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-beta-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 microM (THA) and 7.6 microM (t-PDC), as was the initial rate of [Na+](i) rise (EC(50) values of 14.8 microM for THA and 11.5 microM 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-beta-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of [Na+](i) up to a concentration of 500 microM . TBOA inhibited the [Na+](i) response evoked by 200 microM glutamate in a concentration-dependent manner with IC(50) values of 114 and 63 microM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked [Na+](i) increase by TBOA was approximately 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.     

Methylmercury inhibits cysteine uptake in cultured primary astrocytes, but not in neurons

The maintenance of adequate intracellular glutathione (GSH) concentrations is dependent on the availability and transport of the rate-limiting substrate, cysteine. A suggested mechanism of methylmercury (MeHg) neurotoxicity in brain involves the formation of oxygen radicals, and a decrease in intracellular levels of GSH. Recently, we have characterized various cysteine transport systems (both Na(+)-dependent and -independent) in cerebrocortical astrocytes and hippocampal neurons. The present study was carried out to investigate the effect of MeHg on cysteine uptake in both astrocytes and neurons, and to determine whether cysteine transport is differentially affected in the two cell types by MeHg treatment. Sixty-minute pretreatment with MeHg caused significant concentration-dependent inhibition in cysteine uptake in astrocytes, but not in neurons. As most of the cysteine transport is Na(+)-dependent (80-90% of total), additional studies focused on MeHg's effect on the Na(+)-dependent cysteine transporters X(AG(-)) and ASC. An additive inhibitory effect on cysteine uptake was observed in astrocytes treated with MeHg (5 microM) plus sub-maximal inhibitory concentrations (0.1 and 0.5 mM) of threo-beta-hydroxy-aspartate (THA), a specific inhibitor of the Na(+)-dependent transporter, X(AG(-)), compared to astrocytes treated with MeHg (P<0.001) or THA alone (P<0.05). There was no additive effect of MeHg and maximal inhibitory concentrations of THA (1.0 and 5.0 mM) on astrocytic cysteine uptake inhibition. Additional studies examined the sensitivity of the Na(+)-dependent ASC transport system to MeHg treatment. Maximal inhibitory concentration of L-serine (10 mM) alone had a rather modest inhibitory effect on cysteine uptake, and when applied in the presence of MeHg there was no additive effect. These results suggest that the inhibition of cysteine uptake by MeHg in astrocytes occurs through specific inhibition of both the X(AG(-)) as well as the ASC transport system.     

The effects of ibogaine on dopamine and serotonin transport in rat brain synaptosomes

Ibogaine has been shown to affect biogenic amine levels in selected brain regions. Because of the involvement of these neurotransmitters in drug addiction, the effects of ibogaine on biogenic amine transport may contribute to the potential anti-addictive properties of ibogaine in vivo. With rat brain synaptosomes as our experimental system, we measured the effects of ibogaine on the uptake and release of dopamine (DA) and serotonin (5-HT). Ibogaine competitively blocked both DA and 5-HT uptake with IC50 values of 20 microM at 75 nM 3H-DA and 2.6 microM at 10 nM 3H-5-HT. Ibogaine had no effect on K+-induced release of 3H-DA from preloaded synaptosomes, but 20 microM and 50 microM ibogaine inhibited roughly 40% and 60%, respectively, of the K(+)-induced release of 3H-5-HT from preloaded synaptosomes. In the absence of a depolarizing stimulus, ibogaine evoked a small release of 3H-DA but not 3H-5-HT. These relatively low-potency effects of ibogaine on DA and 5-HT uptake in synaptosomes are consistent with the low binding affinity of ibogaine that has been previously reported for DA and 5-HT transporters. Our results show that if ibogaine modulates DA and 5-HT levels in the brain by directly blocking their uptake, then a concentration of ibogaine in the micromolar range is required. Furthermore, if the anti-addictive effects of ibogaine require this concentration, then ibogaine likely exerts these effects through a combination of neurotransmitter pathways, because binding affinities and functional potencies of ibogaine in the micromolar range have been reported for a variety of neuronal receptors and transporters.     

Demonstration of kynurenine aminotransferases I and II and characterization of kynurenic acid synthesis in cultured cerebral cortical neurons

The present study characterizes the synthesis of kynurenic acid (KYNA) from exogenously added kynurenine and its regulation by extrinsic factors, in cultured cerebral cortical neurons and, for comparison, in astrocytes incubated under identical conditions. The neuronal culture showed positive immunostaining for both kynurenic acid aminotransferase (KAT) isoforms I and II. Neurons synthesized KYNA at a rate about 2.3 times higher than astrocytes. Neuronal, but not astrocytic, KYNA synthesis was lowered approximately 30% by ionotropic glutamate receptor agonists [(R,S)-3-hydroxy-5-methoxyloxasole-4-propionic acid (AMPA; 100 microM) and N-methyl-D-aspartic acid (NMDA; 100 microM)] and depolarizing agents [KCl (50 mM) and 4-aminopyridine (4-AP; 10 microM)]. Neuronal and astrocytic synthesis alike were vulnerable to inhibition exerted by the aminotransferase inhibitor aminooxyacetic acid (AOAA), glutamate (IC50: 31 and 85 microM, respectively), substrates of the L-amino transport system [leucine (Leu); IC50: 19 and 42 microM, respectively] and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH; IC50: 19 and 28 microM, respectively). Glutamine (Gln), which is a metabolic precursor of glutamate in astrocytes and L-system substrate in both cell types, inhibited KYNA synthesis both in neurons and in astrocytes (IC50: 268 and 318 microM, respectively). alpha-Ketoisocaproic acid (KIC), a Leu transamination product that is produced mainly in astrocytes and shuttled to neurons to modulate intraneuronal concentration of glutamate, stimulated KYNA synthesis in neurons but did not affect the synthesis in astrocytes. In conclusion, this study is the first to demonstrate active, regulation-prone KYNA synthesis in neurons.     

Neuron-glia interactions in glutamatergic neurotransmission: roles of oxidative and glycolytic adenosine triphosphate as energy source

Glutamatergic neurotransmission accounts for a considerable part of energy consumption related to signaling in the brain. Chemical energy is provided by adenosine triphosphate (ATP) formed in glycolysis and tricarboxylic acid (TCA) cycle combined with oxidative phosphorylation. It is not clear whether ATP generated in these pathways is equivalent in relation to fueling of the energy-requiring processes, i.e., vesicle filling, transport, and enzymatic processing in the glutamatergic tripartite synapse (the astrocyte and pre- and postsynapse). The role of astrocytic glycogenolysis in maintaining theses processes also has not been fully elucidated. Cultured astrocytes and neurons were utilized to monitor these processes related to glutamatergic neurotransmission. Inhibitors of glycolysis and TCA cycle in combination with pathway-selective substrates were used to study glutamate uptake and release monitored with D-aspartate. Western blotting of glyceraldehyde-3-P dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) was performed to determine whether these enzymes are associated with the cell membrane. We show that ATP formed in glycolysis is superior to that generated by oxidative phosphorylation in providing energy for glutamate uptake both in astrocytes and in neurons. The neuronal vesicular glutamate release was less dependent on glycolytic ATP. Dependence of glutamate uptake on glycolytic ATP may be at least partially explained by a close association in the membrane of GAPDH and PGK and the glutamate transporters. It may be suggested that these enzymes form a complex with the transporters and the Na(+) /K(+) -ATPase, the latter providing the sodium gradient required for the transport process.     

Nitric oxide induces rapid,calcium-dependent release of vesicular glutamate and ATP from cultured rat astrocytes

Nitric oxide (NO; 1 microM) or an NO donor (500 microM diethylenetriamine-nitric oxide, DETA-NONOate) caused rapid glutamate and ATP release from cultured rat cortical astrocytes. NO-induced glutamate release was prevented by calcium chelators (EGTA or BAPTA-AM) and an inhibitor of vesicular exocytosis (botulinum neurotoxin C, BoTx-C), but not by a glutamate transport inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (t-PDC), a cyclooxygenase inhibitor (indomethacin), or an inhibitor of soluble guanylate cyclase 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), and was not induced by mitochondrial respiratory inhibitors (myxothiazol or azide). Similarly to glutamate, NO-induced ATP release was also completely blocked by BAPTA-AM and BoTx-C, suggesting again a vesicular, calcium-dependent mechanism of release. Addition of DETA-NONOate (500 microM) to fura-2-loaded astrocytes induced a rapid, transient increase in intracellular calcium levels followed by a lower, sustained level of calcium entry. The latter was blocked by gadolinium (1 microM), an inhibitor of capacitative Ca(2+) entry. Thus, NO appears to cause rapid exocytosis of vesicular glutamate and ATP from astrocytes by raising intracellular calcium levels. Astrocytes activated by lipopolysaccharide/endotoxin and interferon-gamma to express inducible NO synthase (iNOS) maintained substantially higher extracellular glutamate levels than nonactivated cells or activated cells treated with an iNOS inhibitor (1400W), but the rate of glutamate uptake by these cells was similar. This suggests that NO from inflammatory-activated astrocytes causes release of astrocytic glutamate. NO-induced release of astrocytic glutamate and ATP may be important in physiological or pathological communication between astrocytes and neurons.     

Inhibition of system A-mediated glycine transport in cortical synaptosomes by therapeutic concentrations of clozapine: implications for mechanisms of action

Clozapine is an atypical antipsychotic with particular efficacy in schizophrenia, possibly related to potentiation of brain N-methyl-D-aspartate receptor (NMDAR) -mediated neurotransmission. NMDARs are regulated in vivo by glycine, which is regulated in turn by glycine transporters. The present study investigates transport processes regulating glycine uptake into rat brain synaptosomes, along with effects of clozapine on synaptosomal glycine transport. Amino-acid uptake of amino acids was assessed in rat brain P2 synaptosomal preparations using a radiotransport assay. Synaptosomal glycine transport was inhibited by a series of amino acids and by the selective System A antagonist MeAIB (2-methyl-aminoisobutyric acid). Clozapine inhibited transport of both glycine and MeAIB, but not other amino acids, at concentrations associated with preferential clinical response (0.5-1 microg/ml). By contrast, other antipsychotics studied were ineffective. The novel glycine transport inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) produced biphasic inhibition of [(3)H]glycine transport, with IC(50) values of approximately 25 nM and 25 microM, respectively. NFPS inhibition of [(3)H]MeAIB was monophasic with a single IC(50) value of 31 microM. Clozapine significantly inhibited [(3)H]glycine binding even in the presence of 100 nM NFPS. In conclusion, this study suggests first that System A transporters, or a subset thereof, may play a critical role in regulation of synaptic glycine levels and by extension of NMDA receptor regulation, and second that System A antagonism may contribute to the differential clinical efficacy of clozapine compared with other typical or atypical antipsychotics.     

Inhibition of excitatory amino acid-stimulated phosphoinositide hydrolysis in rat hippocampus by L-aspartate-beta-hydroxamate.

The effect of a series of glutamate uptake inhibitors was tested on ibotenate-stimulated phosphoinositide hydrolysis. The pharmacological profile of the inhibitory effect of these compounds on the ibotenate response was quite different from that on glutamate uptake. Aspartate-beta-hydroxamate was the most potent compound with the L-isomer (IC50 11 +/- 2 microM) being considerably more potent than the D-isomer (IC50 104 +/- 12 microM). The effect of the L-aspartate-beta-hydroxamate was found to be specific for ibotenate and quisqualate-stimulated phosphoinositide hydrolysis; this compound did not affect hydrolysis stimulated by carbachol, K+ or sodium fluoride. The inhibition of the ibotenate response was found to involve a non-competitive and irreversible mechanism.     

Beta-amyloid25-35 inhibits glutamate uptake in cultured neurons and astrocytes: modulation of uptake as a survival mechanism

Glutamate transporters are vulnerable to oxidants resulting in reduced uptake function. We have studied the effects of beta-amyloid(25-35) (beta A(25-35)) on [(3)H]-glutamate uptake on cortical neuron or astrocyte cultures in comparison with a scrambled peptide (SCR) and dihydrokainic acid (DHK), a prototypic uptake inhibitor. beta A(25-35) was more potent than DHK in inhibiting glutamate uptake and the effects of both were more marked on astrocytes than on neurons. At 24 h, beta A(25-35) dose-dependently (0.5-15 microM) increased glutamate levels in media from neuron cultures. DHK only enhanced extracellular glutamate at the highest concentration tested (2500 microM). beta A(25-35) induced gradual neurotoxicity (0.1-50 microM) over time. Exposure to beta A(25-35) resulted in increased uptake in astrocytes (0.25-5 microM) and neurons (0.5-15 microM) surviving its toxic effects. However, exposure to DHK (2.5-2500 microM) did not induce neurotoxicity nor modulated uptake. These results indicate that, while inhibition of glutamate uptake may be involved in the neurotoxic effects of beta A(25-35), enhancement of uptake may be a survival mechanism following exposure to beta A(25-35).     

Characterization of glutamate uptake systems in astrocyte primary cultures from rat brain.

The dependence of 3[H]-L-glutamate uptake on the presence of sodium, chloride, or calcium ions or on a combination of the three was investigated in astrocyte primary cultures. A stimulating effect on glutamate uptake by each of the ions tested was found. In addition to the comparably small effect by calcium alone, calcium exhibits a synergistic effect on the sodium- and chloride-dependent uptake. The sodium-dependent transport accumulates the glutamate analogue D-aspartate as well as L-glutamate. L-aspartate is taken up by about 50% of the values observed for L-glutamate transport. Sodium-dependent glutamate uptake is strongly inhibited by aspartate-beta-hydroxamate (A beta H) and threo-beta-hydroxyaspartate (T beta H). Quisqualate is less potent in inhibiting this uptake. In contrast, the chloride- and the calcium-dependent uptake systems do not handle D- and L-aspartate as substrates. A beta H and T beta H are only poor inhibitors of these transporters while quisqualate reduces glutamate uptake almost completely. Kinetic data of all uptake systems were estimated. High and low affinity components of each individual system are demonstrated by Eadie-Hofstee analysis. Hill plots indicate that high and low affinity uptake may be due either to two respective uptake sites for Na(+)-, Cl(-)-, and Ca(++)-dependent glutamate transport, or to two glutamate binding sites for each single transport system with negative cooperativity.     

Study of the bidirectional transport of choline by blocking choline carriers from outside or inside brain nerve terminals

Membrane carriers can operate bidirectionally. We studied, in rat neocortex synaptosomes, the choline carrier by comparing the ability of the transport inhibitor hemicholinium-3, present outside or inside the nerve terminals, to prevent uptake and release of [(3)H]choline. Because hemicholinium-3 is membrane-impermeable, it was previously entrapped into synaptosomes during homogenization of brain tissue. External and internalized hemicholinium-3 produced similar maximal inhibition (80-90%) of [(3)H]choline uptake. Also comparable (approximately 30 nM) are the potency of externally applied hemicholinium-3 and the estimated potency of the entrapped inhibitor. Exposure to ouabain elicited release of both [(3)H]acetylcholine and [(3)H]choline from synaptosomes prelabeled with [(3)H]choline. The ouabain (300 microM)-evoked release of [(3)H]choline only was blocked by externally added (IC(50) approximately 10 nM) or internalized (estimated IC(50) approximately 5 nM) hemicholinium-3. Release of previously taken up [(3)H]choline elicited by 100 microM external choline (homoexchange) was prevented by external (IC(50) approximately 30 microM) or entrapped (estimated IC(50) approximately 20 microM) hemicholinium-3. The results suggest that the choline carriers fit into the alternating-access model proposed for classical transmitter transport. Entrapping nonpermeant ligands into synaptosomes could allow investigation of the inward-facing conformation of native transporters and how cytoplasmic ligands affect the bidirectional transport of neurotransmitters.     

Activation of gamma-aminobutyric acid GAT-1 transporters on glutamatergic terminals of mouse spinal cord mediates glutamate release through anion channels and by transporter reversal

The effects of gamma-aminobutyric acid (GABA) on the release of glutamate from mouse spinal cord nerve endings have been studied using superfused synaptosomes. GABA elicited a concentration-dependent release of [3H]D-aspartate ([3H]D-ASP; EC50= 3.76 microM). Neither muscimol nor (-)baclofen mimicked GABA, excluding receptor involvement. The GABA-evoked release was strictly Na+ dependent and was prevented by the GABA transporter inhibitor SKF89976A, suggesting involvement of GAT-1 transporters located on glutamatergic nerve terminals. GABA also potentiated the spontaneous release of endogenous glutamate; an effect sensitive to SKF89976A and low-Na+-containing medium. Confocal microscopy shows that the GABA transporter GAT-1 is coexpressed with the vesicular glutamate transporter vGLUT-1 and with the plasma membrane glutamate transporter EAAT2 in a substantial portion of synaptosomal particles. The GABA effect was external Ca2+ independent and was not decreased when cytosolic Ca2+ ions were chelated by BAPTA. The glutamate transporter blocker DL-TBOA or dihydrokainate inhibited in part (approximately 35%) the GABA (10 microM)-evoked [3H]D-ASP release; this release was strongly reduced by the anion channel blockers niflumic acid and NPPB. GABA, up to 30 microM, was unable to augment significantly the basal release of [3H]glycine from spinal cord synaptosomes, indicating selectivity for glutamatergic transmission. It is concluded that GABA GAT-1 transporters and glutamate transporters coexist on the same spinal cord glutamatergic terminals. Activation of these GABA transporters elicits release of glutamate partially by reversal of glutamate transporters present on glutamatergic terminals and largely through anion channels.     

Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: heterogeneity of the quiescent phenotype

Reactive astrocytes display decreased glutamate transporters, such as GLT-1, and as a result synaptic glutamate clearance is impaired. In addition, these activated astrocytes are immunocompetent and release algesic mediators that can sensitize neurons in the spinal cord. Currently, we evaluated the effect of propentofylline (PPF), an experimental antiallodynic agent, on the phenotype and glutamate transporter expression of astrocytes. Primary astrocyte cultures, which represent an activated phenotype with a polygonal morphology and low GLT-1 expression, were treated for 3 or 7 days with 10, 100, or 1,000 microM PPF or dibutyryl-cAMP (db-cAMP), a known inducer of GLT-1 expression. PPF dose-dependently induced astrocytes to display a mature phenotype, with elongated processes and a stellate shape, as well as increased GLT-1 and GLAST immunoreactivity, similar to that seen with db-cAMP. Real time RT-PCR and Western blot analysis clearly demonstrated that PPF caused a potent dose-dependent induction of GLT-1 and GLAST mRNA and protein in these astrocytes. Importantly, the observed increase in glutamate transporters was found to have a functional effect, with significantly enhanced glutamate uptake in astrocytes treated with 100 or 1,000 microM PPF that was sensitive to dihydrokainate inhibition, suggesting it is GLT-1 mediated. Finally, the effect of PPF on lipopolysaccharide-induced chemokine release was investigated. Interestingly, PPF was able to dampen both MCP-1 (CCL2) and MIP-2 (CXCL2) release from astrocytes while db-cAMP significantly enhanced this chemokine expression. These findings suggest that PPF is capable of differentiating astrocytes to a homeostatic, mature phenotype, competent for glutamate clearance and distinct from that induced by db-cAMP.     

The antiapoptotic effect of guanosine is mediated by the activation of the PI 3-kinase/AKT/PKB pathway in cultured rat astrocytes

Guanosine has many trophic effects in the CNS, including the stimulation of neurotrophic factor synthesis and release by astrocytes, which protect neurons against excitotoxic death. Therefore, we questioned whether guanosine protected astrocytes against apoptosis induced by staurosporine. We evaluated apoptosis in cultured rat brain astrocytes, following exposure (3 h) to 100 nM staurosporine by acridine orange staining or by oligonucleosome, or caspase-3 ELISA assays. Staurosporine promoted apoptosis rapidly, reaching its maximal effect (approximately 10-fold over basal apoptotic values) in 18-24 h after its administration to astrocytes. Guanosine, added to the culture medium for 4 h, starting from 1 h prior to staurosporine, reduced the proportion of apoptotic cells in a concentration-dependent manner. The IC50 value for the inhibitory effect of guanosine is 7.5 x 10(-5) M. The protective effect of guanosine was not affected by inhibiting the nucleoside transporters by propentophylline, or by the selective antagonists of the adenosine A1 or A2 receptors (DPCPX or DMPX), or by an antagonist of the P2X and P2Y purine receptors (suramin). In contrast, pretreatment of astrocytes with pertussis toxin, which uncouples Gi-proteins from their receptors, abolished the antiapoptotic effect of guanosine. The protective effect of guanosine was also reduced by pretreatment of astrocytes with inhibitors of the phosphoinositide 3-kinase (PI3K; LY294002, 30 microM) or the MAPK pathway (PD98059, 10 microM). Addition of guanosine caused a rapid phosphorylation of Akt/PKB, and glycogen synthase kinase-3beta (GSK-3beta) and induced an upregulation of Bcl-2 mRNA and protein expression. These data demonstrate that guanosine protects astrocytes against staurosporine-induced apoptosis by activating multiple pathways, and these are mediated by a Gi-protein-coupled putative guanosine receptor.     

Isoflurane enhances glutamate uptake via glutamate transporters in rat glial cells

In this study I examined whether isoflurane, an inhalational anesthetic used commonly in clinical practice, affected glutamate uptake via glutamate transporters, proteins expressed in the plasma membrane of cells in the central nervous system. Isoflurane at clinically relevant concentrations (1-3%) caused a time-, sodium- and concentration-dependent increase of glutamate uptake in primary cultures of rat cerebral mixed glial cells. This enhancement was inhibited by a specific glutamate transporter inhibitor. The study also demonstrated that 2.0% isoflurane significantly increased both Vmax and Km of transporter-mediated glutamate uptake. Thus, isoflurane enhances glutamate uptake by a pathway that requires function of glutamate transporters. This represents a novel pharmacological effect of inhalational anesthetics and may contribute to isoflurane-induced anesthesia and neuroprotective effects.     

Subunit-dependent effects of nickel on NMDA receptor channels

Nickel (Ni2+) is a transition metal that affects different neuronal ionic channels. We investigated its effects on glutamate channels of the NMDA-type in the presence of saturating concentration of glutamate or NMDA (50 microM), in 0 external Mg and in the continuous presence of saturating glycine (30 microM). In neonatal rat cerebellar granule cells, Ni2+ inhibited the current evoked by NMDA at -60 mV with an IC50 close to 40 microM. The inhibition was weakly voltage-dependent and the current at +40 mV was inhibited with IC50=86 microM. Wash out of the metal unmasked a stimulatory effect which persisted for a few seconds. In HEK293 cells transiently transfected with recombinant NR1a-NR2A receptors, Ni2+ inhibited the current elicited by glutamate with an IC50=52 microM at -60 mV and 90 microM at +40 mV. In HEK293 expressing NR1a-NR2B receptors, 0.1-100 microM Ni2+ caused a potentiation of the current, with EC50=4 microM, while with 300 microM, a voltage-dependent block became apparent (IC50=170 microM). As previously reported, the current through both classes of recombinant receptors was steeply dependent on external pH, and in both cases the protonic block had an IC50 close to pH 7.2. Application of Ni2+ showed that stimulation of NR1a-NR2B receptor channels was dependent on external pH, while voltage-independent inhibition of NR1a-NR2A was less sensitive to pH change. These results indicate that Ni2+ has multiple and complex effects on NMDA channels, which are largely dependent on the NR2 subunit.     

Thyroid hormone increases astrocytic glutamate uptake and protects astrocytes and neurons against glutamate toxicity

Thyroid hormone (T(3)) regulates the growth and differentiation of rat cerebellar astrocytes. Previously, we have demonstrated that these effects are due, at least in part, to the increased expression of extracellular matrix molecules and growth factors, such as fibroblast growth factor-2. T(3) also modulates neuronal development in an astrocyte-mediated manner. In the mammalian central nervous system, excitatory neurotransmission is mediated mainly by glutamate. However, excessive stimulation of glutamate receptors can lead to excitotoxicity and cell death. Astrocytic glutamate transporters, GLT-1 and GLAST, play an essential role in the clearance of the neuronal-released glutamate from the extracellular space and are essential for maintaining physiological extracellular glutamate levels in the brain. In the present study, we showed that T(3) significantly increased glutamate uptake by cerebellar astrocytes compared with control cultures. Inhibitors of glutamate uptake, such as L-PDC and DL-TBOA, abolished glutamate uptake on control or T(3)-treated astrocytes. T(3) treatment of astrocytes increased both mRNA levels and protein expression of GLAST and GLT-1, although no significant changes on the distribution of these transporters were observed. The gliotoxic effect of glutamate on cultured cerebellar astrocytes was abolished by T(3) treatment of astrocytes. In addition, the neuronal viability against glutamate challenge was enhanced on T(3)-treated astrocytes, showing a putative neuroprotective effect of T(3). In conclusion, our results showed that T(3) regulates extracellular glutamate levels by modulating the astrocytic glutamate transporters. This represents an important mechanism mediated by T(3) on the improvement of astrocytic microenvironment in order to promote neuronal development and neuroprotection.     

Sodium-independent transport of noradrenaline in mouse and rat astrocytes in primary culture

The uptake of noradrenaline by primary cultures of mouse and rat astrocytes was investigated in order to examine whether an inhibition of extraneuronal noradrenaline uptake was the mechanism whereby some trace biogenic amines potentiate neuronal responses to noradrenaline. In the presence of inhibitors of the enzymes monoamine oxidase and catechol-O-methyl transferase, it was found that astrocytes took up noradrenaline by a temperature-dependent, sodium-independent mechanism that was saturable with a Km = 3.4 x 10(-7) M and a Vmax = 1.6 pmole/mg protein/2 min. This uptake mechanism did not concentrate noradrenaline within the cell. The uptake of noradrenaline was inhibited by ascorbic acid (IC50 = 3.4 x 10(-7) M), adrenaline (IC50 = 7.9 x 10(-7) M), and dopamine (IC50 = 1.5 x 1.0(-6) M). It was not inhibited by the tricyclic antidepressants amitriptyline and desmethylimipramine or the trace biogenic amines beta-phenylethylamine, phenylethanolamine, p- and m-tyramine and p- and m-octopamine. Nor was the uptake inhibited by fluoxetine or 5-hydroxytryptamine. It is concluded that astrocytes take up noradrenaline by a facilitated-diffusion mechanism and that this uptake resembles the extraneuronal uptake described in preparations of brain tissue. It is also concluded that the trace biogenic amines do not potentiate neuronal responses to noradrenaline by inhibiting extraneuronal uptake.     

Methylmercury-mediated inhibition of H- -aspartate transport in cultured astrocytes is reversed by the antioxidant catalase

Astrocytes are essential for removal of glutamate from the extracellular space in the central nervous system. The neurotoxic heavy metal methylmercury potently and specifically inhibits the transport of glutamate in cultured astrocytes by an unknown mechanism. Glutamate transport in astrocytes is also inhibited by reactive oxygen species. A glutamate-induced transporter current is inhibited both by reactive oxygen species and thiol oxidizing agents. These observations suggest that oxidation of the transporter might mediate methylmercury-induced inhibition of glutamate transport. In the present study, we examined the ability of thiol reducing or oxidizing agents to inhibit transport of ³H- -aspartate, a glutamate analog, in primary cultures of neonatal rat astrocytes. To assess if methylmercury-mediated inhibition of ³H-aspartate transport was due to overproduction of reactive oxygen species, we tested the ability of Trolox, α-phenyl-tert-butyl nitrone (PBN), or catalase to attenuate the methylmercury-induced inhibition of aspartate uptake. Neither the thiol reducing agent dithiothreitol (DTT), nor the thiol oxidizing agent 5,5′-dithio-bis(2-nitrobenzoic) acid (DTNB) had any effect on ³H-aspartate transport suggesting that the thiol redox state does not alter transporter function. In contrast, the antioxidant catalase (1000 U/ml) significantly attenuated methylmercury-induced inhibition of ³H-aspartate uptake, suggesting that excess reactive oxygen species, specifically H₂O₂, inhibit the function of an astrocytic excitatory amino acid transporter (EAAT1). Prolonged exposure (6 h) to inhibitors of glutamate transport significantly decreased EAAT1 mRNA levels suggesting that transporter expression is related to function. This study suggests that methylmercury-induced overproduction of H₂O₂ is a mechanism for inhibition of glutamate transport and transporter expression in cultured astrocytes.