i) and the membrane potential (Em) of single, identified neuropile glial cells in the central nervous system of the leech Hirudo medicinalis. Exposed glial cells of isolated ganglia were filled iontophoretically with the Ca2+ indicator dye Fura-2. Application of glutamate (200-500 mumoll-1) caused biphasic membrane potential shifts and increases in [Ca2+]i, which were only partly reduced by either removing extracellular Ca2+ or blocking ionotropic glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50-100 mumol l-1. Metabotropic glutamate receptor (mGluR) ligands had the following rank of potency in inducing a rise in [Ca2+]i: quisqualate (QQ, 200 mumol l-1) > glutamate (200 mumol l-1) > L(+)2-amino-3-phosphonopropionic acid (L-AP3, 200 mumol l-1 > trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 400 mumol l-1). The mGluR-selective antagonist (RS)-alpha-methyl-4-carboxyphenylglycine [(RS)-MCPG, 1 mmol l-1] significantly reduced glutamate-evoked increases in [Ca2+]i by 20%. Incubation of the ganglia with the endoplasmic ATPase inhibitor cyclopiazonic acid (CPA, 10 mumol l-1) caused a significant (53%) reduction of glutamate-induced [Ca2+]i transients, while incubation with lithium ions (2 mmol l-1) resulted in a 46% reduction. The effects of depleting the Ca2+ stores with CPA and of CNQX were additive. We conclude that glutamate-induced [Ca2+]i transients were mediated by activation of both Ca(2+)-permeable ionotropic non-NMDA receptors and of metabotropic glutamate receptors leading to Ca2+ release from intracellular Ca2+ stores.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2006

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells.

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na+ -dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl2) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl2 on glutamate uptake are unknown. This study examines the effects of HgCl2 on the transport of 3H-D-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl2 on mRNA and protein levels of these transporters. The results indicate that (1) HgCl2 leads to significant (p < 0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl2 on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl2 inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.

Animals↗
PubMed2006

Dynamic regulation of synaptic GABA release by the glutamate-glutamine cycle in hippocampal area CA1.

Vesicular GABA and intraterminal glutamate concentrations are in equilibrium, suggesting inhibitory efficacy may depend on glutamate availability. Two main intraterminal glutamate sources are uptake by neuronal glutamate transporters and glutamine synthesized through the astrocytic glutamate-glutamine cycle. We examined the involvement of the glutamate-glutamine cycle in modulating GABAergic synaptic efficacy. In the absence of neuronal activity, disruption of the glutamate-glutamine cycle by blockade of neuronal glutamine transport with alpha-(methylamino) isobutyric acid (MeAIB; 5 mM) or inhibition of glutamine synthesis in astrocytes with methionine sulfoximine (MSO; 1.5 mM) had no effect on miniature IPSCs recorded in hippocampal area CA1 pyramidal neurons. However, after a period of moderate synaptic activity, application of MeAIB, MSO, or dihydrokainate (250 microM; an astrocytic glutamate transporter inhibitor) significantly reduced evoked IPSC (eIPSC) amplitudes. The MSO effect could be reversed by exogenous application of glutamine (5 mM), whereas glutamine could not rescue the eIPSC decreases induced by the neuronal glutamine transporter inhibitor MeAIB. The activity-dependent reduction in eIPSCs by glutamate-glutamine cycle blockers was accompanied by an enhanced blocking effect of the low-affinity GABA(A) receptor antagonist, TPMPA [1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], consistent with diminished GABA release. We further corroborated this hypothesis by examining MeAIB effects on minimal stimulation-evoked quantal IPSCs (meIPSCs). We found that, in MeAIB-containing medium, moderate stimulation induced depression in potency of meIPSCs but no change in release probability, consistent with reduced vesicular GABA content. We conclude that the glutamate-glutamine cycle is a major contributor to synaptic GABA release under physiological conditions, which dynamically regulates inhibitory synaptic strength.

Action Potentials↗
PubMed2005

Deriving the glutamate clearance time course from transporter currents in CA1 hippocampal astrocytes: transmitter uptake gets faster during development.

At many excitatory synapses, the neurotransmitter glutamate diffuses beyond the synaptic cleft to activate extrasynaptic targets. The extent and impact of such transmitter "spillover" on the processing capacity of neuronal networks are unclear, in part because it remains unknown how far transmitter diffuses from its point of release before being removed from the extracellular space by high-affinity glutamate transporters. Synaptically activated, transporter-mediated currents (STCs) recorded in hippocampal astrocytes provide an experimental measure of glutamate uptake, but the time course of the STC may be shaped, or "filtered," by other factors and therefore not represent a direct indication of clearance rate. Here, STCs were recorded from astrocytes in rat hippocampal slices under conditions in which uptake capacity was reduced and the STC decay reflected a slowed rate of glutamate clearance. The temporal characteristics of the filtering mechanisms were extracted from these responses, and the glutamate clearance time course in control conditions was derived. The results indicate that glutamate can be cleared from the extrasynaptic space within 1 ms. Clearance is fastest in adult neuropil, corresponding to a developmental increase in glial transporter expression. Synaptically released glutamate is taken up at the same rate as glutamate released via flash photolysis, indicating that the spatial location of transporters relative to the site of glutamate release does not affect the time course of clearance. Slower clearance in young animals would permit glutamate to diffuse greater distances, indicating a particularly important role for extrasynaptic receptors early in development.

Age Factors↗
PubMed2004

Glutamate signalling and its potential application to tissue engineering of bone.

Mechanical loading of the skeleton is important for maintenance of adequate bone mass and defined mechanical stimuli are highly osteogenic. The identification of mechanoresponsive signalling molecules in bone may allow osteogenic signals to be mimicked. This approach would be useful in the treatment of bone pathologies where the skeleton is too weak to withstand osteogenic forces and to tissue engineering of bone where the mechanical environment of bone cells is disrupted. Glutamate has been implicated as a mediator of mechanical signalling in bone. Evidence for glutamate signalling in bone, its role in mechanotransduction and potential applications of this pathway to tissue engineering of bone is considered in this review. Glutamate receptors, transporters and proteins that regulate glutamate release, are all expressed in bone cells. Glutamate receptor activation affects both osteoblast and osteoclast phenotypes revealing a potential for therapeutic manipulation of glutamate signalling to enhance bone formation. Glutamate transporters contribute to this system by regulating extracellular glutamate concentrations and acting as glutamate-gated ion channels. Artificial regulation of glutamate receptors or transporters may be used to increase the bone forming capacity of osteoblasts. This novel approach may potentially enhance bone tissue engineering strategies.

Animals↗
PubMed1989

Release of glutamate and of free fatty acids in vasogenic brain edema.

The pathophysiological potential of mediator substances in manifestations of secondary brain damage is attracting increased attention. This is particularly true of the excitatory transmitters glutamate and arachidonic acid. Noxious properties of these compounds in central nervous tissue have been demonstrated. The current study was performed to determine whether glutamate and arachidonate are released in brain tissue secondary to focal trauma. For this purpose, a cold injury of exposed cerebral cortex was induced in cats. Marked accumulation of glutamate was observed in interstitially drained edema fluid, reaching 10 to 15 times the level that was assessed in normal cerebrospinal fluid (CSF) prior to trauma. The extracellular release of glutamate was further dramatically enhanced by a critical decrease of the cerebral perfusion pressure due to a malignant increase of intracranial pressure. Under these conditions, glutamate concentrations 1000 to 1500 times normal levels accumulated in vasogenic edema fluid, demonstrating a relationship between the extent of the release of glutamate in damaged brain and the severity of the insult. Although under normal conditions glutamate concentrations in plasma were considerably higher than in the interstitial fluid, the pronounced increase of glutamate in this compartment due to trauma cannot be explained by transport of the compound together with the plasma-like edema from the intravascular space. Corresponding findings were obtained for free fatty acid concentrations in edema fluid. Almost all fatty acids that were studied had a significantly higher concentration in edema fluid than in normal CSF obtained as a control prior to trauma. However, contrary to the findings for glutamate, fatty acid concentrations in edema fluid were lower than in plasma. Accumulation of fatty acids in vasogenic edema fluid might, therefore, have resulted from uptake of the material together with edema fluid through the breached blood-brain barrier. Arachidonic acid was an exception. Its concentrations were significantly higher in edema fluid than in plasma, suggesting that it was released from cerebral parenchyma as the underlying mechanism of its extracellular accumulation. The current observations provide further support for a mediator function of glutamate and arachidonic acid in acute traumatic lesions of the brain. Quantitative assessment of the release of highly active mediator substances in brain tissue may facilitate analysis of the therapeutic efficiency of specific treatment aimed at interfering with the release or pathological function of mediators of secondary brain damage.

Animals↗
PubMed1992

Glutamate transport into synaptic vesicles. Roles of membrane potential, pH gradient, and intravesicular pH.

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)

Adenosine Triphosphate↗
PubMed1992

Aluminum inhibits glutamate release from transverse rat hippocampal slices: role of G proteins, Ca channels and protein kinase C.

Aluminum (Al) has been shown to produce deficits in learning and memory. The present experiments tested the hypothesis that Al-induced inhibition of learning may be due to its effect on glutamate release secondary to changes in calcium channel function and/or intracellular events triggering glutamate release. Calcium-dependent potassium (K)-evoked [14C]-glutamate release from 400 microns transverse rat hippocampal slices was inhibited by Al in a concentration dependent manner (IC50 = 40 microM). Aluminum (30, 100 microM) noncompetitively inhibited Bay K 8644-evoked glutamate release. 4-Aminopyridine (30, 1000 microM) noncompetitively attenuated the Al inhibition of glutamate release, suggesting an Al-induced alteration of Ca channel function. Activation of the Gi protein by R(-)phenylisopropyladenosine (PIA; 1 microM) reduced K-evoked glutamate release 69%, whereas 300 microM Al produced an 84% reduction. These effects were prevented by the Gi protein inhibitor N-ethylmaleimide (NEM; 100 microM), suggesting an effect of Al on the Gi protein to inhibit glutamate release. Phorbol myristate acetate (0.16 microM)-induced glutamate release was inhibited by 300 microM Al and 80 microM polymyxin B, suggesting an Al modulation of protein kinase C (PKC)-evoked glutamate release. These results demonstrate an Al inhibition of glutamate release that may be mediated by multiple, but interconnected mechanisms (e.g., via interactions with Ca systems), providing multiple targets for an Al-induced alteration of neuronal function.

4-Aminopyridine↗
PubMed1988

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver.

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.

Alanine↗
PubMed1995

Activation of a metabotropic glutamate receptor increases intracellular calcium concentrations in neurons of the avian cochlear nucleus.

Metabotropic glutamate receptors have been shown to stimulate phosphatidylinositol metabolism, and subsequently liberate Ca2+ from intracellular stores, in a variety of tissue and cell types. We previously demonstrated that glutamate could stimulate phosphatidylinositol metabolism, generating inositol-1,4,5-trisphosphate (IP3), in isolated cochlear nucleus tissue from the chick. Using the calcium indicator dye fura-2 and ratiometric fluorescent imaging, this study examined the ability of glutamate and its analogs to liberate Ca2+ from intracellular stores of neurons of the avian cochlear nucleus, and qualitatively characterized the pharmacological profile of such an action. In normal, Ca(2+)-containing medium, glutamate, kainate (KA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), NMDA, quisqualate (QUIS), and (+/-)-aminocyclopentane-trans-dicarboxylate (ACPD) elicited increases in intracellular calcium concentrations ([Ca2+]i). In the absence of external Ca2+, glutamate, quisqualate, and ACPD evoked increases in [Ca2+]i. In normal medium, the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV) attenuated but did not abolish the glutamate-evoked response and had no effect on the ACPD-evoked response. The putative metabotropic glutamate receptor antagonist 2-amino-3-phosphonopropionate (AP3) was without effect on the glutamate- and ACPD-evoked increases in [Ca2+]i in Ca(2+)-free medium. We conclude that a metabotropic glutamate receptor (mGluR) is present on cochlear nucleus neurons and is able to stimulate the phosphatidylinositol metabolism--Ca2+ signal transduction cascade.

Animals↗
PubMed1993

Ifenprodil prevents glutamate cytotoxicity via polyamine modulatory sites of N-methyl-D-aspartate receptors in cultured cortical neurons.

Neuroprotective effects of ifenprodil, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, against glutamate cytotoxicity were examined in cultured rat cortical neurons. The viability of the cultures was markedly reduced by a 10-min exposure to glutamate followed by incubation with glutamate-free medium for 60 min. Ifenprodil and its derivative SL 82.0715 dose-dependently prevented cell death induced by glutamate. The NMDA antagonists MK-801 and 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid also prevented glutamate cytotoxicity with a potency similar to that of ifenprodil. Ifenprodil as well as MK-801 prevented NMDA-induced cytotoxicity, but did not affect kainate-induced cytotoxicity. Glutamate cytotoxicity was inhibited by removing extracellular Ca++ during and immediately after glutamate exposure. Ifenprodil and MK-801 reduced NMDA-induced Ca++ influx measured with rhod-2. Either spermidine, a polyamine modulatory site agonist, or glycine, a strychnine-insensitive glycine site agonist, potentiated NMDA- and glutamate-induced cytotoxicity. The protective effects of ifenprodil against NMDA- and glutamate-induced cytotoxicity were significantly reduced by spermidine, but not by glycine. These findings indicate that ifenprodil protects cortical neurons against glutamate cytotoxicity by selective antagonism of the polyamine modulatory site of the NMDA receptor complex.

Animals↗
PubMed1998

Distribution of glutamate receptor subunits at neurochemically characterized synapses in the entopeduncular nucleus and subthalamic nucleus of the rat.

Glutamatergic neurotransmission in the subthalamic nucleus (STN) and in the output nuclei of the basal ganglia is critical in the expression of basal ganglia function, and increased glutamate transmission in these nuclei has been implicated in the pathology of Parkinson's disease. In order to determine the precise spatial relationship of subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) glutamate receptors to nerve terminals enriched in glutamate or gamma-aminobutyric acid (GABA) in one of the output nuclei, the entopeduncular nucleus (EP), and the STN, postembedding immunolabelling for glutamate receptor subunits and for glutamate and GABA was carried out in the rat. Immunolabelling for the AMPA glutamate receptor subunits 1, 2/3, and 4 (GluR1, GluR2/3, and GluR4) and the NMDA receptor subunit 1 (NR1) was localized predominantly within asymmetrical synapses in both the EP and STN. Quantitative analysis revealed that, on average for the whole population, each of the receptor subunits was evenly distributed along the synaptic specialization. Multiple AMPA receptor subunits and the GluR2/3 and NMDA (NR1) subunits were co-localized within individual synapses. The combination of immunolabelling for glutamate and GABA with the receptor immunolabelling revealed that the majority of axon terminals presynaptic to the receptor-immunoreactive synapses were enriched in glutamate immunoreactivity and were GABA-immunonegative. However, at some NR1- and GluR2/3-positive synapses, the level of glutamate immunoreactivity was low in the presynaptic terminal and, in the STN, some of them were GABA-immunopositive. It is concluded that glutamatergic transmission at individual synapses of different origins in the EP and STN is mediated by a combination ofAMPA and NMDA glutamate receptors.

Animals↗