Homeostasis of glutamate in brain fluids: an accelerated brain-to-blood efflux of excess glutamate is produced by blood glutamate scavenging and offers protection from neuropathologies.

L-Glutamate (Glu) homeostasis in brain extracellular fluids and its maintenance at low micromolar concentrations in the face of the extremely high Glu concentrations present in brain cells and synaptic vesicles have been commonly attributed to the very effective action of glutamate transporters present on neuronal and glial cells. This view however does not take into account the fact that the brain is highly vascularized and that the vasculature harbors a high density of glutamate transporters. In this article, we review the accumulated data establishing the existence of an efflux of excess Glu from brain extracellular fluids into blood. We describe plausible mechanisms accounting for this efflux and present evidence that the brain-to-blood Glu efflux is modulated by blood Glu levels and can be accelerated by blood Glu scavenging. The latter procedure shown here to afford brain neuroprotection in a rat model of closed head injury could be applicable, as a first-line therapy, in the various acute brain insults characterized by excess Glu in brain fluids.

Identification of the amino acid subsets accounting for the ligand binding specificity of a glutamate receptor.

In a situation so far unique among neurotransmitter receptors, glutamate receptors share amino acid sequence similarities with the bacterial periplasmic binding proteins (PBPs). On the basis of the primary structure similarity of two bacterial periplasmic proteins (lysine/arginine/ornithine- and phosphate-binding proteins) with the chick cerebellar kainate-binding protein (KBP), a member of the ionotropic glutamate receptor family, we have generated a three-dimensional model structure of the KBP extracellular domain. By an interplay between homology modeling and site-directed mutagenesis, we have investigated the kainate binding properties of 55 different mutants (corresponding to 43 positions) and studied the interactions of some of these mutants with various glutamatergic ligands. As a result, we present here the subsets of amino acids accounting for the binding free energies and specificities of KBP for kainate, glutamate, and CNQX and propose a three-dimensional model, at the microarchitectural level, of the glutamatergic binding domain.

Identification of domains and amino acids involved in GLuR7 ion channel function.

The kainate receptors GluR6 and GluR7 differ considerably in their ion channel properties, despite sharing 86% amino acid sequence identity. When expressed in Xenopus oocytes GluR6 conducts large agonist-evoked currents, whereas GluR7 lacks measurable currents. In the present study, we localized the determinants that are responsible for the functional differences between GluR6 and GluR7 to the extracellular loop domain L3. In addition, we generated several GluR7 point mutants that are able to conduct currents that can be readily measured in Xenopus oocytes. In GluR6, glutamate- and kainate-evoked maximal currents are of the same magnitude when desensitization is inhibited with the lectin concanavalin A. By contrast, all functional GluR7 mutants were found to have glutamate current amplitudes significantly larger than those evoked by kainate. We localized the domain that determines the relative agonist efficacies to the C-terminal half of the L3 domain of GluR7. Our data show that EC(50) values for glutamate (but not for kainate) in GluR7 mutants or chimeras tend to be increased in comparison to the EC(50) values in GluR6. The high EC(50) for wild-type GluR7 reported in the literature appears to be linked to the S1 portion of the agonist-binding domain. Finally, we determined the C-terminal half of the L3 domain plus the far C-terminal domain of GluR7 to be responsible for the recently reported reduction of current amplitude seen when GluR7 is coexpressed with GluR6. We conclude that coexpression of GluR6 and GluR7 leads to nonstochastical assembly of heteromeric receptor complexes.

How well can molecular modelling predict the crystal structure: the case of the ligand-binding domain of glutamate receptors.

The concept that the ligand-binding domain of vertebrate glutamate receptor channels and bacterial periplasmic substrate-binding proteins (PBPs) share similar three-dimensional (3D) structures has gained increasing support in recent years. On the basis of a dual approach that included computer-assisted molecular modelling and functional studies of site-specific mutants, theoretical 3D models of this domain have been proposed. This article reviews to what extent these models could predict the crystal structure of the ligand-binding domain of an ionotropic glutamate receptor subunit recently determined at high resolution by X-ray diffraction studies.

Antigenic cross-reactivity between electrolectins.

The antigenic cross-reactivity between purified chick, eel and mouse electrolectins (endogenous beta-D-galactoside specific lectins) have been studied using a solid phase radioimmunoassay. The immune serum raised against the eel electrolectin crossreacts both with the chick and the mouse electrolectins, while the anti-chick electrolectin anti-serum recognizes only the eel but not the mouse electrolectin. These findings are analyzed in terms of the phylogenetic distance separating the species considered; they suggest that electrolectins fulfil a fundamental biological function.

Presence of N-methyl-D-aspartate-like and kainate-like activities in bovine brain.

Searching for the natural ligands interacting with brain excitatory amino acid receptors, we have isolated from cow brain a low-Mr ampholyte fraction containing molecules with excitatory properties similar to those of N-methyl-D-aspartate and kainate, which cannot be accounted for by any of the known brain excitants. This finding supports the hypothesis of the existence of excitatory neurotransmitters other than L-glutamate and L-aspartate.

A dipeptide derived from kainic and L-glutamic acids: a selective antagonist of amino acid induced neuroexcitation with anticonvulsant properties.

The dipeptide N-[[[2'S-(2' alpha, 3' beta, 4' beta)]-2'-carboxy-4'-(1"-methylethenyl)-3'-pyrrolidinyl)acetyl]-L- glutamic acid (6) has been synthesized by a route that involves the selective protection of the alpha-carboxyl function of kainic acid. This dipeptide inhibits the stimulation of Na+ fluxes induced in brain slices by the neuroexcitant N-methyl-D-aspartic acid. Administered intracerebroventricularly, it is also effective in protecting mice from picrotoxin-induced convulsions with an ED50 of 0.17 mumol.

Bicyclic lactones derived from kainic acid as novel selective antagonists of neuroexcitatory amino acids.

The bicyclic [2S-(2 alpha,3 beta,4 beta)]-2-carboxy-4-(1-hydroxy-1-methylethyl)-3- pyrrolidineacetic acid delta-lactone (4), as well as its 4-[1-hydroxy-1-(iodomethyl)ethyl], 4-[1-hydroxy-1-(hydroxymethyl)ethyl], and 4-[1-hydroxy-1-[(phenyl-thio)methyl]ethyl] analogues, 6, 7, and 9, respectively, were designed and synthesized as potential selective antagonists of neuroexcitatory amino acids. When applied to rat brain slices, these lactones, which are chemically derived from kainic acid, inhibit the stimulation of Na+ fluxes induced by the neuroexcitants kainic acid and N-methyl-D-aspartic acid. Lactone 4 and the hydroxy lactone 7 block preferentially the response to N-methyl-D-aspartic acid, while the iodo lactone 6 and the phenylthio lactone 9 are mainly kainic acid antagonists. Total inhibitions can be obtained, half of the maximal effect being observed at lactone concentrations in the range of 0.2-3 mM.

Properties of kainate receptor/channels on cultured Bergmann glia.

Following the localization, at the electron microscope level, of the immunoreactivity towards a putative kainate receptor on Bergmann glial cells in the chick cerebellar cortex, cultures of Bergmann glia were used to establish the presence of functional kainate receptor/channels and study their properties. Bergmann glia were identified by their fusiform morphology and their ability to bind an anti-kainate binding protein monoclonal antibody, a kainate receptor high affinity ligand--kainyl-bovine serum albumin--and a glial marker--anti-vimentin monoclonal antibody. Membranes prepared from the culture cells displayed, using 25 nM [3H]kainate, the binding of 4.1 pmol of [3H]kainate/mg protein and showed the presence in Western blots of the two polypeptides of 49 and 93 kDa attributed to the kainate binding protein. Kainate, at concentrations above 0.1 mM, was found to increase the influx into cultured Bergmann glia of 22Na+, 86Rb+, 45Ca2+ and 36Cl- ions. The traffic of 22Na+, induced by kainate and glutamate, observed only in the presence of 1 mM ouabain, was blocked by kainate receptor antagonists and by 0.01 mM quisqualate. Analysis of the kinetics of incorporation of 22Na+ and 45Ca2+ ions showed an initial accumulation of 22Na+ and 45Ca2+ ions followed by their total dissipation. The results indicate that the kainate-induced influx of Na+ ions through the kainate receptor/channel causes the reverse transport of Na+ ions, by activation of the Na+/Ca2+ and Na+/H+ exchangers which remove intracellular Na+ ions. Pre-exposure of the cells to 0.5 mM dibutyryl cAMP was found to greatly enhance the kainate-induced 22Na+ ion influx. We propose that the Bergmann glia kainate receptors modulate the efficacy of the glutamatergic synapses between the parallel fibers and Purkinje cell spines and form part of a glial machinery responsible for plastic changes in synaptic transmission.

Subcellular localization of a putative kainate receptor in Bergmann glial cells using a monoclonal antibody in the chick and fish cerebellar cortex.

A monoclonal antibody, IX-50, that was raised against a kainate binding protein (Mr = 49,000) from chicken cerebellum, was used in light and electron microscopic immunocytochemical studies to localize putative kainate receptors. Pre- and postembedding immunoperoxidase and immunogold methods were used in the cerebellar cortices of one to 26-day old chickens and adult rainbow trout. Immunoreactivity was detected only in association with Golgi epithelial/Bergmann glial cells. Intracellular immunoreactivity was present in the granular and agranular endoplasmic reticulum, Golgi apparatus and in lysosomes, representing the sites of synthesis, glycosylation and degradation of the protein. In the fish the granular endoplasmic reticulum was not immunoreactive. Extracellular immunoreactivity was associated with the plasma membrane. In the fish it was established that the epitope is on the outer surface of the membrane. The protein seems to be uniformly distributed along the membrane including the somata, the radial stem processes and the leafy lamellae surrounding Purkinje cell dendrites. Areas of the glial membrane in contact with other glial cells were also immunopositive. High-resolution light microscopy demonstrated all the Bergmann glial plasma membrane in the cortex, providing a "negative" image of Purkinje cell dendrites. It is apparent that Bergmann glial processes selectively outline the dendrites of the Purkinje cells by surrounding the parallel fibre terminal/Purkinje cell spine synaptic complexes. The parallel fiber terminals were highly immunoreactive for glutamate, as shown by an immunogold procedure. The association of Bergmann glial processes, carrying the Mr = 49,000 kainate binding protein, with the Purkinje cell dendrites and spine synapses could provide a basis for neuronal signalling to the Bergmann glia, possibly by glutamate.

In vitro contraction of the pupillary sphincter by substance P and its stable analogs.

The effects of substance P and substance P analogs have been studied quantitatively by use of an isolated preparation of bovine pupillary sphincter muscle. Substance P contracts the pupillary sphincter in a dose-dependent manner with a value for median effective dose (ED50) of 1.0 X 10(-6) M. The effects of substance P result from its interaction with a specific receptor in the pupillary sphincter. These results strengthen the view that substance P is involved in the oculopupillary reflex.

Selective interactions of electrolectins from eel electric organ and mouse thymus with mouse immature thymocytes.

The pure electrolectin, a beta-D-galactoside binding lectin from the electric organ of the electric eel Electrophorus electricus, was found to agglutinate selectively a subpopulation of mouse thymocytes. This cell population could be separated from non-agglutinated cells by 1 g sedimentation over fetal calf serum. The agglutinated cells could be identified as immature thymocytes on the basis of the density of theta-antigen they bear on their surfaces, their mitotic activity and the absence of response to the mitogenic action of phytohemagglutinin. The immature mouse thymocytes were found to bind the endogenous mouse thymic lectin (MTL) a protein that displays the same saccharide specificity as the eel electrolectin and with which it cross-reacts immunologically. MTL is secreted by mouse thymic reticulocytes in tissue culture and its specific activity is markedly increased after depleting the thymus of its thymocytes. This finding is an indication of the possible localization of MTL in the thymic epithelium. These results are discussed in the light of our recent findings that the eel electrolectin has prophylactic and therapeutic actions on the experimental auto-immune myasthenia gravis in rabbits.

CDNA cloning of chick brain alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors reveals conservation of structure, function and post-transcriptional processes with mammalian receptors.

Several types of functional ionotropic glutamate receptor have been cloned in the recent years from the mammalian central nervous system, but till now, none from other vertebrate species. Here, we report the cloning and functional analysis of four chick brain cDNAs, coding for members of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype of glutamate receptors. These receptors are highly homologous to the mammalian GluR1-4 (A-D) receptors ( > 90%), and conserve their post-transcriptional modifications. The flip/flop exons are conserved not only at the amino acid level but also at the nucleotide level, and the intron of GluR4 involved in the RNA editing of the R/G site displays a rat-chick sequence conservation of 95%. Significant sequence differences are found only in the region containing the immunogenic epitope of neuroactive anti-GluR3 antibodies. Chick AMPA receptors are expressed in both the cerebrum and cerebellum. The ion channel activities of chick GluR1-4 were analyzed in Xenopus oocytes and found to be similar to those of mammalian AMPA receptors. Though their contribution to kainate binding activity in the cerebellum is minor, the profile of channel activity of the chick GluR1-4 suggests that they account for the kainatergic channel activity expressed by total chick cerebellar mRNAs.

Organization and expression of the gene encoding chick kainate binding protein, a member of the glutamate receptor family.

The gene encoding chick cerebellar Bergmann glia-specific kainate binding protein (chKBP), has been isolated, characterized and expressed in heterologous systems. The structural gene spans 11.2 kb and contains 11 exons and 10 introns. Several of the exons encode specific receptor domains, including each of the predicted transmembrane regions. Exon/intron boundaries flanking the second, putative channel-forming transmembrane domain are conserved between chKBP and other glutamate/kainate receptor subunits. The putative promoter region 5' to the first exon displays high GC content and TATA, CAAT and AP1 consensus sequences. Transcription of the chKBP gene is evident prior to full cerebellar cortical maturation. Transcripts are abundant in cells consistent with Bergmann glia, as revealed by in situ hybridization. Transfection of 293 kidney cell cultures with chKBP cDNA or chKBP gene expression constructs confers CNQX-sensitive kainate binding with the pharmacological specificity displayed by both chKBP and kainate receptors. However, expression of the same constructs in Xenopus oocytes fails to yield detectable agonist-activated currents.

A ligand binding study of the interactions of guanine nucleotides with non-NMDA receptors.

The interactions of guanine nucleotides, and particularly GTP, with the [3H]-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and [3H]-kainate (KA) binding sites present on brain membranes was studied, using the ligand binding methodology and Scatchard analysis, in order to establish the competitive/non competitive nature of the interaction and determine whether guanine nucleotides, KA and AMPA share common binding sites. GTP was found to block [3H]-AMPA and [3H]-KA binding to rat cortical membranes with IC50 values of 0.4 mM and 1 mM respectively and the [3H] KA-binding to chick cerebellar membranes with a IC50 value of 20 microM. Scatchard analysis of [3H]-KA binding performed in the absence or presence of 1 mM GTP or 0.25 mM AMPA reveals that the high affinity [3H]-KA binding component is not affected by GTP but blocked in a non competitive fashion by AMPA while the low affinity [3H]-KA binding component is not affected by AMPA but blocked by GTP. Scatchard analysis of [3H]-KA binding to chick cerebellar membranes performed in the absence or presence of 33 microM GTP reveals a single binding site blocked in a competitive fashion by GTP. Scatchard analysis of [3H]-AMPA binding performed in the absence or presence of 0.5 mM GTP or 30 microM KA reveals that the high affinity [3H]-AMPA binding component is affected in a non competitive fashion by both GTP and KA while the low affinity [3H] AMPA binding component is affected in a competitive fashion by both GTP and KA.(ABSTRACT TRUNCATED AT 250 WORDS)

A tetrameric subunit stoichiometry for a glutamate receptor-channel complex.

The structure of glutamate receptor-channel (GluR) subunits has recently been shown to differ from that of other ligand-gated channels and to contain a voltage-gated channel-like pore-forming motif. The view that the structure of GluR complexes is similar to the pentameric structure of other ligand-gated channels was questioned here. Studies of the response properties of the GluR1 subunit of the AMPA subtype of GluRs, co-expressed in Xenopus oocytes with its L646A mutant, which differs only by a greatly reduced sensitivity to quisqualate, provide new evidence suggesting that the GluR1 homomeric receptor channel has a tetrameric structure.

Distinct classes of substance P receptors revealed by a comparison of the activities of substance P and some of its segments.

The contracting potency of Substance P and of its C-terminal fragments was studied using four isolated preparations of smooth muscle. The Substance P receptors in the four muscles studied can be differentiated on the basis of their interactions with Substance P and its C-terminal fragments. On the guinea pig ileum, the potency of Substance P is equal to that of the C-terminal octa- and heptapeptide segments and in the rat ileum the potency of Substance P is equal to that of the C-terminal octapeptide and even higher than that of the heptapeptide. In contrast, on the cow pupillary sphincter and guinea pig urinary bladder, Substance P is markedly less potent that the C-terminal octa-, hepta- and hexapeptides. These results suggest the existence of different classes of Substance P receptors and indicate that the N-terminal sequence may be important in regulating Substance P activity.

A microtest plate assay of functional excitatory amino acid receptors.

A microtest plate assay of functional excitatory amino acid receptors present on cultured chick embryo retinal cells has been developed. It is based on measurements of excitatory amino acid-mediated increase in 22Na+ influx into retinal cells adhering to each of the 96 wells of microtest plates. Dose-dependent responses to L-glutamate, kainate and N-methyl-D-aspartate but not to quisqualate can be measured. These responses are selectively inhibited by antagonists of excitatory amino acids. The assay is reliable, fast to perform and parsimonious in terms of the volume and thus of amount of the drug applied. It allows a single investigator to perform, in one day, measurements of the effects of known or putative glutamatergic ligands in more than 100 different conditions.

Barbiturates, alcohols and the CNS excitatory neurotransmission: specific effects on the kainate and quisqualate receptors.

The effects of barbiturates and straight-chain aliphatic alcohols on the responses of rat striatal neurons to excitatory amino acids have been investigated. The responses to N-methyl-D-aspartate, quisqualate, kainate, L-glutamate and L-aspartate were measured by the increase in 22Na+ efflux rate that they produce in brain slices. The responses to quisqualate and kainate, measured in the 22Na+ efflux assay, were found to be partially blocked by barbiturates whereas the responses to N-methyl-D-aspartate, glutamate and aspartate were not. The kainate and quisqualate-induced increases in 22Na+ efflux rate were much more readily blocked by the presence of aliphatic alcohols than were the responses to N-methyl-D-aspartate, glutamate and aspartate. These results strengthen the idea of the existence of 4 distinct receptors for excitatory amino acids in the rat striatum. They are consistent with the presence on the kainate and quisqualate receptors, but not on the N-methyl-D-aspartate and glutamate/aspartate receptors of a hydrophobic domain which would provide a site of interaction for barbiturates and alcohols. They suggest that receptors for excitatory amino acids can be targets for the actions of barbiturates and alcohols on the central nervous system, and may mediate some of the anesthetic and hypnotic effects of these drugs.

An evaluation of selected brain constituents as putative excitatory neurotransmitters.

Searching for the endogenous ligands of the 4 classes of excitatory amino acid receptors detected in the mammalian CNS, we have measured, using a 22Na+ efflux receptor assay, the excitatory activity of 42 brain constituents or analogs and established the receptor specificity of those substances which possess excitatory properties. Among the substances tested were methyltetrahydrofolate and N-acetylaspartylglutamate, two putative ligands of the kainate and glutamate receptors. These compounds were found to have very little or no excitatory activity, respectively. The 8 brain constituents possessing excitatory properties displayed a receptor specificity similar to either that of N-methyl-D-aspartate (e.g. quinolinate) or glutamate (e.g. cysteine sulfinate) but not of kainate or quisqualate. These results are discussed in relation with the problem of the identification of brain excitatory neurotransmitters.