Dihydrokainate Research Articles

Glutamate transporters in platelets: EAAT1 decrease in aging and in Alzheimer’s disease

Platelets release glutamate upon activation and are an important clearance system of the amino acid from blood, through high-affinity glutamate uptake, similar to that described in brain synaptosomes. Since platelet glutamate uptake is decreased in neurodegenerative disorders, we performed a morphological and molecular characterization of platelet glutamate transporters. The three major brain glutamate transporters EAAT1, EAAT2 and EAAT3 are expressed in platelets, with similar molecular weight, although at lower density than brain. A Na +-dependent-high-affinity glutamate uptake was competitively inhibited by known inhibitors but not by dihydrokainic acid, suggesting platelet EAAT2 does not play a major role in glutamate uptake at physiological conditions. We observed decreased glutamate uptake V max, without modification of transporter affinity, in aging, which could be linked to the selective decrease of EAAT1 expression and mRNA. Moreover, in AD patients we found a further EAAT1 reduction compared to age-matched controls, which could explain the decrease of platelet uptake previously described. Platelet glutamate transporters may be used as peripheral markers to investigate the role of glutamate in patients with neuropsychiatric disorders.

Selective blockade of astrocytic glutamate transporter GLT-1 with dihydrokainate prevents neuronal death during ouabain treatment of astrocyte/neuron cocultures.

Glutamate (Glu) is a major excitatory neurotransmitter of the mammalian central nervous system and under normal conditions plays an important role in information processing in the brain. Therefore, extracellular Glu is subject to strong homeostasis. Astrocytes in the brain have been considered to be mainly responsible for the clearance of extracellular Glu. In this study, using mixed neuron/astrocyte cultures, we investigated whether astrocytic Glu transporter GLT-1 is crucial to the survival of neurons under various conditions. Treatment of the mixed cultures with a low concentration of Glu did not produce significant death of neurons. However, cotreatment with dihydrokainate (DHK), a specific blocker of GLT-1, resulted in significant neuronal death that was suppressed by an antagonist of N-methyl-D-aspartate (NMDA) receptors. These results suggested that astrocytic GLT-1 participated in the clearance of extracellular Glu and protected neurons from NMDA receptor-mediated toxicity. When the cultures were treated with ouabain, an inhibitor of Na(+)/K(+)-ATPase, a low concentration of Glu resulted in massive neuronal death that was also suppressed by cotreatment with an antagonist of NMDA receptors. In this case, however, cotreatment with DHK significantly protected neurons from death, suggesting that GLT-1 was responsible for the death of neurons. The present study provides evidence suggesting that astrocytes use their Glu transporter GLT-1 to protect neurons from Glu toxicity, but, ironically, also use GLT-1 to kill neurons through Glu toxicity depending on their status.

Glutamate uptake controls expression of a slow postsynaptic current mediated by mGluRs in cerebellar Purkinje cells.

At the cerebellar parallel fiber-Purkinje cell synapse, isolated presynaptic activity induces fast excitatory postsynaptic currents via ionotropic glutamate receptors while repetitive, high-frequency, presynaptic activity can also induce a slow excitatory postsynaptic current that is mediated by metabotropic glutamate receptors (mGluR1-EPSC). Here we investigated the involvement of glutamate uptake in the expression of the mGluR1-EPSC. Inhibitors of glutamate uptake led to a large increase of the mGluR1-EPSC. D-aspartate (0.4 mM) and L(-)-threo-3-hydroxyaspartate (0.4 mM) increased the mGluR1-EPSC approximately 4.5 and approximately 9-fold, respectively, while dihydrokainic acid (1 mM), had no significant effect on the mGluR1-EPSC. D-aspartate (0.4 mM) shifted the concentration-response curve of the depression of the mGluR1-EPSC by the low-affinity mGluR1 antagonist (S)-a-Methyl-4-carboxyphenylglycine [(S)-MCPG] to higher concentrations and decreased the stimulus intensity and the number of necessary stimuli to evoke an mGluR1-EPSC. Depression of the mGluR1-EPSC by rapid pressure application of (S)-MCPG at varying time intervals after tetanic stimulation of the parallel fibers indicated that the glutamate concentration in the peri- and extrasynaptic space decayed with time constants of 36 and 316 ms under control conditions and with inhibition of glutamate uptake, respectively. These results show that expression of the slow mGluR-mediated excitatory postsynaptic current is controlled by glutamate transporter activity. Thus in contrast to fast glutamatergic synaptic transmission, metabotropic glutamate receptor-mediated transmission is critically dependent on the activity and capacity of glutamate uptake.

Mechanisms of glutamate release in the rat spinal cord slices during metabolic inhibition.

Glutamate toxicity is a viable hypothesis to explain the expanding tissue degeneration occurring after traumatic or ischemic spinal cord injury. One important component in this process is the acute, excessive release of glutamate. In the current communication, the glycolytic inhibitor iodoacetate was used to induce metabolic inhibition in spinal cord slices and thereby provide an in vitro model to study the mechanisms of pathological glutamate release in the spinal cord. The evoked glutamate release was not Ca2+-dependent. Exclusion of NaCl reduced the evoked release of endogenous glutamate by 56%, while excluding Na+ increased release. Glutamate release was also reduced by the PLA2 inhibitors indomethacin (40%), arachidonyltrifluoromethyl ketone (45%) and 4-bromophenacyl bromide (36%). Blocking reverse glutamate transport by preincubation with 1 mM dihydrokainic acid reduced evoked release by 41%. However, when the dihydrokainic acid and arachidonyltrifluoromethyl ketone treatments were combined, no additive effect of the two substances was seen. These findings suggest that glutamate is released by three mechanisms from the energy compromised spinal cord: (1) in response to cellular swelling, most likely by the regulatory volume decrease, (2) by PLA2-mediated breakdown of the cell membrane and diffusion of glutamate down its concentration gradient, and (3) through reversal of the glutamate transporter.

Effect of dihydrokainate on the antitumor activity of doxorubicin

For biochemical modulation, components of green tea have been shown to be useful modulators in combination with doxorubicin (DOX). We have confirmed that theanine enhances the antitumor activity of DOX due to inhibition of DOX efflux from tumor cells. Because theanine is a glutamate analogue, we found that it is associated with a change in the drug transport system on the tumor cell membrane, in particular glutamate transporters. We examined the effect of dihydrokainate (DHK), one of the useful glutamate transporter inhibitors. DHK also inhibits DOX efflux significantly and reduces the glutamate uptake by Ehrlich ascites carcinoma cells. The potential contribution of glutamate transporters not only to glutamate uptake but also to cell membrane export of DOX has been shown. In addition, the combination of DHK with DOX significantly enhances the antitumor activity of DOX, by 1.8-fold ( P<0.001). The DOX concentration in tumors significantly increases on combination with DHK and is correlated with the reduced tumor weight. On the other hand, DHK tends to reduce the DOX concentration in normal tissues. We expect that DHK has different actions in tumor and normal tissues because different isoforms of glutamate transporters are expressed in the two tissues. Thus, the results suggest that DHK is a novel and useful modulator for inducing enhancement of antitumor activity.

Long-term potentiation and contextual fear conditioning increase neuronal glutamate uptake.

Induction and expression of long-term potentiation (LTP) in area CA1 of the hippocampus require the coordinated regulation of several cellular processes. We found that LTP in area CA1 was associated with an N-methyl-D-aspartate (NMDA) receptor-dependent increase in glutamate uptake. The increase in glutamate uptake was inhibited by either removal of Na+ or addition of D,L-threo-beta-hydroxyaspartate. Dihydrokainate (DHK), a specific inhibitor of the glial glutamate transporter GLT-1, did not block the increase in glutamate uptake. LTP was also associated with a translocation of the EAAC1 glutamate transporter from the cytosol to the plasma membrane. Contextual fear conditioning increased the maximum rate (Vmax) of glutamate uptake and membrane expression of EAAC1 in area CA1. These results indicate that regulation of glutamate uptake may be important for maintaining the level of synaptic strength during long-term changes in synaptic efficacy.

Proximity of two oppositely oriented reentrant loops in the glutamate transporter GLT-1 identified by paired cysteine mutagenesis.

Sodium- and potassium-coupled transporters clear the excitatory neurotransmitter glutamate from the synaptic cleft. Their function is essential for effective glutamatergic neurotransmission. Glutamate transporters have an unusual topology, containing eight membrane-spanning domains and two reentrant loops of opposite orientation. We have introduced pairwise cysteine substitutions in several structural elements of the GLT-1 transporter. A complete inhibition of transport by Cu(II)(1,10-phenanthroline)(3) is observed in the double mutants A412C/V427C and A364C/S440C, but not in the corresponding single mutants. No inhibition is observed in more then 20 other double cysteine mutants. The Cu(II)(1,10-phenanthroline)(3) inhibition can be partly prevented by the nontransportable glutamate analogue dihydrokainate. Treatment with dithiothreitol restores much of the transport activity. Moreover, micromolar concentrations of cadmium ions reversibly inhibit transport catalyzed by A412C/V427C and A364C/S440C double mutants, but not by the corresponding single mutants. Inhibition by Cu(II)(1,10-phenanthroline)(3) and by cadmium is only observed when the cysteine pairs are introduced in the same polypeptide. Therefore, in both cases the proximity appears to be intra- rather than intermolecular. Positions 364 and 440 are located on reentrant loop I and II, respectively. Our results suggest that these two loops, previously shown to be essential for glutamate transport, come in close proximity.

Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type horizontal cells

Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or dihydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R-and G-cones to LHCs, respectively.

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.

Effects of glial glutamate transporter inhibitors on intracellular Na + in mouse astrocytes

The effects of inhibitors of the glial Na +/glutamate co-transporter on the intracellular Na + concentration ([Na +] i) were investigated in mouse cortical astrocytes. [Na +] i was monitored by fluorescence microscopy on single astrocytes using the Na +-sensitive probe sodium-binding benzofuran isophtalate. Application of the competitive inhibitors threo-β-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid ( t-PDC) resulted in robust and reversible increases in [Na +] i that were comparable in shape to the response to glutamate but about twice lower in amplitude. As previously observed with glutamate, the amplitude of the [Na +] i response to these compounds was concentration-dependent with EC 50 values of 11.1 μM (THA) and 7.6 μM ( t-PDC), as was the initial rate of [Na +] i rise (EC 50 values of 14.8 μM for THA and 11.5 μM for t-PDC). Both compounds diminished the response to subsequent glutamate applications, possibly because of an inhibitory effect of the intracellularly-accumulated compounds. In comparison, the newly-developed compound threo-β-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of [Na +] i up to a concentration of 500 μM. TBOA inhibited the [Na +] i response evoked by 200 μM glutamate in a concentration-dependent manner with IC 50 values of 114 and 63 μM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked [Na +] i increase by TBOA was ∼70%. The residual response persisted in the presence of a non-NMDA receptor antagonist or the inhibitor of the GLT-1 glutamate transporters, dihydrokainate (DHK). In view of the complete reversibility of its effects, TBOA represents a very useful pharmacological tool for studies of glutamate transporters.

GLIAL GLUTAMATE TRANSPORTER GLT-1 DOES NOT REVERSE ITS FUNCTION IN ISCHEMIC PENUMBRA.

4 Functional glutamate uptake systems are critical for maintenance of normal synaptic transmission. Several studies have shown that in severely ischemic brain regions, massive efflux of excitatory amino acids (EAAs) results, in large portion, from the reversal of Na + -dependent glutamate transporters. This study was designed to examine the function of the predominant glial transporter, GLT-1, in a brain area representative of ischemic penumbra. Spontaneously hypertensive rats were subjected to a 45 min transient focal ischemia by intraluminal occlusion of the middle cerebral artery (MCAO). In this model, the core ischemic region resides in the medial striatum while the surrounding parietal cortex represents a penumbral area. Temporal profiles of extracellular levels of glutamate [Glu] e and aspartate [Asp] e were assessed using microdialysis in the parietal cortex. Preischemic [Glu] e and [Asp] e were constant and no changes were observed in sham operated animals. MCAO induced rapid efflux of EAAs with the peak of 7 and 9 fold increases in [Glu] e and [Asp] e at 20 min post MCAO, respectively. Levels of both EAAs returned to baseline prior to reperfusion. In contrast, a local infusion (reverse microdialysis) of the GLT-1 selective nontransportable inhibitor, dihydrokainate (DHK, 1 mM) during the ischemic insult,prolonged and increased the efflux of both EAAs throughout MCAO interval resulting in peak 9 and 16 fold increases (p>0.05) in [Glu] e and [Asp] e , respectively, at 40 min post MCAO. The total ischemic efflux of Glu and Asp (areas under the curve, AUC) was increased by 70% and 98%. Infusion of the voltage dependent Na + channel blocker tetrodotoxin (TTX, 10 μM) during the ischemic period attenuated the initial MCAO-induced Glu and Asp efflux from 7 to 2 fold and from 9 to 2 fold, respectively. These data provide the first evidence that within the ischemic brain penumbra, GLT-1 remains functional and displays no evidence of reverse transport. In addition, the attenuation of MCAO-induced EAA efflux by TTX indicates that the source of penumbral release is primarily neuronal.

Characterization of endogenous amino acid efflux from hippocampal slices during chemically-induced ischemia.

Using sodium (NaN3)-induced anoxia plus aglycaemia as a model of chemically-induced ischemia, we have characterized the endogenous release of excitatory and inhibitory amino acids from superfused hippocampal slices. Chemical ischemia produced an azide (1-30 mM) dose-dependent increase in the efflux of glutamate, aspartate and GABA. These increases were attenuated to varying degrees by removal of Ca2+, or the addition of the voltage dependent Na+-channel blocker tetrodotoxin (TTX), the selective Ca2+ channel blockers conotoxin MVIIA, MVIIC, and nifedipine, the NMDA antagonist MK801, the AMPA antagonist GYKI-52466. Similarly, addition of the GLT-1 glutamate transport inhibitor dihydrokainate (DHK) and the anti-estrogen/anion channel blocker tamoxifen also attenuated the efflux of glutamate and GABA. It would therefore appear that the increases in amino acid efflux induced by chemical ischemia originates from both the neuronal pool, via conventional exocytotic release, and glial sources via reversal of the GLT-1 transporter and anion channel regulated cell swelling.

Transient increase in the high affinity [ 3H]- l-glutamate uptake activity during in vitro development of hippocampal neurons in culture

The glial GLAST and GLT-1 glutamate transporters are transiently expressed in hippocampal neurons as shown by immunocytochemistry (Plachez et al., 2000. J. Neurosci. Res., 59, 587–593). In order to test if this transient expression is associated to a transient glutamate uptake activity, [ 3H]-glutamate uptake was studied during the in vitro development of embryonic hippocampal neurons cultured in a defined (serum free) medium. In these cultures, the ratio of the number of glial cells to the number of neurons increased from 1.7 to 11.3% during the first 10 days of culture, while 77% of the neurons died. The number of neurons then remains stable up to 23 days of culture. The initial glutamate uptake velocity at 20 and 200 μM [ 3H]-glutamate usually increased about five times between 1 and 10 days in vitro (DIV). Interestingly, at 2 μM [ 3H]-glutamate, the uptake initial velocity showed a biphasic pattern, with a transient peak between 1 and 6 DIV, the maximum being reached at 2 DIV and a delayed regular increase from 8 to 23 DIV. The concentration-dependent curves were best fitted with two saturable sites high and low affinities, at both 2 and 10 DIV. To pharmacologically characterize the transient increased glutamate uptake activity, four uptake inhibitors, l- threo-3-hydroxy-aspartic acid (THA), l- trans-pyrrolidine-2,4-dicarboxylic acid ( l- trans-2,4-PDC), dihydrokainate (DHK), and dl- threo-β-benzyloxyaspartate (TBOA) were tested. THA, l- trans-2,4-PDC and dl-TBOA inhibited glutamate uptake both at 2 and 10 DIV, while the GLT-1 selective uptake inhibitor DHK neither strongly affected the uptake at 2, nor at 10 DIV. These data indicated that, besides the regular increase in the glial-dependent glutamate uptake activity, a transient high-affinity, DHK insensitive, glutamate transport activity in hippocampal neurons in culture is present. This latter activity could potentially be related to the transient expression of the glial GLAST transporter in neurons.

Evidence that reversed glutamate uptake contributes significantly to glutamate release following experimental injury to the rat spinal cord

Released excitatory amino acids contribute significantly to secondary damage following spinal cord injury. Reversal of normal transport due to cell membrane depolarization may contribute to this release. We tested this by administering dihydrokainic acid (DHK), a non-transported glutamate uptake blocker, into the rat spinal cord by microdialysis in association with contusion spinal cord injury. Glutamate release in response to injury was reduced by 34% ( P<0.05) when 3 mM DHK was administered within the microdialysis fiber, suggesting that reversed transport is an important contributor to glutamate release upon spinal cord injury.

The modulation of ecto-nucleotidase activities by glutamate in cultured cerebellar granule cells.

Several enzymes hydrolyze ATP, producing ADP which is hydrolyzed to AMP. Ecto-5'-nucleotidase produces adenosine from AMP. Glutamate (Glu) is an excitatory neurotransmitter and increases extracellular adenosine levels, which is considered an important inhibitory neuromodulator. Here we show that Glu activates ADP and AMP hydrolysis. NMDA and kainic acid (KA) also increased these enzymatic activities, but 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD) had no effect. Dihydrokainate (DHK), an inhibitor of glutamate uptake, also blocked glutamate-evoked activation of ecto-nucleotidases, suggesting that this activation was also Glu transporters dependent. Therefore, we suggest that the Glu-evoked stimulation of ecto-nucleotidases might contribute to the increase of adenosine in extracellular space induced by Glu.

Glutamate transport blockade has a differential effect on AMPA and NMDA receptor-mediated synaptic transmission in the developing barrel cortex

High affinity glutamate transport plays an important role in maintaining a low extracellular glutamate concentration in the CNS. Excitotoxicity due to a loss of glutamate transporter function has been implicated in disease processes such as stroke and amyotrophic lateral sclerosis (ALS). We studied the effects of glutamate transport inhibitors on thalamocortical synapses at developing (postnatal day 3–8) layer IV neurons in the barrel cortex using the thalamocortical slice preparation and whole-cell recordings. Inhibition of glutamate transport by D,L-threo-β-hydroxyaspartate (THA), a combination of THA and dihydrokainate (DHK), or by L- trans-pyrrolidine-2,4-dicarboxylate (tPDC), caused a reversible blockade of AMPA and kainate receptor-mediated dual component excitatory postsynaptic currents (AMPA/KA EPSCs). This effect was not blocked by cyclothiazide (CTZ) indicating that is was not due to desensitisation of AMPARs. Under conditions in which NMDA receptors were unblocked the transport inhibitors caused the massive activation of NMDA receptors leading to the rapid loss of recordings. Previous studies using these transport inhibitors on brain slices from older animals reported no or only modest effects on synaptic transmission. Therefore the data in the present study suggest that neurons in the developing neocortex are particularly sensitive to glutamate transporter function. Furthermore the effects of transport inhibition are dependent upon whether neurons are sufficiently depolarised to relieve the voltage-dependent block of NMDA receptors.

Nicotine stimulation of extracellular glutamate levels in the nucleus accumbens: neuropharmacological characterization.

In the present study, we have characterized the neuropharmacological regulation of nicotine-induced increases in extracellular nucleus accumbens glutamate levels. Sprague-Dawley rats were stereotaxically implanted with 2 mm microdialysis probes in the nucleus accumbens and on the following day in vivo microdialysis experiments were performed in awake, freely moving animals. An acute dose of nicotine (0.3-0.6 mg/kg, s.c.) produced an increase in nucleus accumbens glutamate levels with a maximal increase of approximately 50% following the higher dose. No changes in nucleus accumbens aspartate levels were found. The increase in glutamate levels following nicotine (0.3 mg/kg, s.c.) was blocked by mecamylamine (1 mg/kg, i.p. ) but not by haloperidol (0.2 mg/kg, i.p.) pretreatment. Local perfusion of artificial cerebrospinal fluid (CSF) without calcium did not alter nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. Local perfusion with a selective blocker for the GLT-1 glutamate transporter, dihydrokainic acid (DHKA) (10(-4) M), had no effect, while local perfusion with a nonselective glutamate transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (10(-4) M), blocked nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. In animals previously dopamine denervated by local injections of 6-hydroxydopamine (6-OHDA) into the nucleus accumbens, nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels was enhanced vs. sham-lesioned animals. These findings demonstrate a novel form of nucleus accumbens glutamate release that is dopamine- and calcium-independent. The ability of PDC to block the effects of nicotine suggest that a glutamate transporter may be involved in mediating the stimulation of glutamate release.

Steroid Hormone-Inducible Expression of the GLT-1 Subtype of High-Affinity l-Glutamate Transporter in Human Embryonic Kidney Cells

The cDNA encoding the predominant rat brain high-affinity l-glutamate transporter GLT-1 was isolated and subcloned into the pIND expression vector for the establishment of steroid hormone inducible expression in vitro using the ecdysone-inducible mammalian expression system. Steroid hormone-inducible expression was demonstrable in a stable cell line designated HEK/GLT-1. Treatment of HEK/GLT-1 cells with 10 μM ponasterone A for 24 hincreased the maximum velocity (Vmax) of Na+-dependent l-glutamate uptake by greater than 10-fold, as compared with the uninduced cells. Equivalent levels of l-glutamate transport capacity were observed in the uninduced GLT-1 cell line and the host cell line indicating that the expression of GLT-1 was tightly regulated. To confirm that the increased l-glutamate uptake observed in HEK/GLT-1 cells following induction was attributable to the expression of GLT-1, rather than the up-regulation of the endogenously expressed EAAT3 subtype present in the host cells, we evaluated the effects of the selective GLT-1 inhibitors dihydrokainate (DHK) and kainate. Both DHK and kainate produced concentration-dependent inhibition of the l-glutamate uptake into HEK/GLT-1 cells, and the estimated IC50 values were consistent with those described for the cloned GLT-1. These results demonstrate that the expression of GLT-1 can be tightly regulated in vitro using the ecdysone system.

Na+-dependent Glutamate Transporters (EAAT1, EAAT2, and EAAT3) of the Blood-Brain Barrier: A MECHANISM FOR GLUTAMATE REMOVAL

Na(+)-dependent transporters for glutamate exist on astrocytes (EAAT1 and EAAT2) and neurons (EAAT3). These transporters presumably assist in keeping the glutamate concentration low in the extracellular fluid of brain. Recently, Na(+)-dependent glutamate transport was described on the abluminal membrane of the blood-brain barrier. To determine whether the above-mentioned transporters participate in glutamate transport of the blood-brain barrier, total RNA was extracted from bovine cerebral capillaries. cDNA for EAAT1, EAAT2, and EAAT3 was observed, indicating that mRNA was present. Western blot analysis demonstrated all three transporters were expressed on abluminal membranes, but none was detectable on luminal membranes of the blood-brain barrier. Measurement of transport kinetics demonstrated voltage dependence, K(+)-dependence, and an apparent K(m) of 14 microM (aggregate of the three transporters) at a transmembrane potential of -61 mV. Inhibition of glutamate transport was observed using inhibitors specific for EAAT2 (kainic acid and dihydrokainic acid) and EAAT3 (cysteine). The relative activity of the three transporters was found to be approximately 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively. These transporters may assist in maintaining low glutamate concentrations in the extracellular fluid.

Properties of excitatory amino acid transport in the human U373 astrocytoma cell line

In this study, we describe the presence of Na +-dependent high-affinity l-glutamate transport activity in the human U373 astrocytoma cell line. U373 cells exhibited a robust accumulation of l-glutamate which was predominantly (85%) extracellular Na +-dependent. Kinetic analysis of this transport activity revealed that the uptake followed first-order Michaelis–Menten kinetics and was high-affinity in nature. The kinetic parameters estimated by Eadie–Hofstee transformation of the saturable uptake were 37.3±5.1 μM for K m and 0.13±0.02 nmol min −1 mg −1 protein for V max. A total of 14 known inhibitors of high-affinity l-glutamate transport were examined for their abilities to inhibit l-glutamate uptake by U373 cells. Three compounds, kainate (KA), dihydrokainate (DHK) and α-aminoadipic acid produced less than 30% inhibition at 1 mM. The lack of effect of both KA and DHK indicates that the predominant astroglial l-glutamate transporter EAAT2 (excitatory amino acid transporter 2) does not contribute to the uptake activity present in these cells. The rank order of inhibitory potency for the remaining 11 compounds tested was l-cysteine sulphinate= l-CCG-III= l-cysteate= l-aspartate=threo-β-hydroxyaspartate> trans-PDC> d-aspartate=MPDC>β-glutamate> l-CCG-IV= l-aspartate-β-hydroxamate. Pre-treatment of U373 cells with phorbol ester for 30 min resulted in a 56% decrease in l-glutamate uptake and this effect was blocked in a concentration-dependent manner by the PKC inhibitor bisindolylmaleimide I. Expression of l-glutamate transporters by U373 cells was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western analysis. Transcripts for both the EAAT1 and EAAT3 transporter subtypes were detected but not for EAATs 2, 4, and 5. Immunoblot analysis confirmed the presence of EAAT3 protein, however, we were unable to detect EAAT1 protein. In conclusion, the Na +-dependent high-affinity l-glutamate transport into human U373 astrocytoma cells appears to be mediated predominantly by the EAAT3 subtype.