Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

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.     

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.     

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.     

NMDA and Non-NMDA Receptor-Mediated Release of [3H]GABA from Granule Cell Dendrites of Rat Olfactory Bulb

Abstract: We have studied the effect of glutamate and the glutamatergic agonists N-methyl-d -aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [³H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [³H]GABA release and the release evoked by a high K⁺ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [³H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [³H]GABA release. The presence of glycine and the absence of Mg²⁺ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K⁺ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [³H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [³H]GABA release but not the NMDA-stimulated release. The present results show that in the external plexiform layer of the olfactory bulb, glutamate is stimulating GABA release through a presynaptic, receptor-mediated mechanism as a mixed agonist on NMDA and non-NMDA receptors; glutamate is apparently also able to induce GABA release through heteroexchange.     

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.     

Localization of N-Methyl-d-Aspartate Receptors in the Rat Striatum: Effects of Specific Lesions on the [3H]3-(2-Carboxypiperazin-4-yl)propyl-1-Phosphonic Acid Binding

The binding of [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([3H]CPP), a rigid analogue of 2-amino-7-phosphonoheptanoic acid (AP7) and reported to be a selective N-methyl-D-aspartate (NMDA) antagonist, was studied in rat striatal membranes using a centrifugation procedure to separate bound and free radioligand. [3H]CPP bound with high affinity (KD = 272 nM) in a saturable, reversible, and protein concentration-dependent manner. Specific binding was suggested to involve a single class of noninteracting binding sites. The most potent [3H]CPP binding inhibitors tested were CPP, L-glutamate, 2-amino-5-phosphonovalerate, and AP7. NMDA, L-aspartate, and alpha-aminoadipate were also shown to be efficient in inhibiting the binding, whereas quisqualate, D,L-2-amino-4-phosphonobutyrate, kainate, L-glutamate diethylester, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were found to be essentially inactive. These data are therefore consistent with the view that [3H]CPP selectively binds to NMDA receptors in the rat striatum. Lesions of intrastriatal neurons using local injections of kainic acid revealed a marked decrease in [3H]CPP binding, suggesting an almost exclusively postsynaptic location of binding sites in the striatum. Conversely, bilateral lesion of corticostriatal glutamatergic fibers resulted in an increased number of [3H]CPP striatal binding sites, providing evidence for a putative supersensitivity response to this striatal deafferentation. Interestingly, lesion of the nigrostriatal dopaminergic neurons using intranigral 6-hydroxydopamine injections resulted, 2-3 weeks later, in a similar increase in the number of [3H]CPP striatal binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Binding Sites for l-[3H]Glutamate on Hippocampal Synaptic Membranes: Three Populations Differentially Affected by Chloride and Calcium Ions

The effects of Cl- and Ca2+ were studied on the specific binding of L-[3H]glutamate to multiple sites on rat hippocampal synaptic membranes. Quisqualate (5 microM) or DL-2-amino-4-phosphonobutyrate (2-APB) (300 microM) was used to discriminate two previously identified classes of binding sites. Saturation isotherms and displacement curves constructed under different ionic conditions suggested that the effects of Cl- and Ca2+ could best be explained by postulating the existence of three major binding site populations in this preparation rather than two. The binding of L-glutamate to Glu A sites exhibits an absolute dependence on Cl-, and Ca2+ markedly increases the maximum density of these sites. Glu A sites bind quisqualate and 2-APB with relatively high affinity. Cl- (47 mM) more than doubles the maximum density of Glu B sites, but Ca2+ appears to have no effect. Glu B sites can be discriminated from the other classes by their relatively low affinity for quisqualate and 2-APB. There is reason to think that the Glu B population is heterogeneous. The novel Glu C population can be virtually selectively labeled by exposing 2-APB-sensitive binding sites to radioligand in Tris-HOAc buffer with Ca2+. Binding of L-[3H]glutamate to these sites is enhanced by both Cl- and Ca2+, but requires neither ion. Ca2+ appears to increase both the affinity of Glu C sites for L-glutamate and their maximum binding site density. In the presence of Ca2+ and Cl-, Glu C sites bind the radioligand with micromolar affinity (KD approximately 2 microM) and high capacity (Bmax approximately 160 pmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

Functional Reconstitution of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate (AMPA) Receptors from Rat Brain

Glutamate receptors belonging to the subclass specifically activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were solubilized from rat forebrain membranes with Triton X-100 and partially purified through a series of three chromatographic steps. Specific [3H]AMPA binding increased 30-60-fold during the isolation procedure. A protein band recognized by antibodies against specific amino acid sequences of the glutamate receptor-A subunit was enriched with each purification step; the molecular mass of this band (105 kDa) corresponded to that of cloned AMPA receptor subunits. Photoaffinity labeling of forebrain membranes with 6-cyano-7-[3H]nitroquinoxaline-2,3-dione, a specific antagonist of the AMPA receptor, labeled a single band that comigrated with the immunolabeled protein. On reconstitution of the partially purified material into bilayer patches, single-channel current fluctuations were elicited by 300 nM AMPA and blocked by 1 microM 6,7-dinitroquinoxaline-2,3-dione.     

Solubilization, Characterization, and Partial Purification of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid-, Quisqualate-, Kainate-Sensitive l-Glutamate Binding Sites from Porcine Brain Synaptic Junctions

L-[3H]Glutamate binding sites were solubilized from porcine brain synaptic junctions by Triton X-114 in the presence of KCl. The solubilized binding sites bound L-[3H]glutamate reversibly with KD and Bmax values of 1.48 +/- 0.18 microM and 178.2 +/- 15.9 pmol/mg of protein, respectively. These binding sites appeared to be integral membrane glycoproteins, with sugar moieties recognized by wheat germ agglutinin. A 49.3-fold purification of these binding sites was achieved by Triton X-114 solubilization, anion-exchange chromatography, and affinity chromatography using wheat germ agglutinin-Sepharose. The apparent molecular mass of the partially purified binding sites was 620 +/- 50 kDa. L-[3H]Glutamate bound to the solubilized preparation could be effectively displaced by agonists of non-N-methyl-D-aspartate (NMDA) L-glutamate receptors but not by NMDA or alpha-amino-4-phosphonobutyrate. The rank order for the competitive ligands in displacing L-[3H]glutamate was: quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid greater than L-glutamate greater than kainate.     

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.     

Ontogeny of Receptor Binding Sites for [3H]Glutamic Acid and [3H]Kainic Acid in the Rat Cerebellum

The development of the specific binding sites for L-[3H]glutamic acid (KD = 370 nM) and for [3H]kainic acid (KD = 39 nM) was studied in the rat cerebellum. Specific binding at both sites remains low during the first week after birth but increases markedly during the second and third weeks after birth, when glutamatergic parallel fiber synaptogenesis occurs. The development of the kainate site lags behind that of the glutamate site, indicating their autonomy.     

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.     

Alterations in Cortical [3H]Kainate and α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy

Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Ultraviolet radiation,thiol reagents,and solubilization enhance AMPA receptor binding affinity via a common mechanism

The binding properties of membrane-bound or solubilized AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptors from rat brain were tested following exposure to ultraviolet (UV)_radiation or incubation with the thiol reagent p-chloromercuriphenyl-sulfonic acid (PCMBS). Brief exposure to UV radiation (254 nm) increased [³H]AMPA binding to brain membranes, while binding to soluble fractions decreased. The increase in brain membrane binding was caused by an apparent interconversion of low-affinity [³H]AMPA binding sites into a higher-affinity state. Incubation with PCMBS caused a significant increase in [³H]AMPA binding to brain membranes but had no significant effect on [³HAMPA binding to solubilized receptors. There was an interaction between the PCMBS and UV effects in the brain membranes such that prior exposure to one of the treatments reduced the relative magnitude of the other's effects. The present results suggest that ultraviolet radiation, PCMBS and solubilization all increase AMPA receptor binding affinity via a common mechanism.     

β-N-Oxalylamino-L-Alanine Action on Glutamate Receptors

beta-N-Oxalylamino-L-alanine (L-BOAA) is a non-protein excitatory amino acid present in the seed of Lathyrus sativus L. This excitotoxin has been characterized as the causative agent of human neurolathyrism, an upper motor neuron disease producing corticospinal dysfunction from excessive consumption of the lathyrus pea. Previous behavioral, tissue-culture, and in vitro receptor binding investigations revealed that L-BOAA might mediate acute neurotoxicity through quisqualate (QA)-preferring glutamate receptors. The present study demonstrates the stereospecific action of L-BOAA on glutamate receptor binding in whole mouse brain synaptic membranes. L-BOAA was most active in displacing thiocyanate (KSCN)-sensitive specific tritiated (RS)-alpha-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding (i.e., QA receptor) (Ki = 0.76 microM) with a rank-order potency of QA greater than kainate greater than N-methyl-D-aspartate (NMDA). By contrast, the nonneurotoxic D-BOAA isomer (100 microM) was essentially inactive in displacing radioligands for glutamate receptors, except the NMDA site, where it was equipotent with L-BOAA. Scatchard analysis of L-BOAA displacement of specific [3H]AMPA binding indicated competitive antagonism (KD: control, 135 nM; L-BOAA, 265 nM) without a significant change in QA-receptor density, and Hill plots yielded coefficients approaching unity. Differential L-BOAA concentration-dependent decreases in specific [3H]AMPA binding were observed in synaptic membranes, indicating that the neurotoxin was more potent in displacing specific binding from frontal cortex membranes, followed by that for corpus striatum, hippocampus, cerebellum, and spinal cord. (ABSTRACT TRUNCATED AT 250 WORDS)     

α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid Binding to Human Cerebral Cortical Membranes: Minimal Changes in Postmortem Brains of Chronic Schizophrenics

The binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a selective ligand for the ion channel-linked quisqualate receptor, was evaluated in Triton X-100-treated membranes of human cerebral cortex. The presence of chaotropic ions produced divergent effects on specific [3H]AMPA binding: A twofold increase in the binding was observed with thiocyanide at 100 mM, although iodide (100 mM) and perchlorate (100 mM) reduced the binding. Chemical modifications of the sulfhydryl group with p-chloromercuriphenylsulfonic acid (PCMBS) produced threefold increases in specific [3H]-AMPA binding in the absence of KSCN as well as in the presence of KSCN. Treatment with dithiothreitol restored the enhanced specific [3H]AMPA binding by PCMBS to the basal level. Although specific [3H]AMPA binding in the absence of KSCN showed a single site (KD = 220 nM, Bmax = 235 fmol/mg of protein), curvilinear Scatchard plots of specific [3H]AMPA binding in the presence of 100 mM KSCN can be resolved into two binding sites with the following parameters: KD1 = 5.82 nM, Bmax1 = 247 fmol/mg of protein; KD2 = 214 nM, Bmax2 = 424 fmol/mg of protein. Quisqualate and AMPA were the most potent inhibitors of the [3H]AMPA binding in the presence of KSCN. Potent inhibitors of the binding included beta-N-oxalylamino-L-alanine (L-BOAA), cysteine-S-sulfate, L-glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione, and 6,7-dinitroquinoxaline-2,3-dione. Kainate, L-homocysteine sulfinic acid, and L-homocysteic acid were active with an IC50 value of a micromolar concentration, whereas L-cysteic acid and L-cysteine sulfinic acid were weakly active.(ABSTRACT TRUNCATED AT 250 WORDS)