The ß-lactam clavulanic acid mediates glutamate transport-sensitive pain relief in a rat model of neuropathic pain.

BACKGROUND: Following nerve injury, down-regulation of astroglial glutamate transporters (GluTs) with subsequent extracellular glutamate accumulation is a key factor contributing to hyperexcitability within the spinal dorsal horn. Some ß-lactam antibiotics can up-regulate GluTs, one of which, ceftriaxone, displays analgesic effects in rodent chronic pain models. METHODS: Here, the antinociceptive actions of another ß-lactam clavulanic acid, which possesses negligible antibiotic activity, were compared with ceftriaxone in rats with chronic constriction injury (CCI)-induced neuropathic pain. In addition, the protein expression of glutamate transporter-1 (GLT1), its splice variant GLT1b and glutamate-aspartate transporter (GLAST) was measured in the spinal cord of CCI rats. Finally, protein expression of the same GluTs was evaluated in cultured astrocytes obtained from rodents and humans. RESULTS: Repeated injection of ceftriaxone or clavulanic acid over 10 days alleviated CCI-induced mechanical hypersensitivity, whilst clavulanic acid was additionally able to affect the thermal hypersensitivity. In addition, clavulanic acid up-regulated expression of GLT1b within the spinal cord of CCI rats, whereas ceftriaxone failed to modulate expression of any GluTs in this model. However, both clavulanic acid and ceftriaxone up-regulated GLT1 expression in rat cortical and human spinal astrocyte cultures. Furthermore, clavulanic acid increased expression of GLT1b and GLAST in rat astrocytes in a dose-dependent manner. CONCLUSIONS: Thus, clavulanic acid up-regulates GluTs in cultured rodent- and human astroglia and alleviates CCI-induced hypersensitivity, most likely through up-regulation of GLT1b in spinal dorsal horn. SIGNIFICANCE: Chronic dosing of clavulanic acid alleviates neuropathic pain in rats and up-regulates glutamate transporters both in vitro and in vivo. Crucially, a similar up-regulation of glutamate transporters in human spinal astrocytes by clavulanic acid supports the development of novel ß-lactam-based analgesics, devoid of antibacterial activity, for the clinical treatment of chronic pain.

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions.

Maintenance of low extracellular glutamate ([Glu](O)) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by DL-threo-beta-benzyloxyaspartate (DL-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of DL-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC(50) values ranging from 38 to 48 microM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of DL-TBOA (100 microM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 microM) dose of DL-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.

GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation.

Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.

Metabolic distinction between vesicular and cytosolic GABA in cultured GABAergic neurons using 13C magnetic resonance spectroscopy.

GABA exists in at least two different intracellular pools, i.e., a cytoplasmic or metabolic pool and a vesicular pool. This study was performed to gain information about the quantitative role of the tricarboxylic acid (TCA) cycle in biosynthesis of GABA from glutamine when GABA was selectively released from either one of these two pools. Cultured cerebral cortical neurons (GABAergic) were incubated in a medium containing 0.5 mM [U-13C]glutamine and subsequently depolarized for release of GABA from either the vesicular or the cytoplasmic pool. The vesicular release was induced by 55 mM K+ in the presence of tiagabine, a nontransportable inhibitor of the plasma membrane GABA carriers, whereas the cytoplasmic release via a reversal of the GABA carrier was induced by exposure to N-methyl-D-aspartate (NMDA; 50 microM) in the presence of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA; 50 microM). Cell extracts were analyzed by 13C magnetic resonance spectroscopy subsequent to the incubation or depolarization. The percentage of GABA generated from glutamine via the TCA cycle decreased from 60% to 46% during depolarization, inducing GABA release from the cytoplasmic pool, whereas a significant change in this parameter was not observed after release from the vesicular pool. These observations indicate that, during release from the cytoplasmic pool, the fraction of GABA synthesized directly from glutamine without involvement of the TCA cycle is more pronounced than that occurring during resting conditions and when release occurs from the vesicular pool. This might be explained by differences in the regulation of the two isoforms of glutamate decarboxylase (GAD(65) and GAD(67)), which presumably play different roles in the maintenance of GABA in the two pools. Both isoforms were found in the cultured cerebral cortical neurons, as shown by Western blotting employing an antibody recognizing GAD(65) as well as GAD(67).

The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms.

High-affinity glutamate transporters ensure termination of glutamatergic neurotransmission and keep the synaptic concentration of this amino acid below excitotoxic levels. However, neuronal glutamate transporters, EAAC1 and EAAT4, are located outside the synaptic cleft and contribute less significantly to the glutamate uptake in the brain than two astroglial transporters, GLAST and GLT1. Aberrant functioning of the glutamate uptake system seems to be linked to some neurodegenerative disorders (eg amyotrophic lateral sclerosis, ALS). Expression of glutamate transporters is differentially regulated via distinct cellular mechanisms. GLT1, which is expressed at very low levels in cultured astrocytes, is strongly induced in the presence of neurons. The present immunocytochemical data provide further evidence that neuronal soluble factors, rather than physical contact between neurons and glia, determine the induction of GLT1 in astrocytes. This effect is apparently mediated by yet undefined growth factor(s) via the tyrphostin-sensitive receptor tyrosine kinase (RTK) signalling, that in turn, supports the downstream activation of p42/44 MAP kinases and the CREM and ATF-1 transcription factors. RTK-independent simultaneous activation of the CREB transcription factor suggests a possible involvement of complementary pathway(s). Neuronal soluble factors do not affect expression of GLAST, but induce supporting machinery for differential regulation of GLAST via the astroglial metabotropic glutamate receptors, mGluR3 and mGluR5. Thus, long-term treatment with the group I mGluR agonist, DHPG, causes down-regulation of GLAST, whereas the group II agonist, DCG-IV, has an opposite effect on the expression of GLAST in astrocytes. However, in BT4C glioma cells glutamate or other transportable substrates (D-aspartate and L-2,4-trans-PDC) induced cell-surface expression of EAAT4 in a receptor-independent manner. The activity-dependent trafficking of this transporter which also exhibits properties of a glutamate-gated chloride channel may play functional roles not only in neuronal excitability, but in glioma cell biology as well.

Cellular distribution and kinetic properties of high-affinity glutamate transporters.

L-glutamic acid is a key chemical transmitter of excitatory signals in the nervous system. The termination of glutamatergic transmission occurs via uptake of glutamate by a family of high-affinity glutamate transporters that utilize the Na+/K+ electrochemical gradient as a driving force. The stoichiometry of a single translocation cycle is still debatable, although all proposed models stipulate an inward movement of a net positive charge. This electrogenic mechanism is capable of translocating the neurotransmitter against its several thousand-fold concentration gradient, therefore, keeping the resting glutamate concentration below the treshold levels. The five cloned transporters (GLAST/EAAT1, GLT1/EAAT2, EAAC1/EAAT3, EAAT4, and EAAT5) exhibit distinct distribution patterns and kinetic properties in different brain regions, cell types, and reconstitution systems. Moreover, distinct pharmacological profiles were revealed among the species homologues. GLAST and GLT1, the predominant glutamate transporters in the brain, are coexpressed in astroglial processes, whereas neuronal carriers are mainly located in the dendrosomatic compartment. Some of these carrier proteins may possess signal transducing properties, distinct from their transporter activity. Some experimental conditions and several naturally occurring and synthetic compounds are capable of regulating the expression of glutamate transporters. However, selective pharmacological tools interfering with the individual glutamate carriers have yet to be developed.

Effects of taurine depletion on cell migration and NCAM expression in cultures of dissociated mouse cerebellum and N2A cells.

Cultures of dissociated cerebellum from 5- to 6-day-old mice as well as of the N2A neuronal cell line were exposed to guanidino ethane sulfonate (GES, 2-5 mM) to reduce the cellular taurine content. Control cultures were kept in culture medium or medium containing 2-5 mM GES plus 2-5 mM taurine to restore the intracellular taurine content. Taurine depletion led to changes in the expression of certain splice variants of NCAM mRNA such as the AAG and the VASE containing forms, while no differences were seen in the expression of the three forms of NCAM protein. In the N2A cells taurine depletion led to a decreased migration rate of the cells. The results suggest that the reduced migration rate of neurons caused by taurine depletion may be correlated to changes in expression of certain adhesion molecules such as NCAM. Moreover, taurine appears to be involved in regulation of transcription processes.

Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia.

The glutamate transporters in the plasma membranes of neural cells secure termination of the glutamatergic synaptic transmission and keep the glutamate levels below toxic concentrations. Astrocytes express two types of glutamate transporters, GLAST (EAAT1) and GLT1 (EAAT2). GLT1 predominates quantitatively and is responsible for most of the glutamate uptake activity in the juvenile and adult brain. However, GLT1 is severely down-regulated in amyotrophic lateral sclerosis, a progressive neurodegenerative disease. Furthermore, selective loss of this transporter occurs in cultured astroglia. Expression of GLAST, but not of GLT1, seems to be regulated via the glutamate receptor signalling. The present study was undertaken to examine whether neuronal factors, other than glutamate, influence the expression of astroglial glutamate transporters. The expression of GLT1 and GLAST was examined in primary cultures of cerebellar granule neurons, cortical neurons, and astrocytes under different experimental conditions, including those that mimic neuron-astrocyte interactions. Pure astroglial cultures expressed only GLAST, whereas astrocytes grown in the presence of neurons expressed both GLAST (at increased levels) and GLT1. The induction of GLT1 protein and its mRNA was reproduced in pure cortical astroglial cultures supplemented with conditioned media from cortical neuronal cultures or from mixed neuron-glia cultures. This treatment did not change the levels of GLAST. These results suggest that soluble neuronal factors differentially regulate the expression of GLT1 and GLAST in cultured astroglia. Further elucidation of the molecular nature of the secreted neuronal factors and corresponding signalling pathways regulating the expression of the astroglial glutamate transporters in vitro may reveal mechanisms important for the understanding and treatment of neurological diseases.

High affinity glutamate transporters: regulation of expression and activity.

L-Glutamic acid is a major excitatory neurotransmitter in the mammalian central nervous system. The termination of the glutamatergic transmission and the clearance of the excessive, neurotoxic concentrations of glutamate is ensured by a high affinity glutamate uptake system. Four homologous types of Na/K-dependent high affinity glutamate transporters, glutamate/aspartate transporter, glutamate transporter 1, excitatory amino acid carrier 1, and excitatory amino acid transporter 4, have recently been cloned and were assigned to a separate gene family, together with two neutral amino acid carriers, alanine/serine/cysteine transporter 1/serine/alanine/threonine transporter and adipocyte amino acid transporter. The genomic organization of these transporters is still under investigation. Very little is known about the nature of the factors and molecular mechanisms that regulate developmental, regional, and cell type-specific expression of the glutamate transporters and their aberrant functioning in neurodegenerative diseases (e.g., amyotrophic lateral sclerosis and Alzheimer's disease). Some experimental conditions (e.g., ischemia, corticostriatal lesions, hyperosmolarity, culturing conditions) and several naturally occurring and synthetic compounds (e.g., glutamate receptor agonists, dopamine, alpha1- and beta-adrenergic agonists, cAMP, phorbol esters, arachidonic acid, nitric oxide, oxygen free radicals, amyloid beta-peptide, tumor necrosis factor-alpha, glucocorticosteroids, unidentified neuronal factors) affect the molecular expression and activity of glutamate transporters. Further elucidation of the molecular events that link epigenetic signals with transcriptional and post-transcriptional mechanisms (e.g., alternative splicing, translation and post-translational modifications) is crucial for the development of selective pharmacological tools and strategies interfering with the expression of the individual glutamate transporters.

Glutamate receptor agonists up-regulate glutamate transporter GLAST in astrocytes.

Long-term treatment of astrocytes in primary culture with L-glutamate (0.1-3 mM) resulted in a dose-dependent increase in D-[3H]aspartate uptake. The effect was abolished by an antagonist of kainate/AMPA receptors, CNQX, and mimicked by kainate, but not by AMPA or tACPD. Both glutamate and kainate caused a dramatic up-regulation (82% and 69%, respectively) of GLAST, a predominant glutamate transporter in cultured astroglia, though the mRNA levels appeared unaffected. Long-term treatment of cultures with dBcAMP stimulated D-[3H]aspartate uptake as well as GLAST expression. Apart from glutamate, none of the agonists used was capable of increasing further the uptake capacity of the dBcAMP-treated astroglia. The glutamate receptor-dependent modulation of glutamate transport in astroglial cultures may represent a novel feedback regulatory mechanism for glutamate uptake in the brain.

Two types of amyloid precursor protein (APP) mRNA in rat glioma cell lines: upregulation via a cyclic AMP-dependent pathway.

APP is a multifunctional transmembrane glycoprotein and the only known natural source of beta A4 peptide-the major constituent of senile plaques in Alzheimer's disease (AD). The expression and cAMP-dependent regulation of the APP gene were investigated in primary cultures of rat astrocytes and two related glioma cell lines, BT4C and BT4Cn, which exhibit distinct invasive phenotypes. Besides the well-characterized 3.5 kb APP mRNA class, a robust expression of an unusual 2.8 kb APP mRNA class was revealed by Northern blotting in both glioma cell lines, but not in the astrocytes. Low amounts of the 2.8 kb APP mRNA species were also observed in rat liver and occasionally in aged rat brain. The 2.8 kb APP mRNA contained exons 1-18 and may thus be generated by truncation of the 3' untranslated region. For the first time, regulation of the APP gene via a cAMP-dependent mechanism was shown. Exposure to dBcAMP dramatically upregulated the 3.5 and 2.8 kb transcripts in BT4C cells, and, to a lesser extent, in BT4Cn cells where the constitutive expression of the APP gene was much higher. Elucidation of the factors involved in cAMP-dependent induction of APP mRNA in these cells may shed more light on the molecular mechanisms of APP overexpression.

Expression of amyloid precursor protein (APP) in rat brain and cultured neural cells.

Cell type-specific and developmental patterns of APP expression were investigated in rat brain and cultured neural cells. Nearly all astrocytes were APP-positive, whereas only selected population of neurons appeared to express APP. In these neurons, APP immunoreactivity was preferentially restricted to single processes. mRNAs encoding the major APP isoforms, APP695 and APP770, were co-expressed as 3.4-3.6 kb transcripts both in astrocytes and neurons. In addition, an unusual 2.8 kb mRNA size class was revealed in cultured cerebellar granule neurons by means of the probe recognizing APP770 mRNA. Also, for the first time, APP714 mRNA was detected in rat brain by northern blotting. The steady-state levels of these transcripts were increased from birth up to postnatal day 20, whereas no apparent changes were observed after reaching adulthood. These data hint at the involvement of APP in the major morphogenetic events taking place in rat brain during the first three postnatal weeks.

Cyclic AMP regulates NCAM expression and phosphorylation in cultured mouse astrocytes.

The neural cell adhesion molecule (NCAM) plays a key morphoregulatory role during neural development. Patterns of NCAM expression were investigated in cultured astrocytes exhibiting distinct states of morphological differentiation. In vitro differentiation of confluent primary cultures of astrocytes was enhanced by a long-term (7 days) treatment with dibutyryl cyclic AMP (dBcAMP). The diversity of NCAM mRNAs arising from alternative splicing of a single primary transcript was analyzed by Northern blotting. Synthetic DNA-oligonucleotide probes, designed to recognize selected exons (exons 7, 15, and VASE) or exon combinations (exons 7/8, exons 12/13, and exons 12/a/AAG/13), revealed NCAM mRNA classes of 6.7, 5.2, and 2.9 kilobases (kb) in both control and dBcAMP-treated astrocytes. Although long-term treatment of astrocytes with dBcAMP did not change the qualitative pattern of NCAM transcripts, the relative abundance of individual mRNA species appeared to be altered: in morphologically differentiated astrocytes, a general elevation of NCAM mRNA as well as preferential accumulation of the 6.7 kb message were found. The quantitative rise within this size class may primarily be accounted for by a dramatic upregulation of exon VASE-containing NCAM transcripts. In dBcAMP-treated astrocytes, immunoblot analysis revealed an unexpected expression of the 190 kDa NCAM-A isoform as well as an intense staining of bands corresponding to NCAM-B (135 kDa) and NCAM-C (115 kDa), as compared to the control pattern of untreated astrocytes. Furthermore, NCAM-A and NCAM-B were shown to be phosphorylated in astrocytes subjected to long-term treatment with dBcAMP, whereas no incorporation of [32P]phosphate into NCAM was demonstrated in untreated astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of NCAM diversity in cultured neurons.

A single transcript of the NCAM gene undergoes differential processing resulting in a multiplicity of mRNAs and their translation products. In this study, the diversity of NCAM in rat primary neuronal cultures was investigated utilizing immuno- and Northern blot analyses. NCAM polypeptides of 190 kDa (NCAM-A) and 135 kDa (NCAM-B) were shown to be associated with the neuronal phenotype. These data were confirmed by Northern blotting, which in both neocortical neurons and cerebellar granule neurons revealed mRNA classes of 7.4 kb and 6.7 kb encoding for NCAM-A and -B, respectively. However, oligonucleotide probes, specific for selected exons or exon combinations, revealed special features of cerebellar granule neurons as compared to neocortical neurons: expression of 4.3 kb NCAM mRNA, a relatively low amount of VASE-containing variants, and an apparent lack of mRNA species containing exons alpha and an AAG insert between exons 12 and 13. Distinct patterns of NCAM mRNA may putatively be related to the regional origin and functional specificity of the investigated neurons.