Contribution of Metabotropic Glutamate Receptor mGluR4 to L-2-[3H]Amino-4-Phosphonobutyrate Binding in Mouse Brain

Abstract : The binding of L-2-[³H]amino-4-phosphonobutyrate ([³H]L-AP4) was examined in brain sections of wild-type mice and mice lacking the mGluR4 subtype of metabotropic glutamate receptors (mGluRs). Very high relative densities of [³H]L-AP4 binding were observed in the molecular layer of the cerebellar cortex, the nucleus basalis, the outer layer of the superior colliculus, and the substantia nigra. In mGluR4 knock-out mice, very low levels of binding were observed in these regions. The moderate levels of binding observed with wild-type mice in the molecular layer of the hippocampal dentate gyrus and in the thalamus were absent in mGluR4 knock-out mice. In contrast, the moderate levels observed in most of the cerebral cortex, caudate putamen, and globus pallidus were not different in mGluR4 knock-out mice compared with wild-type. In these regions, mGluR8 is likely to be labeled by [³H]L-AP4 because mGluR8 is expressed in such brain regions and, like mGluR4, has high affinity for L-AP4. We conclude that mGluR4 contributes substantially to the high-affinity binding site for [³H]L-AP4 in several regions of mouse brain, including cerebellar cortex, nucleus basalis, thalamus, superior colliculus, substantia nigra, and hippocampal dentate gyrus.     

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)     

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.     

α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 +/- 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several other excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, L-Glu, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intrastriatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2-3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopaminergic pathway (using nigral injection of 6-hydroxy-dopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.     

Selective Loss of N-Methyl-d-Aspartate-Sensitive l-[3H]Glutamate Binding Sites in Rat Brain Following Portacaval Anastomosis

Excitatory amino acids have been implicated in the pathogenesis of hepatic encephalopathy. In the present study, kainate, quisqualate and N-methyl-D-aspartate (NMDA) subclasses of L-glutamate receptors were measured in adult rat brain by quantitative receptor autoradiography following surgical construction of an end-to-side portacaval anastomosis (PCA). PCA resulted in sustained hyperammonemia and decreased binding of L-glutamate to the NMDA receptor when compared to sham-operated controls. Decreases in binding ranged from 17 to 39% in several regions of cerebral cortex, hippocampus, striatum, and thalamus. Binding to quisqualate and kainate receptor subtypes was not altered. PCA leads to astrocytic changes in brain but does not result in any measurable loss of neuronal integrity. It is therefore proposed that decreased glutamate binding to the NMDA receptor following PCA results from increased extracellular glutamate caused by decreased reuptake into perineuronal astrocytes and a compensatory down-regulation of these receptors. Such changes could be of pathophysiological significance in hepatic encephalopathy.     

Characterization of Two [3H]Glutamate Binding Sites in Rat Hippocampal Membranes

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.     

Characterization of the Binding of [3H]NS 257, a Novel Competitive AMPA Receptor Antagonist, to Rat Brain Membranes and Brain Sections

Abstract: The binding of [³H]NS 257 {1,2,3,6,7,8-hexahydro-3-(hydroxyimino)- N,N -[³H]dimethyl-7-methyl-2-oxobenzo[2,1- b :3,4- c '] dipyrrole-5-sulfonamide} to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [³H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 m M . In the presence of thiocyanate [³H]NS 257 bound to a single population of binding sites with an affinity of 225 ± 8 n M and a binding site density of 0.61 ± 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [³H]NS 257 for both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and l -glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo( f )-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > l -glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [³H]NS 257 binding sites in rat brain was similar to that of [³H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.     

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract: [³H]Diazepam and [³H]flunitrazepam ([³H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, γ -aminobutyric acid (GABA) and NaCl both increased [³H]diazepam binding more than [³H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [³H]diazepam in the absence of NaCl and GABA, the B_max (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [³H]FNP, which dissociates more slowly, had the same B_max in both assays. [³H]Diazepam association had two components, and was faster than [³H]FNP association. [³H]Diazepam dissociation, which also had two components, was faster than that of [³H]FNP, and also had a greater fraction of rapidly dissociating species. [³H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [³H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.     

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.     

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.     

[3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

Abstract: [³H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na⁺ independent, was still largely detectable at low temperature (4°C), and underwent rapid dissociation. Scatchard analysis of [³H]aniracetam binding revealed a single population of sites with an apparent K_D value of ～70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [³H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [³H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [³H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [³H]aniracetam binding in unwashed membranes. High levels of [³H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, specifically bound [³H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.     

Increased Binding of [3H]Muscimol and [3H]Flunitrazepam in the Rat Brain Under Hypoxia

We examined the effects of in vivo hypoxia (10% O2/90% N2) on the gamma-aminobutyric acid (GABA)/benzodiazepine receptors and on glutamic acid decarboxylase (GAD) activity in the rat brain. Male Wistar rats were exposed to a mixture of 10% O2 and 90% N2 in a chamber for various periods (3, 6, 12, and 24 h). The control rats were exposed to room air. The brain regions examined were the cerebral cortex, striatum, hippocampus, and cerebellum. GABA and benzodiazepine receptors were assessed using [3H]muscimol and [3H]flunitrazepam, respectively. Compared with control values, GAD activity was decreased significantly following a 6-h exposure to hypoxia in all four regions studied. On the other hand, the numbers of both [3H]muscimol and [3H]flunitrazepam binding sites were increased significantly. The increase in receptor number tended to return to control values after 24 h. Treatment of the membrane preparations with 0.05% Triton X-100 eliminated the increase in the binding capacity. These results may represent an up-regulation of postsynaptically located GABA/benzodiazepine receptors corresponding to the impaired presynaptic activity under hypoxia.     

Comparison of Excitatory Amino Acid-Stimulated Phosphoinositide Hydrolysis and N-[3H]Acetylaspartylglutamate Binding in Rat Brain: Selective Inhibition of Phosphoinositide Hydrolysis by 2-Amino-3-Phosphonopropionate

Abstract: The activation of phosphoinositide hydrolysis by ibotenate (IBO) in brain slices and the binding of N -[³H]acetylaspartyl-L-glutamate (NAAG) to brain membranes are biochemical parameters previously shown to be selectively inhibited by 2-amino-4-phosphonobutyrate (AP4). We have examined whether the binding of [³H]NAAG and stimulation of phosphoinositide hydrolysis by IBO are indexing the same or different populations of AP4-sensitive excitatory amino acid sites in brain. L-AP4 and D,L-2-amino-3-phosphono-propionate (D,L-AP3) were found to be about equipotent inhibitors of IBO-stimulated phosphoinositide hydrolysis. L-AP4 and D,L-AP3 did not inhibit stimulation of phosphoinositide hydrolysis by the cholinoceptor agonist carbachol. The L-isomers of serine- O -phosphate and α-aminoadipate were selective inhibitors of IBO-stimulated phosphoinositide hydrolysis, but were less potent than L-AP4 or D,L-AP3. When these compounds were examined for their ability to inhibit [³H]NAAG binding to membranes of rat forebrain, the relative order of potency was L-α-aminoadipate = D-α-aminoadipate < L-AP4 < L-serine- O -phosphate < D-AP4 < D,L-AP3. Concentrations of NAAG up to 10⁻² M did not stimulate phosphoinositide hydrolysis. Thus, although both assays are sensitive to L-AP4 inhibition, they appear to represent disparate excitatory amino acid sites in brain. Furthermore, D,L-AP3 appears to be a more selective inhibitor of excitatory amino acid-stimulated phosphoinositide hydrolysis than L-AP4, and might be a more useful pharmacological tool to define the function of these receptor sites in brain.     

Optimal Conditions for [3H]Apomorphine Binding and Anomalous Equilibrium Binding of [3H]Apomorphine and [3H]Spiperone to Rat Striatal Membranes: Involvement of Surface Phenomena Versus Multiple Binding Sites

I. Binding of [³H]apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25°C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [³H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [³H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [³H]spiperone and [³H] apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent K_D, values of spiperone decreased with decreasing tissue concentrations. K_D, values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the ³H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

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.     

Characterization of [3H]Guanine Nucleotide Binding Sites in Brain Membranes

[3H]GTP [guanosine triphosphate] and [3H]GMP-PNP [guanosine 5'-(beta, 8-imino)triphosphate, a nonmetabolized analog of GTP] have been utilized as ligands to characterize binding sites of guanine nucleotides to rat brain membranes. Binding of both [3H]GTP and [3H]GMP-PNP is saturable, with respective KD values of 0.76 and 0.42 microM. The number of binding sites for GMP-PNP (4 nmol/g) is three times greater than for GTP (1.5 nmol/g). This discrepancy is caused by rapid degradation of GTP to guanosine by brain membranes, which can be partially prevented by addition of 100 microM-ATP. The binding of [3H]guanine nucleotides is selective, with approximately equipotent inhibition by GTP, GDP, and GMP-PNP (at 0.2--1.0 microM), but no inhibition by other nucleotides at 100 microM concentrations. The bindings sites for guanine nucleotides in brain membranes appear not to be associated with microtubules, since treatments that reduce [3H]colchicine binding by 65% have no effect on [3H]GTP binding. [3H]Guanine nucleotide binding is widely distributed in various organs, with highest levels in liver and brain and lowest levels in skeletal muscle. The characteristics of these binding sites in brain show specificity properties of sites that regulate neurotransmitter receptors and adenylate cyclase.     

Influence of Sodium and Sulphydryl Groups on [3H]Sulpiride Binding Sites in Rat Striatal Membranes

Abstract: The effect of thiol reagents on the specific binding of the atypical neuroleptic, sulpiride, to rat striatal membranes was examined. Pretreatment of membranes with N -ethylmaleimide (NEM), but not with iodoacetamide or dithiothreitol (DTT), diminishes [³H]sulpiride binding. The effect is dependent on time, temperature, and the concentration of NEM. The reaction proceeds with pseudo-first-order rate kinetics, indicating the involvement of a single essential SH group in the binding of [³H]sulpiride to striatal membranes sites. Both sodium and sulpiride, but not nucleotides, protect the [³H]sulpiride sites from NEM inactivation in a dose-dependent fashion. As sodium is essential for [³H]sulpiride binding it is possible that in the presence of sodium and sulpiride a conformational change takes place that makes the essential SH group less accessible t o alkylation.     

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.     

Characterization of Two [3H]Ketanserin Recognition Sites in Rat Striatum

Two [3H]ketanserin recognition sites are present in the rat striatum. The high-affinity site (KD, 0.39 nM) is similar to the 5-hydroxytryptamine2 (5-HT2) site previously characterized by various investigators. The low-affinity site (KD, 21.8 nM) has a unique pharmacologic specificity and is preferentially localized to rat striatum and septum. Conventional 5-HT2 antagonists as well as 5-HT and 5-HT uptake inhibitors are ineffective at inhibiting [3H]-ketanserin binding to this low-affinity site. Also, chronic treatment with p-chlorophenylalanine, which depletes brain 5-HT, upregulates only the high-affinity site. Thus, in the striatum and septum, [3H]ketanserin labels a unique recognition site. This site has recently been shown to be associated with dopaminergic nerve endings and may regulate biogenic amine release.