Properties of Quisqualate-Sensitive L-[3H]Glutamate Binding Sites in Rat Brain as Determined by Quantitative Autoradiography

Quisqualate, a glutamate analogue, displaced L-[3H]glutamate binding in a biphasic manner, corresponding to "high-affinity" and "low-affinity" binding sites. High-affinity quisqualate sites were termed "quisqualate-sensitive L-[3H]glutamate" binding sites. Quisqualate-sensitive L-[3H]glutamate binding was regionally distributed, with the highest levels present in the cerebellar molecular layer. This binding was stimulated by millimolar concentrations of chloride and calcium. The stimulatory effects of calcium required the presence of chloride ions, whereas chloride's stimulatory effects did not require calcium. All of the L-[3H]glutamate binding stimulated by chloride/calcium was quisqualate sensitive and only weakly displaced by N-methyl-D-aspartate, L-aspartate, or kainate. At high concentrations (1 mM), the anion blockers 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid both reduced, by 41 and 43%, respectively, the stimulatory effects of chloride. At concentrations of 100 microM, kynurenate, L-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and L-2-amino-4-phosphonobutyric acid (L-APB) failed to displace quisqualate-sensitive L-[3H]glutamate binding in the cerebellar molecular layer. In the presence of KSCN, however, 100 microM AMPA displaced 44% of binding. Quisqualate-sensitive L-[3H]glutamate binding was not sensitive to freezing, and, in contrast to other chloride- and calcium-dependent L-[3H]glutamate binding sites that have been reported, quisqualate-sensitive binding observed by autoradiography was enhanced at 4 degrees C compared with 37 degrees C. Quisqualate-sensitive L-[3H]glutamate binding likely represents binding to the subclass of postsynaptic neuronal glutamate receptors known as quisqualate receptors, rather than binding to previously described APB receptors, chloride-driven sequestration into vesicles, or binding to astrocytic membrane binding sites.     

Characterisation of Na+-Independent L-[3H]Glutamate Binding Sites in Human Temporal Cortex

The binding of L-[3H]glutamate to membranes from human temporal cortex was studied in the absence of Na+, Ca2+, and Cl- ions. Pharmacological characterisation revealed that approximately 35% of specific binding at 50 nM L-[3H]glutamate was sensitive to a combination of kainate and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. The remaining approximately 65% of specific binding was to a single population of sites with a KD of 844 nM and a Bmax of 0.92 pmol/mg protein. The pharmacological characteristics were consistent with an interaction at the N-methyl-D-aspartate subclass of excitatory amino acid receptor. The inclusion of Cl- ions revealed additional glutamate binding; this was sensitive to quisqualate and DL-2-amino-4-phosphonobutyrate, but not to kainate, DL-2-amino-7-phosphonoheptanoate, or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid.     

Solubilization of quisqualate-sensitive L-[3H]glutamate binding sites from guinea pig brain membranes using a zwitterionic detergent

L-[3H]Glutamate binding sites were solubilized with a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) plus ammonium thiocyanate from guinea pig synaptosomal membranes. The binding of L-[3H]glutamate to the solubilized binding sites was saturable and reversible. Scatchard analysis suggested the existence of two different classes of binding sites with KDs of 63.8 and 644 nM. The L-[3H]glutamate binding was displaced by excitatory amino acids with such an order of potency that L-glutamate much greater than D-glutamate congruent to L-aspartate greater than D-aspartate. Quisqualate effectively displaced the glutamate binding in biphasic manner. L-Glutamic acid diethyl ester, the quisqualate receptor antagonist, also showed a moderate displacing ability. Other neuroactive amino acid analogues displaced the glutamate binding only weakly, except for L- and D-homocysteic acids which had moderate potency. It is very likely from these results that the glutamate binding sites solubilized in this study are relevant to the physiological glutamate receptors especially of quisqualate-type.     

Biochemical characterization of an autoradiographic method for studying excitatory amino acid receptors using L-[3H]glutamate

A method was developed for radiolabeling excitatory amino acid receptors of rat brain with L-[3H]glutamate. Effective labeling of glutamate receptors in slide-mounted 10-microns sections was obtained using a low incubation volume (0.15 ml) and rapid washing: a procedure where high ligand concentrations were achieved with minimal waste. Saturation experiments using [3H]glutamate revealed a single binding site of micromolar affinity. The Bmax was trebled in the presence of Ca2+ (2.5 mM) and Cl- (20 mM) with no change in the Kd. Binding was rapid, saturable, stereospecific, and sensitive to glutamate receptor agonists. The proportions of [3H]glutamate binding sensitive to N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were 34, 54, and 51%, respectively. NMDA inhibited binding at a distinct subset of L-[3H]glutamate sites, whereas AMPA and kainate competed for some common sites. Labeling of sections with L-[3H]glutamate in the presence of the selective agonists allowed autoradiographic visualization of glutamate receptor subtypes in brain tissue.     

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.     

Displaceable binding of [3H]l-glutamic acid to non-receptor materials

[3H]L-glutamic acid binding to microfuge tubes and glass was investigated in four buffers. Background binding to these materials was negligible, but was increased by centrifugation or suction in Tris-HCl and Tris-citrate buffer. This binding was much less or eliminated when HEPES-KOH, or Tris-acetate buffer was used instead. [3H]L-glutamate binding to microfuge tubes was inhibited by L- but not D-isomers of glutamate and aspartate. DL-2-amino-7-phosphonoheptanoic acid also did not inhibit the binding. Other compounds which showed low to moderate inhibition were: N-methyl-D-aspartate, quisqualate, L-glutamic acid diethyl ester, N-methyl-L-aspartate, kainate, and 2-amino-4-phosphonobutyrate. Binding was inhibited by denatured rat brain membranes. A protein-dependent [3H]glutamate binding was obtained with a repeatedly frozen-thawed membrane preparation when binding was done in Tris-acetate buffer. It is recommended that Tris-acetate or HEPES-KOH buffer should be used in the glutamate binding assay. If Tris-HCl or Tris-citrate buffer is used, appropriate control experiment should be done to correct for binding to microfuge tubes or glass fiber filters.     

Chemoreception in Hydra vulgaris (attenuata): initial characterization of two distinct binding sites for L-glutamic acid.

To elucidate the relationship between L-glutamic acid and the putative chemoreceptor for glutathione, binding of L-[3H]glutamate to a crude membrane fraction from Hydra vulgaris (attenuata) has been characterized. The binding of L-[3H]glutamate was rapid, reversible and saturable. A Scatchard analysis of the specific binding revealed values of 10 microM for the dissociation constant (Kd) and 170 pmol/mg for the maximal capacity of binding sites (Bmax). A maximum of 65% of the specific L-[3H]glutamate binding was inhibited by the chemostimulatory peptide, glutathione. This glutathione-sensitive glutamate binding presumably represents the association of glutamate with a putative chemoreceptor which modulates feeding behavior in hydra. The remaining 35% of the specific L-[3H]glutamate binding may be due to a second class of glutamate binding sites which is insensitive to glutathione. The identification of glutathione-insensitive glutamate binding is the first indication of a putative glutamate receptor, which may mediate an action independent of the glutathione-induced feeding response. The glutathione-insensitive and glutathione-sensitive sites must have similar affinities for glutamate since these sites were indistinguishable by Scatchard analysis. A preliminary characterization of the glutathione-insensitive site, performed in the presence of saturating levels of glutathione, revealed inhibition of glutathione-insensitive glutamate binding by kainate and quisqualate, but not by N-methyl-D-aspartate. A glutathione-insensitive L-[3H]glutamate binding suggests that kainate and alpha-aminoadipate may be selective ligands for the glutathione-insensitive and glutathione-sensitive glutamate binding sites, respectively.     

Pharmacologically distinct glutamate receptors on cerebellar granule cells

Cultured cerebellar granule cells were found to exhibit calcium-dependent release of 3H-D-aspartate when stimulated with excitatory amino acids. L-glutamate and L-aspartate were found to be potent stimulators of 3H-D-aspartate release, D-aspartate was weaker and only minor effects were seen with D-glutamate, quisqualate, kainate, N-methyl-D-aspartate (NMDA) and L-alpha-aminoadipate (L-alpha AA). It was also found that only L-glutamate and L-aspartate showed high affinity for the 3H-L-glutamate binding sites on granule cell membranes. Stimulation by L-glutamate of 3H-D-aspartate release could be blocked by various excitatory amino acid antagonists. From the relative potencies of agonists and antagonists on D-aspartate release it is suggested that cerebellar granule cells express functionally active glutamate receptors with pharmacological characteristics different from all known excitatory amino acid receptors.     

L-Glutamate Receptor Heterogeneity: Labeling of Distinct Receptor Sub-Types Using Radioligand Binding Techniques

Abstract

This study demonstrates (1) that L-[³H]glutamate labels 3 distinct binding sites (types A1, A2 and A4) in isolated rat brain membranes and (2) that only the N-methyl-aspartate (A1) and quisqualate (A2) receptor classes are associated with the postsynaptic density (PSD). L-[³H]glutamate bound to PSDs with K_d 339 nM and B_max 6.1 pmol/mg protein. These sites were resolved into 2 distinct sub-types on the basis of inhibition studies. N-Methyl-aspartate maximally inhibited 57% of PSD-located L-glutamate binding sites (the A1 site) and quisqualate 43% (the A2 site); the effects of both substances were additive. The ligand selectivities of these 2 sites indicated their identity with the N-methyl-D-aspartate and quisqualate receptor classes defined electrophysiologically. The Cl^?-dependent population of L-glutamate binding sites (the A4 site) which predominates in synaptic membranes was absent from PSDs.     

Inhibition of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis in the Neonatal Rat Hippocampus by 2-Amino-3-Phosphonopropionate

The effects of excitatory amino acid agonists and alpha-amino-omega-phosphonocarboxylic acid antagonists on phosphoinositide hydrolysis in hippocampal slices of the 7-day neonatal rat were examined. Significant stimulation of [3H]inositol monophosphate formation was observed with ibotenate, quisqualate, L-glutamate, L-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, L-homocysteate, and kainate. N-Methyl-D-aspartate had no effect. Of these agonists, ibotenate and quisqualate were the most potent and efficacious. Stimulations by ibotenate and quisqualate were partially inhibited by L-2-amino-4-phosphonobutyrate (10(-3) M), but this antagonist had no effect on L-glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, or kainate. At 10(-3) M, D,L-2-amino-3-phosphonopropionate completely inhibited ibotenate and quisqualate stimulations, partially inhibited L-glutamate stimulation, and had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-, kainate-, or carbachol-induced [3H]inositol monophosphate formation. Concentration-effect experiments showed D,L-2-amino-3-phosphonopropionate to be five times more potent as an antagonist of ibotenate-stimulated phosphoinositide hydrolysis than L-2-amino-4-phosphonobutyrate. Thus in the neonatal rat hippocampus, like in the adult rat brain, D,L-2-amino-3-phosphonopropionate is a selective and relatively potent inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis. Because this glutamate receptor is uniquely sensitive to D,L-2-amino-3-phosphonopropionate, these studies provide further pharmacological evidence for the existence of a novel excitatory amino acid receptor subtype that is coupled to phosphoinositide hydrolysis in brain.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Characterization of a glutamic acid neurotransmitter binding site on neuroblastoma hybrid cells

Glutamate is thought to be a major excitatory neurotransmitter in the central nervous system. To study the glutamate receptor and its regulation under carefully controlled conditions, the specific binding of [3H]glutamate was characterized in washed membranes isolated from a neuroblastoma X retina hybrid cell line, N18-RE-105. [3H]Glutamate bound in a saturable and reversible fashion with an apparent dissociation constant, KD, of 650 nM and a maximum binding capacity, Bmax, of 16 pmol/mg of protein. Pharmacologic characterization of the site indicates that it closely resembles the Na+-independent binding site for glutamate found on brain membranes and thought to be an excitatory amino acid neurotransmitter receptor. Thus, while kainate, N-methyl-DL-aspartate, and nonamino acid ligands did not displace [3H]glutamate, quisqualate and ibotenate were potent inhibitors of specific binding. Furthermore, this binding site is regulated by ions in a manner which resembles that described in the hippocampus (Baudry, M., and Lynch, G. (1979) Nature (Lond.) 282, 748-750). Calcium (10 mM) increased the number of binding sites 2.6-fold with no change in receptor-ligand affinity. Lanthanum (1 mM) was the only other cation added which enhanced (3-fold) the binding of [3H]glutamate. Monovalent cations resulted in a decrease in the number of glutamate binding sites. Incubation of membranes in the presence of chloride ions caused a marked increased in [3H] glutamate binding, an effect which was synergistic with that of calcium incubation. Thus, N18-RE-105 cells possess a binding site for [3H]glutamate pharmacologically similar to an excitatory neurotransmitter binding site in brain and which exhibits regulatory properties resembling those previously described in hippocampal membranes, providing an excellent model for mechanistic studies.     

l-Glutamate Binding Site on N18-RE-105 Neuroblastoma Hybrid Cells Is Not Coupled to an Ion Channel

We studied the properties of the N18-RE-105 neuronal cell line to determine if its glutamate binding site represents a neurotransmitter receptor. In immunocytochemical experiments, these cells stained strongly for neurofilament, but not for glial fibrillary acidic protein. In whole-cell patch clamp experiments, cells exhibited voltage-dependent Na+, Ca2+, and K+ currents characteristic of neurons. However, perfusion with L-glutamate or other excitatory amino acids did not evoke the inward current expected of a receptor/channel complex. In binding studies, the maximum accumulation of L-[3H]glutamate by washed membrane vesicles at 37 degrees C was 69 pmol/mg protein, and half-maximal accumulation occurred at 0.64 microM. This accumulation was blocked completely by quisqualate, partially by DL-2-amino-4-phosphonobutyric acid and L-cystine, but not at all by 1 mM kainate or N-methylaspartate. L-[3H]Glutamate accumulation was stimulated by Cl-, but reduced by Na+, 0.01% digitonin, or hyperosmotic (400 mM glucose) assay medium. The release of L-[3H]glutamate from vesicles was much faster in the presence of 100 microM unlabelled glutamate than 100 microM unlabelled quisqualate or DL-2-amino-4-phosphonobutyric acid. Thus, although N18-RE-105 cells possess many neuronal properties, the results obtained are not those expected from reversible binding of L-glutamate to a receptor/channel complex, but are consistent with a Cl- -stimulated sequestration or exchange process.     

6,7-Dinitroquinoxaline-2,3-Dione Blocks the Cytotoxicity of N-Methyl-D-Aspartate and Kainate, but Not Quisqualate, in Cortical Cultures

Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N-methyl-D-aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor-channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor-channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate-evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate-preferring receptor insensitive to DNQX may mediate quisqualate toxicity.     

Changes in Excitatory Amino Acid Receptor Binding in the Intact and Decorticated Rat Neostriatum Following Insulin-Induced Hypoglycemia

An involvement of excitatory amino acid (EAA) transmitter-receptor interactions in the development of hypoglycemia-induced neuronal damage has been suggested. We report here on the binding to EAA receptors in the rat caudate nucleus and cerebral cortex, during and following severe insulin-induced hypoglycemia with an isoelectric EEG of 10 or 30 min duration. The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [( 3H]AMPA) to quisqualate receptors, [3H]kainic acid (KA) to kainate receptors, and [3H]glutamate to N-methyl-D-aspartate (NMDA)-sensitive sites was determined by quantitative autoradiography. During EEG isoelectricity, AMPA binding was reduced by approximately 40%, which could represent quisqualate receptor desensitization. One hour following glucose-induced recovery, AMPA binding was no longer different from control level. As the recovery period was prolonged to 1 or 4 weeks, AMPA binding decreased. The decrease was more pronounced in the dorsolateral than in the ventromedial part of the striatum. This correlates with the distribution of neuronal damage, and probably reflects loss of receptor binding sites due to cell death. During the period of EEG silence there was a tendency toward an increase in NMDA displaceable glutamate binding. Following 4 weeks of recovery, binding to NMDA receptors was significantly decreased. Glutamate binding to NMDA-sensitive sites was remarkably resistant to neuronal necrosis and was not significantly different from control values in the dorsolateral caudate 1 week following the hypoglycemic coma. No changes in KA binding were found until 1 week posthypoglycemia, when a significant reduction in binding was noted in the lateral striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitatory Amino Acid Receptor Potency and Subclass Specificity of Sulfur-Containing Amino Acids

The sulfur-containing amino acids, L- and D-cysteate, L-cysteine, L- and D-cysteine sulfinate, L- and D-cysteine-S-sulfate, L-cystine, L- and D-homocysteate, L- and D-homocysteine sulfinate, L-homocysteine, L-serine-O-sulfate, and taurine were tested in two excitatory amino acid receptor functional assays and in receptor binding assays designed to label specifically the AA1/N-methyl-D-aspartate (NMDA), AA2/quisqualate, and AA3/kainate receptor recognition sites, as well as a CaCl2-dependent L-2-amino-4-phosphonobutanoate site, and a putative glutamate uptake site. Agonist efficacies were determined by chick retinal excitotoxicity and stimulated sodium efflux from rat brain slices. D-Homocysteine sulfinate, L-homocysteate, and L-serine-O-sulfate had affinities most selective for the NMDA binding site, whereas the binding affinities of D-cysteate, D-cysteine sulfinate, D-homocysteate, and L-homocysteine sulfinate were less selective. However, the correlation of agonist activity sensitive to blockade by D-2-amino-7-phosphonoheptanoate or D-2-amino-5-phosphonopentanoate in the functional assays with affinity in the NMDA binding assay (r = 0.87, p less than 0.005 and r = 0.98, p less than 0.005 for excitotoxicity and sodium efflux, respectively) allows characterization of these sulfur-containing amino acids as acting at NMDA subclass receptors. L-Homocysteate, which has been found in the brain, and L-serine-O-sulfate are selective agonists and could serve as endogenous neurotransmitters at the NMDA receptor.     

L-[3H]Glutamate Binding to a Membrane Preparation from Crayfish Muscle

The binding of L-[3H]glutamate to an isolated membrane preparation from crayfish tail muscle has been studied. The muscle homogenate was osmotically shocked, frozen and thawed, and thoroughly washed before incubation with L-[3H]glutamate. The preparation showed high specific binding of L-glutamate with a KD of 0.12 microM and Bmax of 4.7 pmol/mg protein measured in Tris/HCl pH 7.3 and at 4 degrees C. Nonspecific binding was 5-10% of total binding. The glutamate binding was highly stereospecific [K0.5 (D-glutamate), 270 microM] and showed a high degree of discrimination between L-glutamate and L-aspartate [K0.5 (L-aspartate), 54 microM]. In mammalian CNS preparations potent agonists of L-glutamate such as kainate and N-methyl-D-aspartate had no effect at 1 mM, and quisqualate was a weak inhibitor of L-glutamate binding [K0.5 (quisqualate), 162 microM]. Ibotenate was the most potent inhibitor [K0.5 (ibotenate), 0.27 microM], and various esters of L-glutamate were of intermediate potency as displacers of L-[3H]glutamate binding (K0.5 values from 6 to 60 microM). The glutamate binding site from crayfish muscle is clearly different from any of the subclasses of glutamate receptors in mammalian CNS. A possible physiological function of the binding site is a postsynaptic receptor for glutamate, either an extra-junctional or a junctional receptor.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Specific binding of [3H]+/- 2-amino-7-phosphono heptanoic acid to rat brain membranes in vitro

The specific binding of [3H]+/- 2-amino-7-phosphono heptanoic acid (3H-APH), a potent N-methyl-D-aspartate (NMDA) antagonist, to extensively washed, previously frozen crude mitochondrial fractions of rat brain is described. Binding was optimal at physiological pH and temperature and, in Triscitrate buffer, attained equilibrium within 60 minutes. Scatchard analysis of the equilibrium data for forebrain revealed a single, non-interacting population of binding sites (BMapp = 15 picomoles/mg protein; KDapp = 3.6 uM; Hill coefficient = 0.92, r = 0.99; N = 5). Specific binding of the ligand was readily reversible by unlabeled APH and was absent in peripheral tissues including heart, lung, kidney, liver, spleen and striate muscle and in heat treated brain sonicates. An 8-fold variation in the amount of ligand bound to brain membranes prepared from different regions was observed with binding being greatest in the hippocampal formation and least in the midbrain. Kainic acid, NMDA and aspartic acid exhibited negligible affinity for the [3H]-APH site; in contrast, quisqualic acid, ibotenic acid, glutamatic acid, homocysteic acid and 2-amino-4-phosphono butyric acid were moderately potent displacers. The results indicate that [3H]-APH labels a quisqualate preferring site in vitro. Unlike the receptor labeled by [3H]-glutamate however, [3H]-APH binding was attenuated in the presence of chloride ions suggesting that this ligand may label a subpopulation of excitatory amino acid receptors.     

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.