l-aspartate binding sites in rat cerebellum: A comparison of the binding ofl-[H]aspartate andl-[H]glutamate to synaptic membranes

The binding ofl-[³H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (K_d = 874nM and B_max = 44pmol/mg protein), which displayed no cooperativity (Hill coefficient approx.= 1). Specific [³H]aspartate was readily and reversibly displaced by unlabelledl-aspartate (thed-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [³H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists,dl-α-aminoadipate,dl-α-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-d-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate.While freezing or cold storage of membranes resulted in diminished [³H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site.Differential effects of various cations on [³H]aspartate binding were observed — monovalent cations reduced, while divalent cations enhancedl-[³H]aspartate binding.     

Effect of aspartame on N-methyl-d-aspartate-sensitive l-[H]glutamate binding sites in rat brain synaptic membranes

Aspartame (l-aspartyl-l-phenylalanine methyl ester), an artificial low-calorie sweetener, was shown to dose-dependently inhibitl-[³H]glutamate binding to its N-methyl-d-aspartate-specific receptors.l-Aspartic acid, a major endogenous metabolite of aspartame, inhibited the binding more stronger than aspartame, while the other metabolites,l-phenylalanine and methanol, had no effect at the same concentration. Aspartame caused a significant change in the affinities ofl-[³H]glutamate binding without altering the V_max values of the binding, suggesting the inhibition is competitive. These in vitro findings suggested that aspartame may act directly on the N-methyl-d-aspartate-sensitive glutamate recognition sites in the brain synaptic membranes.     

l-[H]Glutamate binds to kainate-, NMDA- and AMPA-sensitive binding sites: an autoradiographic analysis

The anatomical distribution ofl-[³H]glutamate binding sites was determined in the presence of various glutamate analogues using quantitative autoradiography. The binding ofl-[³H]glutamate is accounted for the presence of 3 distinct binding sites when measured in the absence of Ca²⁺, Cl⁻ and Na⁺ ions. The anatomical distribution and pharmacological specificity of these binding sites correspond to that reported for the 3 excitatory amino acid binding sites selectively labeled byd-[³H]2-amino-5-phosphonopentanoate (d-[³H]AP5), [³H]kainate ([³H]KA) and [³H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([³H]AMPA) which are thought to be selective ligands for the N-methyl-d-aspartate (NMDA), KA and quisqualate (QA) receptors, respectively.     

Characterization of specific, high-affinity binding sites for l-[H]glutamic acid in rat brain membranes

l-[³H]Glutamic acid binds reversibly to rat brain membranes with high affinity. Specific binding is linear with tissue concentration and has a pH optimum at neutrality. Saturation isotherms reveal anomalous kinetics of specific binding with an high affinity site with a K_D of 11 nM and a lower affinity site with a K_D of 80 nM; the Scatchard plots intercept at a common bound/free ratio. Hill plots of the complete saturation isotherms have a slope of 1.0. There are marked regional differences in the distribution of binding sites in rat brain: parietal cortex, frontal cortex, hippocampus striatum thalamus cerebellum, pons-medulla and hypothalamus. Except for a small amount of specific binding in heart, other peripheral tissues do not exhibit specific binding of l-[³H]glutamic acid. Several amino acids with neuroexcitatory effects inhibit the specific binding: l-glutamic acid l-aspartic acid and d,l-homocysteic acid d-glutamic acid and l-cysteine sulfinic acid; related amino acids without neuroexcitatory effects do not inhibit specific binding. Reputed antagonists of glutamate-induced neuronal depolarization block specific binding: α-aminoadipic acid 2-amino,4-phosphonobutyric acid glutamate diethylester. Prior kainate lesion of the neurons intrinsic to the striatum results in a 45% decrement in specific binding of l-[³H]glutamic acid whereas cortical ablation, which causes degeneration of a cortical-striatal glutamatergic projection and reduces striatal glutamate synaptosomal uptake, does not affect specific binding. These results are compatible with the interpretation that the binding of [³H]glutamic acid occurs at excitatory receptors on neurons.     

Release of [H]l-glutamine acid (l-Glu) and [H]d-aspartic acid (d-Asp) in the area of nucleus tractus solitarius in vivo produced by stimulation of the vagus nerve

We have investigated the effects of bilateral electrical stimulation of the vagus nerves in anesthetized, paralyzed rats on the release of exogenously administered [³H]l-glutamic acid ([³H]l-Glu) or [³H]d-aspartic acid ([³H]d-Asp) from the intermediate portion of the nucleus tractus solitarius (NTS). Electrical stimulation of afferent fibers with the frequency, pulse, duration, and intensity required to activate C-fibers, elicted hypotension and bradycardia. Such stimuli induced the release of [³H]l-Glu, or its stable analogue [³H]d-Asp, from the NTS into perfusate collected through push-pull cannulae. The release of radioactive materials, calculated as a percent of increase in radioactivity above the prestimulation level, was for [³H]l-Glu 114.4 ± 25.1% (n= 20) during bilateral vagal stimulation, and45.6 ± 11.3% (n= 9) (P < 0.001) during unilateral stimulation. The release of [³H]d-Asp induced by bilateral vagal stimulation was100.4 ± 31.9%. The release, which was anatomically specific and restricted to the NTS, was directly related to stimulus (and hence reflex) intensity. Overflow of the inert substances [¹⁴C]urea or [¹⁴C]sucrose, co-administered with the [³H]amino acids, did not increase at the same time. Local depolarization of the cells in the NTS by K⁺ (53 mM) increased the overflow of [³H]l-Glu, as well as [¹⁴C]urea, and was able to induce the release of [³H]l-Glu when electrical stimulation failed to have an effect. The results are consistent with the hypothesis thatl-Glu is a neurotransmitter of neurons in the NTS mediating vasodepressor response from vagal afferents, including those from systemic baroreceptors.     

A l-[H]glutamate binding site on glia: an autoradiographic study on implanted astrocytes

In the present study cultured astrocytes were implanted into the inferior colliculus of rats to create an astrocyte-enriched field that could be examined autoradiographically. The presence of the astrocytes was confirmed with anti-glial fibrillary acidic protein (GFA) immunocytochemistry. We report the presence of a chloride-dependent glutamate binding site on the implanted astrocytes. In the presence of chloride, the specific glutamate binding detected in the implant area was 5-fold greater than that found in a corresponding contralateral region. When the chloride was replaced with acetate, glutamate binding to the astrocytes decreased by more than 80%. The chloride-dependent binding to the astrocytes was insensitive to inhibition by kainic acid (KA) and N-methyl-d-aspartate (NMDA) and sensitive to quisqualate, l-aspartate, l-2-amino-4-phosphonobutyrate, and l-α-aminoadipate. The pharmacology of the binding was very similar to that of the in vitro glutamate binding to membranes from cultured astrocytes and to that of a chloride-dependent transport system identified in a glioma cell line. We conclude that the interaction of glutamate with astrocytes is an important component of the total glutamate binding observed in brain slices.     

Reduced d-[H]aspartate binding in Down's syndrome brains

The binding of d-[³H]aspartate to glutamate uptake sites was measured in post-mortem brains from subjects with Down's syndrome (DS) and age-matched controls. DS brains had substantially reduced d-[³H]aspartate binding in the frontal and temporal cortex, hippocampus and caudate nucleus. There was no correlation between the numbers of Alzheimer-like plaques and tangles or clinically-assessed dementia and d-[³H]aspartate binding in DS brains. The binding of [³H]N-(1-[2-thienyl]cyclohexyl)piperidine([³H]TCP) to postsynaptic N-methyl-d-aspartate sites was normal in DS brains. This study suggests that the reduction in glutamate uptake sites in DS is more substantial and widespread than in Alzheimer's disease.     

l-aspartate binding sites in rat cerebellum: A comparison of the binding ofl-[3H]aspartate andl-[3H]glutamate to synaptic membranes

The binding ofl-[³H]aspartate to sonicated, extensively washed and preincubated cerebellar synaptic membranes was investigated. Binding was optimal under physiological conditions of pH and temperature, and attained equilibrium within 10 min. Binding was saturable, and Eadie-Hofstee analysis revealed interaction with a single population of binding sites (K_d = 874nM and B_max = 44pmol/mg protein), which displayed no cooperativity (Hill coefficient approx.= 1). Specific [³H]aspartate was readily and reversibly displaced by unlabelledl-aspartate (thed-isomer being less than half as active) with a half-life of dissociation of 32 sec. Quisqualate, 4-fluoroglutamate and 2-amino-4-phosphonobutyrate, which are good displacers of [³H]glutamate binding, were only weakly active against the aspartate system. The excitatory amino acid antagonists,dl-α-aminoadipate,dl-α-aminosuberate and HA-966 were effective displacers, but the proposed aspartate receptor-preferring agonist, N-methyl-d-aspartate was inactive. Kainic acid exhibited negligible affinity for the aspartate binding site, in common with that for glutamate.While freezing or cold storage of membranes resulted in diminished [³H]-aspartate binding, lyophilization was not only able to confer substantial stability, but induced a marked increase in affinity of the binding site.Differential effects of various cations on [³H]aspartate binding were observed — monovalent cations reduced, while divalent cations enhancedl-[³H]aspartate binding.     

Cells in the anteroventral cochlear nucleus are insensitive to l-glutamate and l-aspartate; excitatory synaptic responses are not blocked by d-α-aminoadipate

Auditory nerve fibers transmit signals from the cochlea to the 3 regions of the cochlear nuclear complex, the anteroventral (AVCN), posteroventral, and dorsal cochlear nucleus in the brainstem. It has been suggested that the amino acids l-aspartate and l-glutamate might serve as a neurotransmitter in auditory nerve fibers^{6–10,13,17–20}. The sensitivity of postsynaptic cells in the cochlear nuclei to these amino acids has been tested by iontophoretic techniques^4,9,10. One difficulty with these experiments is that responses were recorded only extracellularly. A second difficulty is that the concentrations needed to affect cells could not be determined. To avoid these difficulties a brain slice preparation was used to test the sensitivity of cells in the AVCN to bath applied l-glutamate and l-aspartate at concentrations ranging from 10⁻⁵ to 10⁻² M. All cells that were tested in the cochlear nuclear complex were insensitive at all concentrations used; the resting potentials and the input resistances remained unchanged and the synaptic responses to electrical stimulation of the auditory nerve were not desensitized. All cells that were tested in the hippocampus, however, depolarized in the presence of 10⁻⁴ M l-glutamate and l-aspartate. The synaptic responses to electrical stimulation of the auditory nerve were not blocked by d-α-aminoadipate, an amino acid which has been shown to block excitation of cells in the cochlear nuclei by auditory nerve fibers¹⁰. The results are not consistent with l-glutamate and l-aspartate serving as neurotransmitters in the AVCN.     

l-[H]carnosine binding in the olfactory bulb. II. Biochemical and biological studies

During the first 10 days after peripheral deafferentation of the mouse olfactory bulb stereoselective binding ofl-[³H]carnosine declines markedly. The initial phase of this decline is due to a decrease in binding site stereoselectivity, which is then followed by a loss of assayable binding sites. The specificity of inhibition ofl-[³H]carnosine binding by various peptides is also altered after denervation. Competitive inhibitors of carnosine binding become less potent after denervation, while analogues which are not competitive inhibitors remain equipotent before and after denervation. Several carnosine analogues that are normally poor inhibitors become more potent after denervation. Treatment of bulb membranes with trypsin, RNase and hyaluronidase, but not DNase or collagenase, resulted in significant alterations in carnosine binding.l-, but notd-carnosine, protected the binding site from trypsin digestion, and induced additional binding in bulb membranes in a dose-and temperature-dependent fashion. Preincubation of membranes withl-carnosine also led to the induction of additional carnosine binding in membranes from cerebral cortex, cerebellum and deafferentated bulbs but not from muscle. Bulbs from newborn mice contain about one-half of the adult levels of binding and no significant sex differences in carnosine binding were detected in bulbs from adult rats.l-[³H]carnosine binding was two-fold higher in the anterior compared to the posterior portion of the bulb, but there were no significant differences in binding of opiate, GABA, α-adrenergic, muscarinic cholinergic, benzodiazepine or glutamic acid receptor ligands.     

The distribution and function of DL-[H]2-Amino-4-Phosphonobutyrate binding sites in the rat striatum

The binding of the glutate-like radioligand,DL-[³H]2-amino-4-phosphhonobutyrate (DL-[³H]APB), to L-glutamate-sensitive sites in the rat striatum was investigated. A single, saturable population of binding sites, indistinguishable from that characterized previously on rat whole brain synaptic membranes, was identified. The effects of specific lesions of the striatum: (a) decortication; (b) striatal injection of kainic acid; and (c) 6-hydroxydopamine injections into the substantia nigra, were also examined. SpecificDL-[³H]APB binding in the striatum was elevated significantly following decortication. An increase in the number of binding sites was found to be responsible for this enhancement in binding. Lesions of the postsynaptic tartets of corticostriatal fibres reduced the number of DL-[³H]APB binding sites in the striatum without affecting binding site affinity. This finding suggests thatL-APB sensitive excitatory amino acid receptors are located predominantly on membranes derived from structures postsynaptic with regard to the glutamatergic innervation. The possible physiological role of these receptors was examined using an in vitro release technique. BothL-glutamate and L-APB were found to facilitate potassium evoked [³H]dopamine release from striatal slices. This finding supports the proposed existence of functional acidic amino acid receptors on dopaminergic terminals in the striatum. These receptors may play an important role in the control of motor function.     

Increased [H]glutamate receptor binding in aged rats

Na-independent [³H]glutamate binding to rat hippocampal membranes increases progressively as a function of age. The increased binding represents an increased number of binding sites without changes in their apparent affinity for glutamate. However, [³H]glutamate binding, measured with a saturating concentration of calcium does not change at various ages. This does not reflect a change in the apparent affinity of calcium ions to stimulate [³H]glutamate binding, but a decrease in their maximal stimulatory effect. These results are discussed in relationship to age-related chages in certain physiological and behavioral functions.     

Irreversibility and time course of calcium stimulated [H]glutamate binding to rat hippocampal membranes

Previous studies have indicated that calcium ions induce an irreversible increase of [³H]glutamate binding to rat hippocampal membranes. The present study was intended to provide an estimate of the time-course for this effect. The calcium chelator EGTA and the proteinase inhibitor leupeptin were introduced at various times during the incubation of hippocampal membranes with calcium and the binding of [³H]glutamate subsequently assayed. Both compounds totally blocked the calcium-induced increase in binding when added at the beginning of the incubation period but produced progressively less inhibition when added after longer delays. This method indicates that calcium produces its maximal effect between 5 and 10 min of incubation, a time-course compatible with the hypothesis that calcium stimulates a membrane-bound proteinase which results in the unmasking of [³H]glutamate binding sites.     

Increased [3H]glutamate receptor binding in aged rats

Na-independent [3H]glutamate binding to rat hippocampal membranes increases progressively as a function of age. The increased binding represents an increased number of binding sites without changes in their apparent affinity for glutamate. However, [3H]glutamate binding, measured with a saturating concentration of calcium does not change at various ages. This does not reflect a change in the apparent affinity of calcium ions to stimulate [3H]glutamate binding, but a decrease in their maximal stimulatory effect. These results are discussed in relationship to age-related changes in certain physiological and behavioral functions.     

Effect ofd- andl-α-aminoadipate on the efflux ofl-aspartate,l-glutamate and γ-aminobutyrate from superfused rat brain slices

d-α-Aminoadipate (d-AA) andl-α-aminoadipate (l-AA) were found to significantly reduce spontaneous efflux of [¹⁴C]l-aspartate from preloaded rat brain slices. Onlyd-AA significantly reduced spontaneous efflux of [¹⁴C]l-glutamate and [³H]γ-aminobutyric acid (GABA);l-AA reduced but not significantly the efflux of these 2 labeled amino acids.d-AA reduced K⁺-stimulated release of [¹⁴C]l-aspartate and [^14]C]l-glutamate significantly, andl-AA that of [³H]GABA significantly. Since bothd-AA andl-AA inhibit the uptake ofl-aspartate,l-glutamate and GABA, their effects on the efflux of these amino acids are more specific. These results also suggest that it is unlikely that the depressant effect ofd-AA, and the excitant effect ofl-AA on neurons when applied locally by iontophoresis are secondary to the accelerated or decelerated release of more specific transmitter amino acids from neighboring cells.     

GABA and benzodiazepine-induced modification of [C]l-glutamic acid release from rat spinal cord slices

The spontaneous and potassium-evoked release of [¹⁴C]-label from rat spinal cord slices preloaded with [¹⁴C]l-glutamic acid and its modification by GABA and related drugs, such as flurazepam, was studied as a possible indirect measure of presynaptic inhibition and of the ability of benzodiazepines to augment it. GABA (100 μM) reduced the spontaneous release of [¹⁴C]-label (glutamate) provided that GABA metabolism was blocked by amino-oxyacetic acid (AOAA), but failed to reduce the potassium-evoked release of glutamate, although muscimol (10 μM) had some effect. In contrast, flurazepam (1–100 μM) did not affect spontaneous release but produced some inhibition of the evoked release (through a system insensitive to 10 μM bicuculline). This inhibition became more marked in the presence of both GABA and AOAA, and was then overcome by bicuculline. It is concluded that either some benzodiazeophine receptors must be occupied for GABA to produce an effect on evoked release and/or, that the benzodiazepines can only augment GABA function once a certain amount has been released. Studies of the rapid distribution of [¹⁴C]-label from glutamate, to GABA, glutamine and other amino acids, using high voltage electrophoresis, showed the importance of blocking metabolic pathways in studies of this kind.