Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics of the cell lines in the FMP assay were in good agreement with previous findings in electrophysiology studies of the transporters. The FMP assay was capable of distinguishing between substrates and non-substrate inhibitors and to discriminate between "full" and "partial" substrates at the transporters. Taking advantage of the prolific nature of the FMP assay, interactions of the EAATs with substrates and inhibitors were studied in some detail. This is the first report of a high throughput screening assay for EAATs. We propose that the assay will be of great use in future studies of the transporters. Although conventional electrophysiology set-ups might be superior in terms of studying sophisticated kinetic aspects of the uptake process, the FMP assay enables the collection of considerable amounts of highly reproducible data with relatively little labor. Furthermore, considering that the number of EAAT ligands presently available is limited, and that almost all of these are characterized by low potency and a low degree of subtype selectivity, future screening of compound libraries at the EAAT-cell lines in the FMP assay could help identify structurally and pharmacologically novel ligands for the transporters.     

Targeting glial physiology and glutamate cycling in the treatment of depression

Accumulating evidence indicates that dysfunction in amino acid neurotransmission contributes to the pathophysiology of depression. Consequently, the modulation of amino acid neurotransmission represents a new strategy for antidepressant development. While glutamate receptor ligands are known to have antidepressant effects, mechanisms regulating glutamate cycling and metabolism may be viable drug targets as well. In particular, excitatory amino acid transporters (EAATs) that are embedded in glial processes constitute the primary means of clearing extrasynaptic glutamate. Therefore, the decreased glial number observed in preclinical stress models, and in postmortem tissue from depressed patients provides intriguing, yet indirect evidence for a role of disrupted glutamate homeostasis in the pathophysiology of depression. More direct evidence for this hypothesis comes from studies using magnetic resonance spectroscopy (MRS), a technique that non-invasively measures in vivo concentrations of glutamate and other amino acids under different experimental conditions. Furthermore, when combined with the infusion of ¹³C-labeled metabolic precursors, MRS can measure flux through discrete metabolic pathways. This approach has recently shown that glial amino acid metabolism is reduced by chronic stress, an effect that provides a link between environmental stress and the decreased EAAT activity observed under conditions of increased oxidative stress in the brain. Furthermore, administration of riluzole, a drug that enhances glutamate uptake through EAATs, reversed this stress-induced change in glial metabolism. Because riluzole has antidepressant effects in both animal models and human subjects, it may represent the prototype for a novel class of antidepressants with the modulation of glial physiology as a primary mechanism of action.     

Roles and regulation of glutamate transporters in the central nervous system

1. Glutamate transporters (also known as excitatory amino acid transporters or EAAT) are solely responsible for the removal of the excitatory neurotransmitter l-glutamate (Glu) from the extracellular space and, thus, permit normal transmission, as well as preventing cell death due to the excessive activation of Glu receptors. 2. Five subtypes of glutamate transporter (EAAT1-5) exist, possessing distinct pharmacology, cellular localization and modulatory mechanisms. 3. Experimental inhibition of EAAT activity in vitro and in vivo results in increased extracellular concentrations of Glu and in neuronal death via excitotoxicity, highlighting the importance of EAAT in normal excitatory neurotransmission. 4. Dysfunction of EAAT may contribute to the pathology of both acute neuronal injury and chronic neurodegenerative conditions, so correction of EAAT function under these conditions may provide a valuable therapeutic strategy. 5. The present review describes basic pharmacological studies that allow new insights into EAAT function and suggest possible strategies for the therapeutic modulation of EAAT.     

Excitatory amino acid transporters as emerging targets for central nervous system therapeutics

In the mammalian central nervous system (CNS) the excitatory amino acid transporter (EAAT) family of proteins are responsible for the high-affinity sodium-dependent uptake of glutamate into both astroglial cells and neurones. Normal EAAT function is required both for the efficient termination of glutamatergic neurotransmission and for the maintenance of low extracellular glutamate concentrations, thereby preventing glutamate excitotoxicity. It is widely believed that a dysfunction of glutamate transmission participates in the aetiology of a number of neurodegenerative and neuropsychiatric disorders and diseases. This review introduces the EAATs as a new family of emerging therapeutic targets for CNS disorders by virtue of their central role in maintaining glutamate homeostasis. We examine recent findings on the modulation and regulation of EAATs and review the changes in both EAAT function and expression which have been described in a number of neuropathological conditions.     

Expression of GFP-tagged neuronal glutamate transporters in cerebellar Purkinje neurons

Of the five excitatory amino acid transporters (EAATs) identified, two genes are expressed by neurons (EAAT3 and EAAT4) and give rise to transporters confined to neuronal cell bodies and dendrites. At an ultrastructural level, EAAT3 and EAAT4 proteins are clustered at the edges of postsynaptic densities of excitatory synapses. This pattern of localization suggests that postsynaptic EAATs may help to limit spillover of glutamate from excitatory synapses. In an effort to study transporter localization in living neurons and ultimately to manipulate uptake at intact synapses, we have developed viral reagents encoding neuronal EAATs tagged with GFP. We demonstrate that these fusion proteins are capable of Na(+)-dependent glutamate uptake, that they generate ionic conductances indistinguishable from their wild-type counterparts, and that GFP does not alter their glutamate dose-dependence. Two-photon microscopy was used to examine fusion protein expression in Purkinje neurons in acute cerebellar slices. Both EAAT3-GFP and EAAT4-GFP were observed at high levels in the dendritic spines of transfected Purkinje neurons. These findings indicate that functional EAAT fusion proteins can be synthesized and appropriately trafficked to postsynaptic compartments. Furthermore, they validate a powerful system for looking at EAAT function in situ.     

Functional and morphological characterization of glutamate transporters in the rat locus coeruleus

Background and Purpose

Excitatory amino acid transporters (EAATs) in the CNS contribute to the clearance of glutamate released during neurotransmission. The aim of this study was to explore the role of EAATs in the regulation of locus coeruleus (LC) neurons by glutamate.

Experimental Approach

We measured the effect of different EAAT subtype inhibitors/enhancers on glutamate- and KCl-induced activation of LC neurons in rat slices. EAAT2–3 expression in the LC was also characterized by immunohistochemistry.

Key Results

The EAAT2–5 inhibitor DL-threo-β-benzyloxaspartic acid (100 μM), but not the EAAT2, 4, 5 inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (100 μM) or the EAAT2 inhibitor dihydrokainic acid (DHK; 100 μM), enhanced the glutamate- and KCl-induced activation of the firing rate of LC neurons. These effects were blocked by ionotropic, but not metabotrobic, glutamate receptor antagonists. DHK (100 μM) was the only EAAT inhibitor that increased the spontaneous firing rate of LC cells, an effect that was due to inhibition of EAAT2 and subsequent AMPA receptor activation. Chronic treatment with ceftriaxone (200 mg·kg⁻¹ i.p., once daily, 7 days), an EAAT2 expression enhancer, increased the actions of glutamate and DHK, suggesting a functional impact of EAAT2 up-regulation on the glutamatergic system. Immuhistochemical data revealed the presence of EAAT2 and EAAT3 surrounding noradrenergic neurons and EAAT2 on glial cells in the LC.

Conclusions and Implications

These results remark the importance of EAAT2 and EAAT3 in the regulation of rat LC by glutamate. Neuronal EAAT3 would be responsible for terminating the action of synaptically released glutamate, whereas glial EAAT2 would regulate tonic glutamate concentrations in this nucleus.     

Regulation of transmitter release by high-affinity group III mGluRs in the supraoptic nucleus of the rat hypothalamus

We analyzed the subtypes of group III metabotropic glutamate receptors (mGluRs) modulating inhibitory and excitatory transmission in the rat supraoptic nucleus. Bath application of the agonist l-AP4 at 200 microM, a concentration that activates all group III mGluR subtypes, inhibited the frequency but not the amplitude of miniature inhibitory and excitatory postsynaptic currents, indicating a presynaptic site of action. l-AP4 at low concentrations (10 microM), as well as ACPT-1 (50 microM), a specific mGluR III agonist, inhibited transmission at GABAergic and glutamatergic synapses to the same extent as 200 microM l-AP4. Because the potency of l-AP4 and ACPT-1 is much higher on mGluR4 and mGluR8 than on mGluR7, these results are consistent with the presence of high-affinity group III mGluRs regulating transmitter release in this nucleus. In agreement with these findings, DCPG (30 microM), a selective mGluR8 agonist, induced a significant depression of inhibitory and excitatory synaptic currents. Group III mGluRs such as mGluR8, because of their high affinity for glutamate, are particularly well suited to detect small changes in the concentration of this excitatory amino acid in the extracellular space. Their presence, therefore, may favor the negative feedback control exerted by glutamate on its own release as well as the intersynaptic crosstalk mediated by glutamate spillover on adjacent synapses.     

Abolition of chondral mineralization by group III metabotropic glutamate receptors expressed in rodent cartilage

1 Previous studies have demonstrated the functional expression by osteoblasts of glutamate (Glu) signaling machineries responsible for the stimulation of cell proliferation and differentiation in bone, while there is no information available on the expression of the Glu signaling system by cartilage to date. 2 In cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely inhibited in the presence of the group III metabotropic Glu receptor (mGluR) agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, with the total length being unchanged. 3 A group II mGluR agonist was similarly more effective in inhibiting the mineralization than a group I mGluR agonist, while none of ionotropic GluR agonists drastically affected the mineralization. 4 Both histological and in situ hybridization analyses revealed that L-AP4 specifically inhibited chondral mineralization, without apoptotic cell death, in cultured metatarsals. 5 In addition to the constitutive expression of mRNA for particular mGluRs in both cultured mouse metatarsals and rat costal chondrocytes, L-AP4 significantly inhibited the accumulation of cyclic AMP by forskolin and parathyroid hormone in a manner sensitive to a group III mGluR antagonist in cultured chondrocytes. 6 Moreover, L-AP4 drastically inhibited the expression of osteopontin mRNA in both cultured metatarsals and chondrocytes. 7 These results suggest that Glu may at least in part play a role as a signal mediator in mechanisms associated with chondral mineralization through the group III mGluR subtype functionally expressed by chondrocytes in rodent cartilage.     

Chronic exposure of nicotine modulates the expressions of the cerebellar glial glutamate transporters in Rats

Rats were given nicotine (25 ppm) in their drinking water at the start of their mating period in order to study the expressions of glutamate transporter subtypes in cerebellar astrocytes following the chronic exposure of nicotine after mating. After the offspring were delivered, each group was divided into two subgroups. One group, the control group, was given distilled water only and the other group, the experimental group, was given distilled water containing nicotine. The cerebellar astrocytes were prepared from 7 day-old pups at each group. Ten days after the cells were cultured, the expression of the glutamate transporter subtypes (GLAST and GLT-1) was determined using immunochemistry and immunoblotting. During the continuous treatments, the developmental expression patterns of the GLAST and GLT-1 in the cerebellum were also determined from 2, 4 and 8 week-old rats. The expression levels of GLAST in cultured astrocytes of both the pre- or post-natally exposed groups were higher than those of the control group. However, these expression levels of the continuously exposed group were lower than those of the control group. Compared to those of the control group, the GLT-1 expression levels of all the nicotine-treated groups were higher, particularly in the continuously treated group. According to the results from the immochemistry procedure, the cerebellar GLAST and GLT-1 expression levels of all nicotine-treated groups were lower than those of the control group at each age. However, the immunoblotting procedure showed that the cerebellar GLT-1 expression levels of all the nicotine-treated groups were higher than those of the control group, except for the rats that were continuously exposed for 8 weeks using immunoblotting. These results suggest that the expression of the glial GLAST and GLT-1 are altered differently depending on the initial exposure time and the particular period of nicotine exposure. In addition, nicotine exposure during gestation has persistent effects on glial cells.     

The lathyrus toxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x(c)(-) and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrus excitotoxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons >50-fold to L-AP6 and >10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM alpha-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x(c)(-) and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.     

Among the twenty classical L-amino acids, only glutamate directly activates metabotropic glutamate receptors

Under pathophysiological conditions, cellular amino acids can be profusely released from cells into the cerebral interstitial space. Because several class-C G protein coupled receptors (GPCRs) display a broad natural ligand spectrum, being sensitive to more than one endogenous ligand, we wondered whether the related metabotropic glutamate (mGlu) receptors could be modulated by various types of L-amino acids, allowing them to sense large increase in extracellular amino acid concentration. Here, the agonist, antagonist and allosteric effects of the twenty classical L-amino acids were evaluated on the eight mGlu receptor subtypes. We show that, in addition to glutamate (Glu), cysteine, aspartate and asparagine also lead to the activation of mGlu3, 4 and 5. Interestingly, our data demonstrate that the effect of these three amino acids did not result from a direct activation of the receptors, but from an indirect action involving Glu-transporters/exchangers. These data first demonstrate that mGlu receptors, unlike other class-C GPCRs, display an extremely high selectivity towards one ligand. Moreover, our results also show that Glu transport systems allow mGlu receptors to sense large increase in the extracellular concentration of some amino acids. Such a system will certainly lead to a large increase in some mGlu receptor activity under pathological conditions, such as seizure, ischemia or other brain injuries.     

Down-regulation of the glial glutamate transporter GLT-1 in rat hippocampus and striatum and its modulation by a group III metabotropic glutamate receptor antagonist following transient global forebrain ischemia

Our goals were to identify biochemical markers for transient global ischemia-induced delayed neuronal death and test possible drug therapies against this neuronal damage. Four-vessel occlusion (4-VO) for 20 min was used as a rat model. The temporal expression profiles of three glutamate transporters (GLT-1, GLAST and EAAC1) were evaluated in the CA1 region of the hippocampus and the striatum. The protein levels of the GLT-1 were significantly down-regulated between 3 and 6 h after ischemia-reperfusion in the CA1 region and striatum, returned to the control (2-VO) levels 24 h after reperfusion and remained unchanged for up to 7 days. The levels of GLAST in the CA1 region and striatum, and EAAC1 in the CA1 region did not change after ischemia from 1 h to 7 days. Pretreatment with group III metabotropic glutamate receptor antagonist s-alpha-MCPA (20 microg/5 microl) 30 min prior to 4-VO significantly restored the GLT-1 levels in the CA1 region caused by global ischemia at both 3 and 6 h after reperfusion. The loss of pyramidal neurons in the CA1 region due to ischemia-reperfusion could also be prevented by intraventricular pretreatment with s-alpha-MCPA. The current findings pinpoint the significance of GLT-1 during ischemia/reperfusion and suggest a potential application of group III metabotropic glutamate receptor antagonist against ischemic/hypoxic neuronal damage.     

Effects of synthetic sphingosine-1-phosphate analogs on arachidonic acid metabolism and cell death

Sphingolipid metabolites such as sphingosine regulate cell functions including cell death and arachidonic acid (AA) metabolism. D-erythro-C18-Sphingosine-1-phosphate (D-e-S1P), a sphingolipid metabolite, acts as an intracellular messenger in addition to being an endogenous ligand of some cell surface receptors. The development of S1P analogs may be useful for studying and/or regulating S1P-mediated cellular responses. In the present study, we found that several synthetic S1P analogs at pharmacological concentrations stimulated AA metabolism and cell death in PC12 cells. D-erythro-N,O,O-Trimethyl-C18-S1P (D-e-TM-S1P), L-threo-O,O-dimethyl-C18-S1P (L-t-DM-S1P) and L-threo-O,O-dimethyl-3O-benzyl-C18-S1P (L-t-DMBn-S1P) at 100 microM stimulated [(3)H]AA release from the prelabeled PC12 cells. L-t-DMBn-S1P at 20 microM increased prostanoid formation in PC12 cells. L-t-DMBn-S1P-induced AA release was inhibited by D-e-sphingosine, but not by the tested PLA(2) inhibitors. L-t-DMBn-S1P did not stimulate the activity of cytosolic phospholipase A(2alpha) (cPLA(2alpha)) in vitro and the translocation of cPLA(2alpha) in the cells, and caused AA release from the cells lacking cPLA(2alpha). These findings suggest that L-t-DMBn-S1P stimulated AA release in a cPLA(2alpha)-independent manner. In contrast, D-e-S1P and D-erythro-N-monomethyl-C18-S1P caused cell death without AA release in PC12 cells, and the effects of D-e-TM-S1P, L-t-DM-S1P and L-t-DMBn-S1P on cell death were limited. Synthetic S1P analogs may be useful tools for studying AA metabolism and cell death in cells.     

Functional calcium coupling with the human metabotropic glutamate receptor subtypes 2 and 4 by stable co-expression with a calcium pathway facilitating G-protein chimera in Chinese hamster ovary cells

The objective of the current study was to facilitate functional calcium assays, compatible with the fluorometric imaging plate reader platform, for the human metabotropic glutamate receptor (mGluR) subtypes 2 and 4, by co-expressing each receptor with a G-protein chimera comprising Galphaq with the C-terminal five amino acids replaced with those from Galphai3 (GqGi3). Transfection of GqGi3 into previously validated stable CHO cell lines expressing mGluR2 or mGluR4 allowed for the selection of new double transfectants in which application of L-glutamate and other mGluR agonists resulted in calcium coupling with a high signal:noise ratio (maximal changes in relative fluorescence units up to 20,000). The rank order of agonist potency for the stimulation of calcium mobilization in the mGluR2/GqGi3 stable cell line was LY354740>L-CCG-I=DCG-IV>L-glutamate>/=(2R,4R)-APDC>/=(1S,3R)-ACPD. In the mGluR4/GqGi3 stable cell line the rank order of agonist potency was L-AP4>L-SOP>/=ACPT-I=L-CCG-I>/=L-glutamate=(R,S)-PPG. By comparison, equivalent potency orders and a significant correlation in functional activities were observed when the same compounds were profiled in [35S]GTPgammaS binding assays for each mGluR subtype. These results validate the use of functional calcium assays, amenable to high-throughput applications on the fluorometric imaging plate reader, for the mGluR2 and mGluR4 subtypes when co-expressed in stable cell lines with the GqGi3 chimera.     

Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes

Aim:

To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552).

Methods:

Methods: Primary astrocyte cultures were prepared from the cortex of postnatal rat pups. An astrocytes model of Huntington''s disease was established using the astrocytes infected with adenovirus carrying coden gene of N-terminal 552 residues of Huntingtin. The protein levels of glutamate transporters GLT-1 and GLAST, the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the autophagy substrate p62 in the astrocytes were examined using Western blotting. The mRNA expression levels of GLT-1 and GLAST in the astrocytes were determined using Real-time PCR. ³H]glutamate uptake by the astrocytes was measured with liquid scintillation counting.

Results:

The expression of mutant Htt-552 in the astrocytes significantly decreased both the mRNA and protein levels of GLT-1 but not those of GLAST. Furthermore, Htt-552 significantly reduced ³H]glutamate uptake by the astrocytes. Treatment with the autophagy inhibitor 3-MA (10 mmol/L) significantly increased the accumulation of mutant Htt-552, and reduced the expression of GLT-1 and ³H]glutamate uptake in the astrocytes. Treatment with the autophagy stimulator rapamycin (0.2 mg/mL) significantly reduced the accumulation of mutant Htt-552, and reversed the changes in GLT-1 expression and ³H]glutamate uptake in the astrocytes.

Conclusion:

Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.     

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.     

Co-administration of ultra-low dose naloxone attenuates morphine tolerance in rats via attenuation of NMDA receptor neurotransmission and suppression of neuroinflammation in the spinal cords

Although mechanisms underlying ultra-low dose naloxone-induced analgesia have been proposed, possible interactions with glutamatergic transmission and glial cell activation have not been addressed. In the present study, we examined the effect of ultra-low dose naloxone on spinal glutamatergic transmission and glial cell activity in rats chronically infused with morphine.In male Wistar rats, intrathecal morphine infusion (15 μg/h) for 5 days induced (1) antinociceptive tolerance, (2) downregulation of glutamate transporters (GTs) GLT-1, GLAST, and EAAC1, (3) increasing of NMDA receptor (NMDAR) NR1 subunit expression and phosphorylation, (4) upregulation of protein kinase C gamma (PKCγ) expression, and (5) glial cell activation. On day 5, morphine challenge (15 μg/10 μl) caused a significant increase in the concentration of the excitatory amino acids (EAAs) aspartate and glutamate in the spinal CSF dialysates of morphine-tolerant rats. Intrathecal co-infusion of ultra-low dose naloxone (15 pg/h) with morphine attenuated tolerance development, reversed GTs expression, inhibited the NMDAR NR1 subunit expression and phosphorylation, and PKCγ expression, inhibited glial cell activation, and suppressed the morphine-evoked EAAs release. These effects may result in preservation of the antinociceptive effect of acute morphine challenge in chronic morphine-infused rats. Ultra-low dose naloxone infusion alone did not produce an antinociceptive effect. These findings demonstrated that attenuation of glutamatergic transmission and neuroinflammation by ultra-low dose naloxone co-infusion preserves the lasting antinociceptive effect of morphine in rats chronically infused with morphine.     

Glutamate transporters in brain ischemia： to modulate or not？

In this review, we briefly describe glutamate （Glu） metabolism and its specific transports and receptors in the central nervous system （CNS）. Thereafter, we focus on excitatory amino acid transporters, cystine/glutamate antiporters （system xc-） and vesicular glutamate transporters, specifically addressing their location and roles in CNS and the molecular mechanisms underlying the regulation of Glu transporters. We provide evidence from in vitro or in vivo studies concerning alterations in Glu transporter expression in response to hypoxia or ischemia, including limited human data that supports the role of Glu transporters in stroke patients. Moreover, the potential to induce brain tolerance to ischemia through modulation of the expression and/or activities of Glu transporters is also discussed. Finally we present strategies involving the application of ischemic preconditioning and pharmacological agents, eg β-lactam antibiotics, amitriptyline, riluzole and N-acetylcysteine, which result in the significant protection of nervous tissues against ischemia.     

Neuroprotective effects of N-acetylaspartylglutamate in a neonatal rat model of hypoxia-ischemia

Neuroprotective effects of N-acetylaspartylglutamate (NAAG), the precursor of glutamate and a selective agonist at the Group II metabotropic glutamate (mGlu) receptor, against hypoxic-ischemic brain injury were examined in a neonatal rat model of cerebral hypoxia-ischemia. The neonatal hypoxia-ischemia procedure (unilateral carotid artery ligation followed by exposure to an 8% oxygen hypoxic condition for 1.5 h) was performed in 7-day-old rat pups. Following unilateral carotid artery ligation, NAAG (0.5 to 20 mg/kg, i.p.) was administered before or after the hypoxic exposure. Brain injury was examined 1-week later by weight reduction in the ipsilateral brain and by neuron density in the hippocampal CA1 area. In the saline-treated rat, neonatal hypoxia-ischemia resulted in severe brain injury as indicated by a 24% reduction in the ipsilateral brain weight. Low doses of NAAG (2-10 mg/kg, but not 0.5 mg/kg), administered before or even if 1 h after the hypoxic exposure, greatly reduced hypoxia-ischemia-induced brain injury (3.8-14.2% reduction in the ipsilateral brain weight). A high dose of NAAG (20 mg/kg) was ineffective. While L(+)-2-Amino-4-phosphonobutyric acid (L-AP4) and trans-[1S,3R]-1-Amino-cyclopentane-1, 3-dicarboxylic acid (t-ACPD) were unable to provide protection against hypoxic-ischemic brain injury, 2-(phosphonomethyl) pentanedioic acid (2-PMPA), an inhibitor of N-acetylated alpha-linked acidic dipeptidase (NAALADase), which hydrolyzes endogenous NAAG into N-acetyl-aspartate and glutamate, significantly reduced neonatal hypoxia-ischemia-induced brain injury. (alphaS)-alpha-Amino-alpha-[(1S, 2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), a selective antagonist at the mGlu2/3 receptor, prevented the neuroprotective effect of NAAG. Neuron density data measured in the hippocampal CA1 area confirmed that ipsilateral brain weight reduction was a valid measure for hypoxic-ischemic brain injury. Neonatal hypoxia-ischemia stimulated an elevation of cyclic AMP (cAMP) concentration in the saline-treated rat brain. NAAG, L-AP4 and t-ACPD all significantly decreased hypoxia-ischemia-induced elevation of cAMP. LY341495 blocked the effect of NAAG, but not of L-AP4 or t-ACPD, on hypoxia-ischemia-stimulated cAMP elevation. The overall results suggest that the neuroprotective effect of NAAG is largely associated with activation of mGlu2/3 receptor.