Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal.
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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. (+info)
Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters.
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Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells. (+info)
Glutamate induces rapid upregulation of astrocyte glutamate transport and cell-surface expression of GLAST.
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Glutamate transporters clear glutamate from the extracellular space by high-affinity binding and uptake. Factors that regulate glutamate transporter expression and activity can thereby influence excitatory neurotransmission. Transporter function in GABAergic and other systems has been shown to be regulated by transporter substrates. Here, glutamate regulation of glutamate transport was studied using primary murine astrocyte cultures that express the GLAST (EAAT1) and GLT-1 (EAAT2) transporter subtypes. Glutamate was found to stimulate glutamate transport capacity (V(max)) in a dose- and time-dependent manner. The maximal increase was 100%, with an ED(50) of 40 microM glutamate and with onset beginning approximately 15 min after onset of glutamate exposure. The uptake stimulation was reproduced by D-aspartate, which is also a transporter substrate, but not by nontransported glutamate receptor agonists. Moreover, glutamate incubation did not stimulate transport when performed in a sodium-free medium, suggesting that the stimulatory effect of glutamate is triggered by increased transporter activity rather than receptor activation. Treatment with the actin-disrupting agents cytochalasin B or cytochalasin D prevented the glutamate-induced increase in glutamate uptake. Biotinylation labeling of membrane surface proteins showed that glutamate incubation produced an increase in GLAST expression at the astrocyte cell surface. These results suggest that cell-surface expression of GLAST can be rapidly regulated by glutamate through a process triggered by GLAST activity and involving the actin cytoskeleton. This feedback loop provides a mechanism by which changes in extracellular glutamate concentrations could rapidly modulate astrocyte glutamate transport capacity. (+info)
Diffuse bipolar cells provide input to OFF parasol ganglion cells in the macaque retina.
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Parasol retinal ganglion cells are more sensitive to luminance contrast and respond more transiently at all levels of adaptation than midget ganglion cells. This may be due, in part, to differences between bipolar cells that provide their input, and the goal of these experiments was to study these differences. Midget bipolar cells are known to be presynaptic to midget ganglion cells. To identify the bipolar cells presynaptic to parasol cells, these ganglion cells were intracellularly injected with Neurobiotin, cone bipolar cells were immunolabeled, and the double-labeled material was analyzed. In the electron microscope, we found that DB3 diffuse bipolar cells labeled by using antiserum to calbindin D-28k were presynaptic to OFF parasol cells. In the confocal microscope, DB3 bipolars costratified with OFF parasol cell dendrites and made significantly more appositions with them than expected due to chance. Flat midget bipolar cells were labeled with antiserum to recoverin. Although they made a few appositions with parasol cells, the number was no greater than would be expected when two sets of processes have overlapping distributions in the inner plexiform layer. DB2 diffuse bipolar cells were labeled with antibodies to excitatory amino acid transporter 2, and they also made appositions with OFF parasol cells. These results suggest that DB2 bipolar cells are also presynaptic to OFF parasol ganglion cells, but midget bipolar cells are not. We estimate that midperipheral OFF parasol cells receive approximately 500 synapses from 50 DB3 bipolar cells that, in turn, receive input from 250 cones. (+info)
Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter.
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1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system. (+info)
Selective high-affinity transport of aspartate by a Drosophila homologue of the excitatory amino-acid transporters.
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Excitatory amino-acid transporters (EAATs) are structurally related plasma membrane proteins that mediate the high-affinity uptake of the acidic amino acids glutamate and aspartate released at excitatory synapses, and maintain the extracellular concentrations of these neurotransmitters below excitotoxic levels [1] [2] [3] [4]. Several members of the EAAT family have been described previously. So far, all known EAATs have been reported to transport glutamate and aspartate with a similar affinity. Here, we report that dEAAT2 - a nervous tissue-specific EAAT homologue that we recently identified in the fruit fly Drosophila [5] - is a selective Na(+)-dependent high-affinity aspartate transporter (K(m) = 30 microM). We found that dEAAT2 can also transport L-glutamate but with a much lower affinity (K(m) = 185 microM) and a 10- to 15-fold lower relative efficacy (V(max)/K(m)). Competition experiments showed that the binding of glutamate to this transporter is much weaker than the binding of D- or L-aspartate. As dEAAT2 is the first known EAAT to show this substrate selectivity, it suggests that aspartate may play a specific role in the Drosophila nervous system. (+info)
Isolation of current components and partial reaction cycles in the glial glutamate transporter EAAT2.
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The kinetic properties of the excitatory amino acid transporter EAAT2 were studied using rapid applications of L-glutamate to outside-out patches excised from transfected human embryonic kidney 293 cells. In the presence of the highly permeant anion SCN(-), pulses of glutamate rapidly activated transient anion channel currents mediated by the transporter. In the presence of the impermeant anion gluconate, glutamate pulses activated smaller currents predicted to result from stoichiometric flux of cotransported ions. Both anion and stoichiometric currents displayed similar kinetics, suggesting that anion channel gating and stoichiometric charge movements are linked to early transitions in the transport cycle. Transporter-mediated anion currents were recorded with ion and glutamate gradients favoring either unidirectional influx or exchange. Analysis of deactivation and recovery kinetics in these two conditions suggests that, after binding, translocation of substrate is more likely than unbinding under physiological conditions. The kinetic properties of EAAT2, the dominant glutamate transporter in brain astrocytes, distinguish it as an efficient sink for synaptically released glutamate. (+info)
Depression of retinal glutamate transporter function leads to elevated intravitreal glutamate levels and ganglion cell death.
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PURPOSE: Elevated levels of extracellular glutamate have been implicated in the pathophysiology of neuronal loss in both central nervous system and ophthalmic disorders, including glaucoma. This increase in glutamate may result from a failure of glutamate transporters (molecules that ordinarily regulate extracellular glutamate; E:xcitatory A:mino A:cid T:ransporter; EAAT). Elevated glutamate levels can also lead to alterations in glutamate receptor expression. It was hypothesized that selective blockade of glutamate transporters would be toxic to retinal ganglion cells. METHODS: Glutamate transporters were blocked either pharmacologically or with subtype-specific antisense oligonucleotides against EAAT1. Glutamate levels, transporter levels and ganglion cell survival were assayed. RESULTS: Pharmacological inhibition of glutamate transporters with either an EAAT2 specific inhibitor or a nonspecific inhibitor of all the subtypes of transporters was toxic to ganglion cells. Treatment with oligonucleotides against the glutamate transporter EAAT1 decreased the levels of expression of the transporter, increased vitreal glutamate, and was toxic to ganglion cells. CONCLUSIONS: These results demonstrate that normal function of EAAT1 and EAAT2 is necessary for retinal ganglion cell survival and plays an important role in retinal excitotoxicity. Manipulation of retinal glutamate transporter expression may become a useful tool in understanding retinal neuronal loss. (+info)