A Novel Chloride-Dependent L-[3H]Glutamate Binding Site in Astrocyte Membranes

Membrane fractions prepared from astrocytes grown in culture exhibit a specific binding site for L-[3H]glutamate that is Cl--dependent and Na+-independent. The binding site is a single saturable site with a KD of about 0.5 microM, is inhibited by L-aspartate, L-cysteate, and quisqualate, and is insensitive to kainate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, and 2-amino-4-phosphonobutyrate. The pharmacological characteristics of the binding site indicate that it is distinct from any site previously described in synaptic membrane preparations. Comparisons of ionic requirements, ligand specificity, and inhibitor sensitivities, however, suggest the described binding is the first step in a Cl--dependent high-affinity glutamate uptake system. Such binding studies provide a useful model system in which to investigate the close association between excitatory amino acids, astrocytes, the termination of glutamate's excitatory action by high-affinity uptake, and the excitotoxic action of acidic amino acids in membranes of a single cell type.     

Characterization of Glutamate Binding Sites in Receptors Assembled from Transfected NMDA Receptor Subunits

Abstract: Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using l -[³H]glutamate and [³H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg²⁺ can alter [³H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [³H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [³H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.     

Glutamate Binding to Brain Membranes Is Increased in Pentylenetetrazole-Kindled Rats

Abstract: The specific binding of L-[³H]glutamate to its receptors was investigated on crude membrane preparations from different brain regions of pentylenetetrazole-kindled rats using a binding assay technique. Pentylenetetrazole kindling induced by 10 intraperitoneal applications of 45 mg/kg over a period of 20 days resulted in a significant increase of both the convulsive susceptibility of animals to the convulsant and the specific L-[³H]glutamate binding in hippocampus and in motor, frontal, and inferiotemporal (acoustic) cortex tested with a L-[³H]glutamate concentration of 50 n M . No differences were observed in the other brain structures studied. Kinetic studies indicated that the enhanced L-[³H]glutamate binding to hippocampal membranes from kindled rats reflects changes in the density of the glutamate binding sites rather than an increase in receptor affinity. To study the effect of acute generalized convulsions on L-[³H]glutamate binding to synaptosomal membranes of hippocampus and visual cortex, rats were treated 24 h before the experiment with 60 mg/kg of pentylenetetrazole, i.p. Under these conditions, no differences between treated and control rats were observed. From these findings, it is concluded that the increase in glutamate receptor density demonstrated in hippocampus and several neocortical brain structures of pentylenetetrazole-kindled rats may be the expression of a specific enhancement of susceptibility of glutamatergic systems to this excitatory amino acid developing in the course of formation of pentylenetetrazole-induced kindling.     

Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Synaptosomes Is Mediated by Glutamate Uptake and Not by Receptors

L-Glutamate (10 microM-1 mM) released endogenous adenosine from rat cortical synaptosomes. Studies with excitatory amino acid antagonists, (+)-5-methyl-16,11,dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), 6,7-dinitroquinoxaline-2,3-dione (DNQX), Mg2+, and agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate, indicated that this release was not receptor mediated. D,L-2-Amino-4-phosphonobutanoic acid (APB) also did not affect glutamate-evoked adenosine release. Inhibition of glutamate uptake by dihydrokainate or replacement of extracellular Na+ blocked glutamate-evoked adenosine release. D-aspartate, which is a substrate for the glutamate transporter but is not metabolized, also released adenosine, suggesting that release was due to amino acid transport and not to its subsequent metabolism. D-Glutamate, a relatively poor substrate for the transporter, was correspondingly less potent than L-glutamate at releasing adenosine. Glutamate-evoked adenosine release was not Ca2+ dependent or tetrodotoxin sensitive and did not appear to occur on the bidirectional nucleoside transporter. Inhibition of ecto-5'-nucleotidase virtually abolished glutamate-evoked adenosine release, indicating that adenosine was derived from extracellular metabolism of released nucleotide(s). However, L-glutamate did not release ATP and did not appear to release cyclic AMP. Therefore, transport of glutamate into presynaptic terminals releases some other nucleotide which is converted extracellularly to adenosine. This adenosine could act at P1-purinoceptors to modulate glutamatergic neurotransmission.     

Glutamate Uptake by Squid Nerve Fiber Sheaths

Abstract: The squid giant nerve exhibits neuron-Schwann cell interactions that appear to involve glutamate as a mediator; however, there is no information available about the possible fate of the released glutamate. In this study, it is demonstrated that the periaxonal sheaths of the extrasynaptic regions of squid giant nerves (where the glial cells are located) possess the capacity to transport glutamate. In whole intact nerves incubated with low-glutamate concentrations for long periods of time, the majority of the glutamate incorporated into the tissue was found in the sheaths. Axoplasm-free sheaths incubated for long periods of time with low concentrations of glutamate were able to accumulate this amino acid against a large apparent concentration gradient. Sheath glutamate uptake occurred in a sodium-dependent fashion over a wide concentration range and displayed both high- and low-affinity components. Glutamate uptake at concentrations below the K _m of the high-affinity component was independent of homoexchange and displayed a specificity that is similar to that described for high-affinity glutamate transport in mammalian brain. It is proposed that the sheath transport systems may be involved in the regulation of glutamate levels in the intercellular clefts of the nerve fiber, as part of the glutamatergic neuron-glial signaling mechanisms in the squid giant nerve fiber.     

Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca2+-Dependent Manner in Rat Brain Synaptic Plasma Membranes

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylgluc osyl ceramide (GM1) on high-affinity binding of L-[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40-50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H-labeled GM1 proved trypsin-stable insertion of GM1 into SPM, with a maximum incorporation of four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+-stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 microliters/mg of protein, in ganglioside-treated as well as in control membranes. From the data, it is concluded that a special, Na+- and Cl- -independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.     

Effect of Monocular Deprivation on Uptake and Binding of [3H]Glutamate in the Visual System of Rat Brain

Abstract: [³H]Glutamate uptake and binding studies were performed in the visual cortices, lateral geniculate nuclei (LGN), and superior colliculi of 3-month-old rats with one eyelid surgically closed from postnatal day 10 (monocular deprivation). Uptake and binding were highest in the lateral geniculate nucleus followed by the visual cortex (69% and 15%, respectively compared to LGN values) and the superior colliculus (32% and 59% of LGN values). Monocular deprivation did not affect [³H]glutamate uptake in any of the visual regions examined. However, a 46% decrease in [³H]glutamate binding in the lateral geniculate nucleus ipsilateral to the sutured eye was detected. Binding levels in other regions were not affected.     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

Glutamine, Glutamate, and Other Possible Regulators of α-Ketoglutarate and Malate Uptake by Synaptic Terminals

Abstract: The uptake of a-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of γ-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in V_max. In contrast, glutamine did not appear to affect the V_max of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-as-partate were comparatively strong inhibitors of γ-keto-glutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of γ-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited γ-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca²⁺ exhibited a variable effect but usually enhanced the uptake of γ-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of γ-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, γ-aminobutyric acid, and several other amino acids was not affected. The enhancement of γ-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-^-14C] γ-ketoglutarate to glutamate, as-partate, glutamine, and aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.     

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500–508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of β-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.     

周质结合蛋白依赖的转运系统研究进展

周质结合蛋白依赖的转运系统是复杂的、多组分的透酶系统,具有广泛的生理功能。在从微生物到人类的所有物种中都可发现这种转运系统或与之相关的结构。本以细菌为例描述了这类系统的基本结构和功能以及转运机制。     

Solubilization of Quisqualate-Sensitive [3H]Glutamate Binding Activity from Rat Retina

Binding activity of a putative central neurotransmitter, L-glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P-40. [3H]Glutamate binding activity increased linearly with increasing concentrations of the solubilized proteins up to 15 micrograms. The binding activity reached an equilibrium within 10 min at 2 degrees C, while increasing with incubation time up to 60 min at 30 degrees C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30 degrees C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 microM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to L-glutamate, and was abolished by quisqualate, L-glutamate diethyl ester, and DL-2-amino-3-phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation-exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30 degrees C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate-sensitive membranous enzyme with a stereospecific and structure-selective high affinity for the central neurotransmitter.     

Effects of Organic and Inorganic Lead on Synaptosomal Uptake, Release, and Receptor Binding of Glutamate in Young Rats

Chemical changes in central glutamate neurotransmission were assessed by measuring synaptic membrane receptor binding, the uptake and release by synaptosomes of glutamate in rats treated acutely with tetraethyl lead and chronically with lead acetate. The activity of Na+, K+-ATPase in synaptosomes was measured to correlate with the changes in uptake/release studies. The affinity of receptor binding and uptake systems was significantly reduced although the number of receptor sites and the capacity of uptake systems were increased. The activity of Na+, K+-ATPase was also found to be increased in synaptosomes. The changes were more marked in inorganic lead toxicity, and all three regions studied--cerebral cortex, cerebellum, and brainstem--showed significant alterations.     

Modulation of the Mesolimbic Dopamine System by Glutamate

Glutamate has been shown to modulate motor behavior, probably via N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that are involved in the control of the mesolimbic dopamine (DA) system, that is, the ventral tegmental area (VTA)-nucleus accumbens (NAC). In the present study, we investigated the effects of uncompetitive (MK-801) and competitive [DL-2-amino-5-phosphonopentanoic acid (AP-5), CGP 40116] NMDA receptor antagonists and NMDA and AMPA on DA release in the mesolimbic system and on motor behavior. Systemic injection and intrategmental infusion of MK-801 increased DA levels in the VTA, but the systemic administration enhanced DA exclusively in the NAC and increased motor behavior. In contrast, intrategmental infusion of AP-5, but not the systemic administration of its lipophilic analogue CGP 40116, decreased the DA release in the two regions without affecting motor behavior. NMDA and AMPA infusion into the VTA increased DA levels in both areas. This increase was accompanied by a strong motor behavioral stimulation after NMDA but only a moderate increase after AMPA infusion. The present results indicate that mesolimbic DA neurons are controlled by the glutamatergic system and that the effects of uncompetitive and competitive NMDA receptor antagonists on DA release are mediated by an interaction with different brain areas. These findings may account for the different effects of NMDA receptor ligands on motor behavior.     

L-[3H]Glutamate Binding to Hippocampal Synaptic Membranes: Two Binding Sites Discriminated by Their Differing Affinities for Quisqualate

The excitatory glutamate analogs quisqualate and ibotenate were employed to distinguish multiple binding sites for L-[3H]glutamate on freshly prepared hippocampal synaptic membranes. The fraction of bound radioligand that was displaceable by 5 microM quisqualate was termed GLU A binding. That which persisted in the presence of 5 microM quisqualate, but was displaceable by 100 microM ibotenate, was termed GLU B binding. GLU A binding equilibrated within 5 min and remained unchanged for up to 80 min. GLU B binding appeared to equilibrate at least as rapidly, but incubation with ligand unmasked latent binding sites. Saturation binding curves were best fitted by single exponentials, which yielded KD values of about 200 nM (GLU A) and 1 microM (GLU B). On the average, GLU B binding sites were about twice as abundant in these membranes as were GLU A sites. Rapid freezing of the membranes, followed by storage at -26 degrees C and rapid thawing markedly diminished GLU A binding, but nearly tripled GLU B binding. Both site bound L-glutamate with 10-30 times the affinity of D-glutamate. The GLU A site also bound L-glutamate with about 10 times the affinity of L-aspartate and discriminated poorly between L- and D-aspartate. In contrast, the GLU B site bound L-aspartate with an affinity similar to that for L-glutamate, and with an order-of-magnitude greater affinity than D-aspartate. The structural specificities of the GLU A and GLU B binding sites suggest that these sites may correspond to receptors on hippocampal pyramidal cell dendrites that are activated by iontophoretically applied L-glutamate.     

Binding of L-[3H]Glutamate to Fresh or Frozen Synaptic Membrane and Postsynaptic Density Fractions Isolated from Cerebral Cortex and Cerebellum of Fresh or Frozen Canine Brain

Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[³H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the B_max value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl^? increased the number of L-glutamate binding sites and the effect was enhanced by Ca²⁺; Ca²⁺ alone had no significant effect; (b) the Cl^?/Ca²⁺ -sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na⁺ ion was biphasic: low concentration of Na⁺ (< 5 mM) decreased Cl^?7Ca²⁺ -de-pendent L-glutamate binding sites, whereas at higher concentrations of Na⁺ the binding of glutamate was found to increase either in the presence or absence of Ca²⁺ and Cl^?. In addition, the K⁺ ion (50 mM) was found to decrease the Na⁺-independent and Cl^?/Ca^2--independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na^?-dependent and Cl^?/Ca²⁺-independent L-glutamate binding by 93%; in the presence of Cl, ^?/Ca²⁺, the K⁺ ion decreased the Na⁺-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na⁺-dependent L-glutamate binding sites assayed in the absence of Ca²⁺ and Cl^?. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na^?-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na⁺-dependent nor APB (or Cl^?/Ca²⁺)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX-SPM or CTX-PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L-glutamate. Furthermore, binding of L-glutamate to CTX-SPM or CTX-PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX-PSD fraction with 0.5% deoxycholate, 1.0% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L-glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L-glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ-aminobutyric acid and flunitrazepam binding sites, of the Ca²⁺-dependent K⁺ channel, and of the voltage-dependent Ca²⁺ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.     

Specificity and Reversibility of the Inhibition by HgCl2 of Glutamate Transport in Astrocyte Cultures

Inhibition of glutamate transport is a potential indirect cause of excitotoxic damage by glutamate in the CNS. The mercuric ion, the form in which metallic mercury vapor is believed to exert its neurotoxic action, is a known inhibitor of amino acid transport. This study examines the specificity with which HgCl2 inhibits glutamate transport in mouse cerebral astrocytes by means of comparative measurements of 2-deoxyglucose uptake. Uptake of 2-deoxyglucose is an index of glucose utilization that reflects the function of Na+,K+-ATPase and hexokinase, and is sensitive to Na+ entry. The kinetic parameters, ionic dependence, and substrate specificity of glutamate transport in these astrocyte cultures were consistent with the commonly occurring system designated X-AG. Acute exposure to 0.5 microM HgCl2 inhibited by 50% the initial rate of glutamate transport but did not affect 2-deoxyglucose uptake. Glutamate transport was not detectably inhibited by Al2+, Pb2+, Co2+, Sr2+, Cd2+, or Zn2+ (10 microM as chlorides). The inhibitory action of 0.5 microM HgCl2 on glutamate transport was rapidly reversible. The action of 1-2 microM HgCl2 was progressive when exposures were extended to 1-3 h, and was more slowly reversible. These results suggest that Hg2+ can impair glial glutamate transport reversibly at exposure levels that do not compromise some other vital cell functions.     

Metabotropic Glutamate Receptor Activation Produces Extrapyramidal Motor System Activation That Is Mediated by Striatal Dopamine

Little is known about the in vivo function of the GTP-binding protein-coupled "metabotropic" excitatory amino acid (EAA) receptor. In vitro studies on agonist-induced brain phosphoinositide hydrolysis have shown that (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid is a highly selective and efficacious metabotropic EAA agonist. We have recently reported that in vivo unilateral intrastriatal injection of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid induces transient extrapyramidal motor activation that manifests itself as contralateral turning. In this study, we fully characterized the onset of turning behavior following intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid injection and the possible involvement of striatal dopamine neurons in the mediation of this effect. Rats were anesthetized with the short-acting agent halothane to allow for rapid surgical recovery and thus early behavioral measurements. Intrastriatal (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1 mumol/2 microliters) produced an incremental increase in contralateral turning starting at 1 h and plateauing 3-6 h after injection (peak effect, 39.1 +/- 6.7 rotations per 5 min). Dopamine depletion with alpha-methyl-DL-p-tyrosine (250 mg/kg i.p., 80% depletion) resulted in greater than 85% inhibition of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced contralateral turning. The dopamine antagonist haloperidol (0.3 mg/kg i.p.) produced 48% inhibition of the (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid response. In time course studies, turning behavior correlated with increases in levels of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid. These results suggest a functional interaction between the metabotropic EAA receptor and the dopaminergic system in the striatum.     

Stimulation of Cultured Cerebellar Granule Cells via Glutamate Receptors Induces TRE- and CRE-Binding Activities Mediated by Common DNA-Binding Complexes

By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-mobility assay, exogenous N-methyl-D-aspartate (NMDA) or kainate was shown to increase both 12-O-tetradecanoylphorbol 13-acetate-responsive element (TRE)- and cyclic AMP-responsive element (CRE)-binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The increase of TRE-binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE-binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA-binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE-binding activity could be mediated by the TRE-binding activity. Thus, the TRE-binding activity cross-binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c-Fos or Fos-related proteins in the TRE- and CRE-binding complexes was shown by a supershift gel-mobility assay using anti-c-Fos antiserum.