Differentiation of the Ca2+-stimulated binding from the Cl- -dependent binding of [3H]glutamate in synaptic membranes from rat brain

The effect of Ca2+ as well as Cl- ions on [3H]glutamate (Glu) binding was re-examined using rat brain synaptic membranes frozen at -80 degrees C in 0.32 M sucrose. The inclusion of 20 mM ammonium chloride or 20 mM ammonium chloride plus 2.5 mM calcium acetate disclosed the Cl- -dependent binding or Ca2+-stimulated binding even at 2 min after the initiation of incubation at 30 degrees C and each binding reached a plateau within 30 min. In contrast, the binding reached its maximal value within 10 min followed by a progressive decline up to 60 min in the presence of 100 mM sodium acetate. Scatchard analysis revealed that Cl- as well as Cl-/Ca2+ ions invariably caused a significant increment of the number of binding sites without altering their affinity, whereas Na+ ions induced a prominent increment of the density of binding sites with a concomitant lowering of their affinity. DL-2-Amino-4-phosphonobutyric acid selectively abolished the Cl- -dependent and Ca2+-stimulated bindings without significantly affecting the basal or Na+-dependent binding. Quisqualic acid induced a profound inhibition of both Cl- -dependent and Ca2+-stimulated bindings, to a significantly greater extent than that of the basal and Na+-dependent bindings. D-Aspartic acid exhibited a potent inhibition of the Na+-dependent binding with a significantly less potent displacement of the basal, Cl- -dependent and Ca2+-stimulated bindings. An inhibitor of anion transport, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), not only eliminated the Cl- -dependent binding, but also completely abolished the Ca2+-stimulated binding. Scatchard analysis revealed that DIDS (0.1 mM) prevented the Cl- - and Cl-/Ca2+-induced increment of the density of binding sites with no significant change of their affinity. Pretreatment of the membranes with hydrophilic SH-reactive agents such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) invariably resulted in a more sensitive inhibition of the Ca2+-stimulated binding than that of the Cl- -dependent binding, while hydrophobic reagent p-chloromercuribenzoic acid produced a similarly potent elimination of the Cl- -dependent and Ca2+-stimulated bindings. Calcium-stimulated binding was also found to be sensitively diminished by dithiothreitol and dithioerythritol as compared with the Cl- -dependent binding. In vitro addition of L-ascorbic acid (10(-6)-10(-3) M) attenuated the Ca2+-stimulated binding to a significantly greater extent than the inhibition of the Cl- -dependent binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of [3H]phenobarbital binding to rat brain membranes

The binding of [3H]phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. (Kd = 700 microM) and very high density (Bmax = 2.7 nmol/mg protein). It was unaffected by temperature changes from 0 degrees C to 95 degrees C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of [3H]phenobarbital is a rather non-specific interaction with the plasma membrane.     

Age-dependent increase in [3H]verapamil binding to rat cortical membranes

[3H]Nitrendipine bound to cerebral cortex membranes is displaced more efficiently by verapamil in old rats (24 months old) compared to young ones (3 months old). In addition, [3H]verapamil binding was studied in detail in 3-, 12- and 24-month-old rats. Aging increases the Bmax of [3H]verapamil, leaving the affinity unchanged. These observations further indicate that aging may affect calcium channels leading to a derangement of calcium movements which in turn alter neuronal activity.     

Glycine potentiates N-methyl-D-aspartate-induced [3H]TCP binding to rat cortical membranes

In extensively washed preparations of rat cortical membranes, N-methyl-D-aspartate (NMDA) increases the specific binding of [3H]TCP by over 4-fold in a concentration dependent manner (EC50 = 3.1 microM). Glycine (1 microM) potentiates the maximal effect of NMDA by a factor of 1.7. The effect of glycine is concentration dependent (EC50 = 380 nM) and strychnine insensitive. These data are discussed with reference to the recently reported effects of glycine on the NMDA operated cation channel and the relationship between the PCP and NMDA receptors.     

3H]glutamate binding sites in the rat pituitary

A significant activity of [3H]glutamate binding was detected in homogenate particulate preparations obtained from the rat pituitary, in addition to central structures including the cerebral cortex. In contrast to the cerebral binding, the pituitary binding was significantly inhibited by Na+ ions. It was also found that neurohypophysis possessed more than a two-fold higher binding activity than that found in adenohypophysis. These results suggest a possible significance of glutamate in rodent pituitary.     

Possible presence of [3H]glutathione (GSH) binding sites in synaptic membranes from rat brain

Reduced as well as oxidized forms of glutathione exhibited a significant displacement of the specific binding of [3H]L-glutamic acid (Glu), a potential candidate for the central excitatory neurotransmitter, to the rat brain synaptic membranes. In order to elucidate these findings, an attempt was made to determine whether or not the synaptic membranes contained the binding sites for this peptide using [3H]glutathione (GSH) as a ligand. The specific binding activity was detected in the synaptic membranous preparations and found to be dependent on the incubation temperature and incubation time. The binding reached a plateau within 60 min of incubation at 2 degrees C and 30 degrees C. [3H]GSH binding increased linearly with increasing concentrations of membranous proteins employed. Scatchard analysis revealed that the binding sites consisted of two separate independent components rather than being comprised of a single constituent. A significant and concentration-dependent displacement of the binding was induced not only by the addition of GSH, but also by the inclusion of some GSH derivatives without SH-moiety, such as the oxidized form of glutathione, S-methyl-glutathione and S-hexyl-glutathione. The binding was also significantly inhibited by various alpha- and gamma-peptides containing L-Glu, but not by those containing D-Glu. Amongst 4 different agonists and antagonists used for the subclassification of the central Glu receptors, an agonist, quisqualic acid, and an antagonist, 2-amino-4-phosphonobutyric acid, exhibited a significant inhibition of the binding at the highest concentration employed. These results suggest that the rat brain synaptic membranes may contain structure-selective, temperature-dependent, high affinity and saturable binding sites for glutathione.     

Inhibition of [3H] glutamate release from rat hippocampal slices by L-phenylisopropyladenosine

Hippocampal slices were labelled with [3H]glutamate. Evoked release, which was calcium-dependent and inhibited by tetrodotoxin, could be reduced concentration-dependently by L-PIA. The effect of L-PIA was blocked by 10 microM 8-phenyltheophylline. The results indicate that there are adenosine receptors modulating the efflux of glutamate in field stimulated hippocampal slices.     

Evidence for interactions between [3H]glutamate and [3H]kainic acid binding sites in rat striatal membranes. Possible relevance for kainic acid neurotoxicity

The projection of the substantia nigra-ventral tegmental area (SN-VTA) to the ipsilateral and contralateral caudate-putamen was studied with double-labeling fluorescence techniques in the albino rat. By combining glyoxylic acid histofluorescence with retrograde fluorescence tracers, we were able to determine that 93% of the minor (1–2%) contralateral mesostriatal pathwayis catecholaminergic. In addition, combined immunofluorescence and retrograde fluorescence tracer studies showed that 50% of the crossed mesostriatal pathway also arises from cholecystokinin-containing neurons. These results provide evidence for crossed catecholamine- and peptide-containing pathways in the mesostriatal system which is part of the ‘prefrontal system’ that links the prefrontal cortex through the caudate-putamen, with the thalamus and tectum of each hemisphere.     

Specific binding of [3H]inositol trisphosphate to rat cerebral cortical microsomal membranes

Specific binding sites for inositol-(1,4,5)-trisphosphate (IP3) were demonstrated in rat cerebral cortical microsomal membranes using [3H]IP3 as the ligand. The binding was displaceable (IC50 for IP3 = 28 nM), high affinity (Kd = 20 nM) and saturable (Bmax = 7.75 pmol/mg protein). Kinetic studies showed an extremely rapid time-course of [3H]IP3 binding with half-maximal binding achieved in less than 1 min and equilibrium binding was attained by around 5 min. These results are in accord with the proposed function of IP3 in mobilizing intracellular calcium from endoplasmic reticulum.     

Gangliosides modulate glutamate receptor binding in rat brain synaptic plasma membranes

The influence of gangliosides on the binding of L-[3H]glutamate (Glu) to its receptor(s) on isolated rat brain synaptic plasma membranes was investigated in two different buffer systems using an in vitro filter binding assay. It was found that the tested pure monosialogangliosides GM1 and GM2 as well as the polysialogangliosides GT1b and GD1a enhanced binding by up to about 100%, but only in the presence of 5 mM calcium. The binding site involved was sensitive to quisqualate but insensitive to N-methyl-D-aspartate and kainate. GM1 did exert its stimulating effect by increasing the number of binding sites on the membranes whereas the receptor's affinity for L-Glu was unchanged. Regional differences in rat brain were found: while the hippocampus and cortex exhibited significant Ca/GM1-induced stimulation of Glu binding, the cerebellum was unaffected. Our results suggest modulation of at least one subtype of the Glu receptor by gangliosides.     

Autoradiographic visualisation of [3H]DTG binding to sigma receptors, [3H]TCP binding sites, and L-[3H]glutamate binding to NMDA receptors in human cerebellum.

The autoradiographic distributions of [3H]1,3-di-ortho-tolyguanidine ([3H]DTG), [3H]1-[1-(2-thienyl) cyclohexyl] piperidine ([3H]TCP) and L-[3H]glutamate were studied in the human cerebellum. [3H]DTG is a selective label for the sigma receptor, while L-[3H]glutamate binding was carried out under conditions selective for the N-methyl-D-aspartate (NMDA) receptor. [3H]TCP binding sites and sigma receptors showed marked enrichment in the Purkinje cell layer, while L-[3H]glutamate-labelled NMDA receptors showed virtually no binding in the Purkinje cell layer. The results confirm the existence of [3H]TCP binding sites which are not linked to NMDA receptors in the human cerebellum, having a distribution which is more similar to that of the haloperidol-sensitive sigma receptor.     

Quantitative autoradiographic characterization of l-[3H] glutamate binding sites in rat vestibular nuclei

Summary Quantitative autoradiography has been used to characterize l-[³H] glutamate binding sites and to describe their distribution in frozen sections of rat vestibular nuclei. Scatchard plots and Hill coefficients of glutamate binding suggest that glutamate interacts with a single population of sites having a K_D of about 126 nM and a capacity of 2.5 pmol/mg of protein. Although the level of glutamate binding was not very high compared to the highest levels described for some other brain regions, it was nonetheless substantial. The sites were distributed unevenly in the four vestibular nuclei and their distribution correlated well with the projection areas of the vestibular nerve, which has been described as a glutamate-mediated pathway. The highest numbers of glutamate binding sites were observed in the medial vestibular nuclei. This technique provides a very sensitive assay for characterizing the pharmacological subtypes of glutamate binding in the vestibular nuclei and for analyzing changes in these sites during development or after deafferentation of the vestibular nuclei.     

Brain ischemia increased high affinity binding of [3H]muscimol into synaptic plasma membrane receptor

The authors investigated the high affinity binding of [3H] muscimol to the receptor of synaptic plasma membranes (SPM) isolated from a normoxic and ischemic brain. Brain ischemia enhanced the [3H] muscimol binding to the receptor, located in native (Triton X-100 untreated) membranes. Scatchard's analysis showed that the total number of binding sites (BMAX) and the KD value increased by about 60%. The higher KD value persisted during 20 min of the reperfusion period. Concomitantly, ischemia stimulated the activity of phospholipase C and phospholipase A2, acting against phosphatidylinositol (PI). The degradation of PI and a transient accumulation of docosahexaenoic and arachidonic acids may be important factors involved in the modification of high affinity agonist binding to the GABAA receptor of SPM isolated from the brain submitted to ischemia.     

Distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in rat hippocampal long-term slice cultures isolated from external afferents

The distribution of ionotropic glutamate receptors in transverse hippocampal sections and along the septotemporal hippocampal axis can be correlated to hippocampal connectivity, in particular to area- and layer-specific termination zones of afferents. However, in isolated organotypic hippocampal slice cultures developing without extrinsic afferent input no systematic studies exist about the distribution of glutamate receptors. In the present study we used receptor autoradiography to examine [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites in hippocampal slice cultures prepared from 6-day-old rats. After 24 days in vitro layer-specific concentrations of [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites were compared to age-matched hippocampi in situ (P30 rats). An obvious difference between hippocampal slice cultures and hippocampi in situ was a changed distribution of binding sites among the hippocampal areas showing a relative increase of [³H]MK-801 and [³H]AMPA binding sites in CA3 as compared to CA1 and to the dentate gyrus in the cultures. In CA1, however, the relative layer-specific distributions of [³H]MK-801 and [³H]AMPA binding sites were identical in hippocampal slice cultures and in hippocampi in situ. Interestingly, layer-specific binding of [³H]Kainate in the cultures exceeded that in the hippocampi in situ 3–5 times. Moreover, in the cultures the binding of the three ligands varied systematically showing gradients along the ”superficiomembranal’’ axis. Cultures taken from different positions along the hippocampal axis differed with respect to concentrations of [³H]MK-801 and [³H]Kainate binding sites, but not of [³H]AMPA binding sites. The results suggest a massive sprouting and reorganisation of intrinsic projections in long-term hippocampal slice cultures.     

Loss of hippocampal [3H]TCP binding in Alzheimer's disease

We have previously demonstrated a marked loss in N-methyl-D-aspartate (NMDA) receptors in the hippocampus and cerebral cortex of patients dying with dementia of the Alzheimer type (DAT). In addition, we have found that the dissociative anesthetic N-(1-[2-thienyl]cyclohexyl)3,4-piperidine ([3H]TCP) binds to a site whose regional distribution is highly correlated with that of NMDA receptor sites. We studied the binding of [3H]TCP to sections of hippocampi from 8 controls, 12 patients with DAT and 7 patients with other dementias. [3H]TCP binding was significantly reduced in strata pyramidalia of CA1/CA2, CA3 and subiculum of DAT hippocampal formation compared to that of control. Labelled dissociative anesthetics could potentially be used with positron emission tomography in the diagnosis of DAT.     

Ligand-binding characteristics of [3H]QNB, [3H]prazosin, [3H]rauwolscine, [3H]TCP and [3H]nitrendipine to cerebral cortical and hippocampal membranes of senescence accelerated mouse

The senescence accelerated mouse (SAM) is known as a murine model of aging and memory dysfunction. In the cerebral cortical membranes of male 9-month-old SAM mice, the Bmax values of [3H]rauwolscine and [3H]nitrendipine binding, and the values of both Kd and Bmax of [3H]TCP binding in the accelerated aging strain SAM-P/8, were significantly increased compared with the values in the control strain SAM-R/1. In hippocampal membranes, however, the Bmax values of [3H]quinuclidinyl benzilate and [3H]nitrendipine binding were significantly decreased in SAM-P/8 compared with those in SAM-R/1. These results suggest that muscarinic acetylcholine receptors, alpha 2-adrenoceptors, N-methyl-D-aspartate receptor channels and L-type Ca2+ channels are changed in cerebral cortex and hippocampus in SAM-P/8 at 9 months.     

Ammonia-induced alterations in glutamate and muscimol binding to cerebellar synaptic membranes

Binding of glutamate and muscimol (an agonist for GABA_A receptors) to their respective receptors has been studied in the cerebellum of normal and hyperammonemic rats. There was a decrease in both high- and low-affinity binding of glutamate in the cerebellum during hyperammonemia. Kinetic studies revealed that the decrease is due to a reduction in the number of binding sites, but not due to changes in the binding affinities. Further studies also revealed that the decrease was only in the (NMDA)-specific binding sites without any alterations in the binding to non-NMDA sites represented by kianic acid (KA)- and quisqualic acid (QQ)-sensitive receptor sites. These effects were also mimicked when the membrane preparations from the cerebellum of normal animals were incubated with ammonium acetate. Enhancement of muscimol binding was observed in animals injected with ammonium acetate. It is concluded that hyperammonemic states, even in the presence of a functional liver, are capable of altering amino acid neurotransmission and this might play an important role in cerebral dysfunction under these conditions.     

Stereospecific interaction of bicuculline with specific [3H]strychnine binding to rat spinal cord synaptosomal membranes

The gamma-aminobutyric acid (GABA) antagonist (+/-)-bicuculline inhibits specific [3H]strychnine binding to postsynaptic glycine receptor sites in rat spinal cord synaptosomal membranes with an inhibition constant of about 5 microM, which is fairly similar to its inhibition constant reported for the GABA receptor. This effect is highly stereospecific, since the affinity of (-)-bicuculline for the specific [3H]strychnine binding sites is more than ten times less than that of the pharmacologically active (+)-bicuculline. Besides an unspecific effect at the glycine receptor, the results could suggest that the glycine and the GABA receptors are located close together in spinal cord membranes, so that the antagonist states of both receptors may be able to interfere with each other in some mechanistic way.     

Septotemporal distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in the rat hippocampus

The distribution of glutamate receptors in transverse hippocampal sections has been well investigated. However, in spite of the known septotemporal gradients of hippocampal connectivity no systematic studies exist about the distribution of glutamate receptors along the septotemporal (longitudinal) hippocampal axis. Therefore, in the present study this issue was investigated using receptor autoradiography for the [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites. Hippocampi from 30-day-old rats were sectioned perpendicularly to their longitudinal axis, yielding a total of 25–30 equidistantly spaced autoradiographs for each hippocampus. For each section layer-specific concentrations of binding sites were calculated by the aid of a computerized image analysing system. The dependency of concentrations of binding sites on the septotemporal position was evaluated by regression analysis. Gradients of binding were confined to distinct hippocampal layers. Significant septotemporal gradients of [³H]MK-801 binding were observed in selected layers of CA1 and the dentate gyrus, a septal to temporal decrease of binding in the oriens and radiatum layers of CA1 being most prominent. For [³H]AMPA, significant septotemporal gradients of binding were restricted to layers of CA3, CA4 and the dentate gyrus, with values generally increasing from septal to temporal levels. The observed septotemporal gradients possibly reflect functional segregations along the longitudinal hippocampal axis and could be important for the comparability of ligand binding studies using transverse hippocampal sections or hippocampal slice cultures.