[3H]Propyl β-Carboline-3-Carboxylate Binds Specifically to Brain Benzodiazepine Receptors

Ethyl beta-carboline-3-carboxylate has recently been isolated from human urine and it was proposed that derivatives of this compound might be related to an endogenous ligand for benzodiazepine receptors. In the present study we investigated high-affinity binding of [3H]propyl beta-carboline-3-carboxylate ([3H]PrCC) to rat brain membranes. [3H]PrCC binds specifically and with high affinity (half-maximal binding at ca. 1nM) to rat brain membranes. The regional and subcellular distributions of specific [3H]PrCC binding are similar, but not identical, to the distributions of [3H]flunitrazepam or [3H]-diazepam binding. The total numbers of binding sites labelled by [3H]PrCC and [3H]flunitrazepam in rat cerebellum are closely similar, and both ligands bind to cerebellar membranes in a mutually exclusive way. The pharmacological selectivity of [3H]PrCC and [3H]diazepam binding is almost identical. Binding of [3H]PrCC like binding of [3H]diazepam, can be increased in vitro by muscimol, GABA and SQ 20.009. Although subtle differences in binding characteristics were observed, these results indicate that [3H]PrCC and benzodiazepines bind to a common recognition site on benzodiazepine receptors.     

Increased γ-Aminobutyric Acid Receptor Function in the Cerebral Cortex of Myoclonic Calves with an Hereditary Deficit in Glycine/Strychnine Receptors

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Binding of the benzodiazepine inverse agonist [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate [( 3H]DMCM) and the agonist [3H]flunitrazepam [( 3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three- to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37 degrees C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37 degrees C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through gamma-aminobutyric acid (GABA)-gated chloride channels. Two stress paradigms (10 min of immobilization or ambient-temperature swim stress), previously shown to increase significantly the magnitude of halide-stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.     

Decreased High-Affinity Binding of [3H]Muscimol to Cerebral Synaptic Membranes of Scrapie-Infected Mice

Scrapie is a transmissible disease that results in progressive degeneration of the central nervous system and death. Although scrapie has been studied histopathologically, relatively little is known concerning neurotransmitter alterations. Specific [3H]muscimol binding to whole brain crude synaptic membranes (CSM) from mice clinically affected with scrapie was significantly (p less than 0.01) reduced, to approximately 73% of that of the controls. Of the brain regions examined, binding to only cerebral CSM was significantly (p less than 0.0001) decreased. Scatchard analyses of saturation curves revealed that the high-affinity (KD = 8 +/- 3 nM) site for muscimol was abolished in cerebral CSM from scrapie-infected mice, while the low-affinity site was unaffected. Binding of [3H]flunitrazepam to cerebral CSM was unaffected by scrapie and was stimulated by GABA to the same extent in both scrapie and control mice. These results suggest that scrapie agent 139A in C57BL/6J mice manifests a portion of its CNS pathology via a high-affinity GABA binding site that is unassociated with the benzodiazepine receptor.     

Characteristics of Flunitrazepam Binding to Intact Primary Cultured Spinal Cord Neurons and Its Modulation by GABAergic Drugs

The interaction of [3H]flunitrazepam and its modulation by various drugs was studied in intact primary cultured spinal cord neurons. In the intact cells, the [3H]-flunitrazepam binding was rapid and saturable. The benzodiazepine binding sites exhibited high affinity and saturability, with an apparent KD of 6.1 +/- 1.6 nM and Bmax of 822 +/- 194 fmol/mg protein. The association and dissociation of [3H]flunitrazepam binding exhibited monoexponential kinetics. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by benzodiazepines like flunitrazepam, clonazepam, diazepam, Ro 15-1788, and beta-carbolines like methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3'-carboxylate. Specific [3H]flunitrazepam binding to intact cells was enhanced in a concentration-dependent manner by gamma-aminobutyric acid (GABA) agonists and drugs which facilitate GABAergic transmission like etazolate, (+)-etomidate, and pentobarbital. The enhancing effect of GABA agonists was antagonized by bicuculline and picrotoxinin. These results suggest that the intact cultured spinal cord neurons exhibit the properties of benzodiazepine GABA receptor-ionophore complex. Since these cells can also be studied in parallel for characterizing GABA-induced 36Cl-influx, they provide an ideal in vitro assay preparation to study GABA synaptic pharmacology.     

Ion and Temperature Effects on the Binding of γ-Aminobutyrate to Its Receptors and the High-Affinity Transport System

A set of procedures was developed to study the binding of gamma-[3H]aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na(+)-dependent transport carrier, at 25 and 37 degrees C in the presence of physiological concentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze-thawing or treatment with Triton X-100. Isoguvacine, (-)-baclofen, and (-)-nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high-(KD less than 30 nM), middle- (KD = 100-500 nM), and low-affinity (KD greater than 5 microM) binding sites. However, the binding data in the middle-affinity region (100-1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl- at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl-. Apparent binding to the transport carrier required both Na+ and Cl-. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high-affinity site of the GABAA receptor and the transport binding site.     

Solubilisation of the Benzodiazepine Receptor from Rat Cerebellum by the Detergent n-Octylpentaoxyethylene

The detergent n-octylpentaoxyethylene is one in the series of tenside detergents developed for membrane solubilisation. We have used this detergent to solubilise benzodiazepine receptors from rat cerebellum. The soluble receptor has an affinity (KD) for [3H]flunitrazepam of 1.8 nM +/- 0.2, which is not significantly different from that observed in synaptic membranes. Under optimal conditions (0.6% wt/vol), the number of soluble flunitrazepam binding sites (Bmax) of 0.35 pmol/mg protein suggests an apparent solubilisation of 40% of sites from the membrane. However, the absence of the characteristic facilitation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), cartazolate, and pentobarbital in this soluble receptor preparation suggests that such a preparation is unlikely to be a useful preparation for further studies on the molecular mechanisms underlying GABAA receptor function.     

Solubilisation of the γ-Aminobutyric Acid/Benzodiazepine Receptor from Rat Cerebellum: Optimal Preservation of the Modulatory Responses by Natural Brain Lipids

We have solubilised the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat cerebellum using 3-[(3-cholamidopropyl)dimethylammonio] 1-propane sulphonate (CHAPS) in the presence of a natural brain lipid extract and cholesteryl hemisuccinate. The soluble material shows a homogeneous [3H]flunitrazepam ([3H]FNZ) binding population with an equilibrium dissociation constant (KD) of 4.4 +/- 0.2 nM compared to a KD of 2.3 +/- 0.2 nM in cerebellar synaptosomal membranes. The receptor complex in solution retains the characteristic facilitation of [3H]flunitrazepam binding induced by GABA, the pyrazolopyridine cartazolate, and the depressant barbiturate pentobarbital to the same extent as that observed in synaptosomal membranes. Furthermore, these responses are retained both quantitatively and qualitatively when this preparation is stored for 48 h at 4 degrees C. This is contrary to the results obtained with a CHAPS-soluble preparation including asolectin in which these responses are anomalous and extremely labile on storage.     

Possible Involvement of Pertussis Toxin-Sensitive G Proteins and D2 Dopamine Receptors in the A1 Adenosine Receptor-Adenylate Cyclase System in Rat Cerebral Cortex

To identify the involvement of dopamine receptors in the transmembrane signaling of the adenosine receptor-G protein-adenylate cyclase system in the CNS, we examined the effects of pertussis toxin (islet-activating protein, IAP) and apomorphine on A1 adenosine agonist (-)N6-R-[3H]phenylisopropyladenosine ([3H]PIA) and antagonist [3H]xanthine amine congener ([3H]XAC) binding activity and adenylate cyclase activity in cerebral cortex membranes of the rat brain. Specific binding to a single class of sites for [3H]XAC with a dissociation constant (KD) of 6.0 +/- 1.3 nM was observed. The number of maximal binding sites (Bmax) was 1.21 +/- 0.13 pmol/mg protein. Studies of the inhibition of [3H]XAC binding by PIA revealed the presence of two classes of PIA binding states, a high-affinity state (KD = 2.30 +/- 1.16 nM) and a low-affinity state (KD = 1.220 +/- 230 nM). Guanosine 5'-(3-O-thio)triphosphate or IAP treatment reduced the number of the high-affinity state binding sites without altering the KD for PIA. Apomorphine (100 microM) increased the KD value 10-fold and decreased Bmax by approximately 20% for [3H]PIA. The effect of apomorphine on the KD value increase was irreversible and due to a conversion from high-affinity to low-affinity states for PIA. The effect was dose dependent and was mediated via D2 dopamine receptors, since the D2 antagonist sulpiride blocked the phenomenon. The inhibitory effect of PIA on adenylate cyclase activity was abolished by apomorphine treatment. There was no effect of apomorphine on displacement of [3H]quinuclidinyl benzilate (muscarinic ligand) binding by carbachol. These data suggest that A1 adenosine receptor binding and function are selectively modified by D2 dopaminergic agents.     

Avermectin Bla Modulation of γ-Aminobutyric Acid/Benzodiazepine Receptor Binding in Mammalian Brain

The anthelminthic natural product avermectin B1a (AVM) modulates the binding of gamma-aminobutyric acid (GABA) and benzodiazepine (BZ) receptor ligands to membrane homogenates of mammalian brain. The potent (EC50 = 40 nM) enhancement by AVM of [3H]diazepam binding to rat or bovine brain membranes resembled that of barbiturates and pyrazolopyridines in being inhibited (partially) by the convulsants picrotoxin, bicuculline, and strychnine, and by the anticonvulsants phenobarbital and chlormethiazole. The maximal effect of AVM was not increased by pentobarbital or etazolate. However, AVM affected BZ receptor subpopulations or conformational states in a manner different from pentobarbital. Further, unlike pentobarbital and etazolate, AVM did not inhibit allosterically the binding of the BZ receptor inverse agonist [3H]beta-carboline-3-carboxylate methyl ester, nor did it inhibit, but rather enhanced, the binding of the cage convulsant [35S]t-butyl bicyclophosphorothionate to picrotoxin receptor sites. AVM at submicromolar concentrations had the opposite effect of pentobarbital and etazolate on GABA receptor binding, decreasing by half the high-affinity binding of [3H]GABA and related agonist ligands, and increasing by over twofold the binding of the antagonist [3H]bicuculline methochloride, an effect that was potentiated by picrotoxin. AVM also reversed the enhancement of GABA agonists and inhibition of GABA antagonist binding by barbiturates and pyrazolopyridines. These overall effects of AVM are unique and require the presence of another separate drug receptor site on the GABA/BZ receptor complex.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Enhancement of gamma-aminobutyrate-receptor binding by the anaesthetic propanidid.

The binding of [3H]muscimol, a gamma-aminobutyrate (GABA) receptor agonist, to a membrane preparation from pig cerebral cortex was enhanced by the anaesthetic propanidid in a concentration-dependent manner. At 0 degrees C, binding was stimulated to 220% of control values, with 50% stimulation at 60 microM-propanidid. At 37 degrees C, propanidid caused a more powerful stimulation of [3H]muscimol binding (340% of control values). Propanidid (1 mM) exerted little effect on the affinity of muscimol binding (KD approx. 10 nM), but increased the apparent number of high-affinity binding sites in the membrane by 2-fold. Enhancement of [3H]muscimol binding was observed only in the presence of Cl- ions, half-maximal activation being achieved at approx. 40 mM-Cl-. Picrotoxinin inhibited the stimulation of [3H]muscimol binding by propanidid with an IC50 (concentration causing 50% inhibition) value of approx. 25 microM. The enhancement of [3H]muscimol binding by propanidid was not additive with the enhancement produced by secobarbital. Phenobarbital inhibited the effect of propanidid and secobarbital. The GABA receptor was solubilized with Triton X-100 or with Chaps [3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate]. Propanidid and secobarbital did not stimulate the binding of [3H]muscimol after solubilization with Triton X-100. However, the receptor could be solubilized by 5 mM-Chaps with retention of the stimulatory effects of propanidid and secobarbital. Unlike barbiturates, propanidid did not stimulate the binding of [3H]flunitrazepam to membranes. It is suggested that the ability to modulate the [3H]muscimol site of the GABA-receptor complex may be a common and perhaps functional characteristic of general anaesthetics.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological and Biochemical Properties of the γ-Aminobutyric Acid–Benzodiazepine Receptor Protein from Codfish Brain

The gamma-aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of codfish GABA receptor photoaffinity-labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7-1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver-stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (greater than 1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA-BZ receptor is generally similar to that of mammalian brain, including GABA-BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo-oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero-oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ-GABA coupling.     

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.     

Characterization of a Novel Serotonin Receptor Subtype (5-HTls) in Rat CNS: Interaction with a GTP Binding Protein

Three pharmacologically distinct high-affinity [3H]serotonin ([3H]5-HT) binding sites were identified in spinal cord synaptosomes. [3H]5-HT competition studies using selective 5-HT1A receptor ligands indicated that approximately 25% of high-affinity synaptosomal [3H]5-HT binding was inhibited by 5-HT1A-selective compounds, an estimate consistent with [3H](+-)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) saturation experiments in which 5-HT1A receptors were directly labeled. [3H]5-HT competition studies using high-affinity 5-HT1B compounds performed in the presence of 100 nM 8-OH-DPAT (to block 5-HT1A receptors) indicated that approximately 26% of all specific, high-affinity [3H]5-HT binding to spinal cord synaptosomes was to 5-HT1B receptors. [3H]5-HT competition studies performed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (to block 5-HT1A and 5-HT1B receptors, respectively) indicated that the remaining 49% of [3H]5-HT binding did not possess the pharmacologic profile previous reported for 5-HT1C, 5-HT1D, 5-HT1E, 5-HT2, or 5-HT3 receptors. This residual 49% of [3H]5-HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8-OH-DPAT and 10 nM RU 24969 (subsequently referred to as "5-HT1S") displayed high affinity and saturability (KD = 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 microM GTP or the nonhydrolyzable form of GTP, 5'-guanylylimidodiphosphate, inhibited [3H]5-HT binding to 5-HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the Binding of [3H]SR 95531, a GABAA Antagonist, to Rat Brain Membranes

A synthetic derivative of gamma-aminobutyric acid (GABA), SR 95531 [2-(3'-carboxy-2'-propyl)-3-amino-6-p-methoxyphenylpyridazinium bromide], has recently been reported, on the basis of biochemical and in vivo microiontophoretic studies, to be a potent, selective, competitive, and reversible GABAA antagonist. In the present study, the binding of [3H]SR 95531 to washed, frozen, and thawed rat brain membranes was characterized. Specific binding was linear with tissue concentrations, had a pH optimum at neutrality, and was maximal at 4 degrees C after 30 min of incubation. Pretreatment of the membranes with Triton X-100 resulted in a 50% decrease of specific binding. Addition of iodide, thiocyanate, or nitrate to the incubation mixture decreased the affinity of [3H]SR 95531 for its binding site; Na+ had no effect. Subcellular fractionation showed that 74% of the P2 binding was in synaptosomes; 31% of the total homogenate binding was in P2 and 50% in P3. The binding of [3H]SR 95531 was saturable; Scatchard analysis of the saturation isotherm revealed two apparent populations of binding sites (KD of 6.34 nM and Bmax of 0.19 pmol/mg of protein; KD of 32 nM and Bmax of 0.81 pmol/mg of protein). The binding of [3H]SR 95531 was reversible, and association and dissociation kinetics confirmed the existence of two binding sites. Only GABAA ligands were effective displacers of [3H]SR 95531. GABAA antagonists were relatively more potent in displacing [3H]SR 95531 than [3H]GABA; the inverse was true for GABAA agonists. There were marked regional differences in the distribution of binding sites: hippocampus = cerebral cortex greater than thalamus = olfactory bulb = hypothalamus = amygdala = striatum greater than pons-medulla and cerebellum. The surprisingly low density of binding sites in the cerebellum was owing to a marked reduction of Bmax values at both the high- and the low-affinity binding sites. In conclusion, the present results demonstrate specific, high-affinity, saturable, and reversible binding of [3H]SR 95531 to rat brain membranes and strongly suggest that this radioligand labels the GABAA receptor site in its antagonist conformation.     

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.     

Characterization of [3H]Clonidine Binding to Two Sites in Calf Brain Membranes

Kinetic studies showed that under appropriate conditions, [3H]clonidine binds to two distinct receptor sites in calf cortex membranes. At 23 degrees C, binding was obtained at a low-affinity site (dissociation constant, KD = 5.4 nM) and a high-affinity site (KD = 1.1 nM). In contrast, at 0 degree C, selective binding occurred to the low-affinity site only. Consequently, at 0 degree C, it was possible to evaluate the interaction of drugs with the low-affinity receptor directly. On the other hand, competition with the high-affinity receptor could be ascertained by generating displacement curves representing the differential between specific binding values obtained at 23 and 0 degree C. Guanine nucleotides selectively decreased binding to the high-affinity site without apparent influence on the low-affinity [3H]clonidine binding. The activities of various pharmacological agents at the low- and high-affinity clonidine receptors are discussed and compared with WB-4101 binding data.