Enhanced binding of radioligands to receptors of γ-aminobutyric acid and benzodiazepine by a new anticonvulsive agent,LY81067

A diaryltriazine, LY81067, effectively protects against pentylenetetrazole- and picrotoxin-induced convulsions in mice, with ED₅₀ values of 5.7 and 5.8 mg/kg i.p., respectively. LY81067 enhances the binding of both ³H-GABA and ³H-flunitrazepam to specific sites in rat brain membranes. The degree of enhancement by LY81067 varies from one brain region to another and is different for the binding of ³H-GABA and ³H-flunitrazepam. In cortical membranes, LY81067 increases the affinity of ³H-GABA for both high and low affinity sites and increases the number of sites. LY81067 increases the affinity of ³H-flunitrazepam for its binding sites without greatly increasing the number of sites. Like the pyrazolopyridines, the enhancement of ³H-flunitrazepam binding by LY81067 is dependent on chloride or related anions and is reversed by picrotoxin, suggesting that LY81067 exerts its anticonvulsant effects by binding to or near picrotoxin binding sites. The differential effects of LY81067 on the enhancements of ³H-GABA and ³H-flunitrazepam binding in several brain regions suggest extensive multiplicity of GABA/benzodiazepine/picrotoxin/anioin receptor complexes.     

Species dependent dual modulation of the benzodiazepine/GABA receptor chloride channel by dihydroergosine

Dihydroergosine (50 and 100 mg/kg) enhanced the incidence of bicuculline (3 mg/kg)-induced convulsions in female rats, while 100 mg/kg of dihydroergosine given to female mice made 45% convulsive dose of bicuculline (2.5 mg/kg) to be subconvulsive. The same dose of dihydroergosine enhanced in mice the latency of bicuculline (4 mg/kg)-induced convulsions. Although, in in vitro experiments dihydroergosine showed very weak ability to prevent the binding of 3H-muscimol, the drug was able to diminish and to augment the IC50 of bicuculline and GABA when added to crude synaptosomal pellet of the rat and mouse brain respectively. Lower concentrations of dihydroergosine stimulated and higher inhibited 3H-TBOB binding to the crude synaptosomal pellet of the rat brain. In the preparation of mouse brain dihydroergosine produced only inhibition of 3H-TBOB binding. Only slight quantitative differences were observed in bicuculline-induced stimulation and in GABA- and diazepam-induced inhibition of 3H-TBOB binding between the two species. The results suggest that the opposite species-dependent effects of dihydroergosine on bicuculline-induced convulsions are due to the ability of this drug to modulate species-dependently the benzodiazepine/GABA receptor chloride channel complex.     

In vitro interaction of premazepam with benzodiazepine receptors in rat brain regions

Premazepam (PRZ) in vitro competitively displaced 3H-diazepam (DIA), 3H-flunitrazepam (FLU) and 3H-RO 15-1788 from their binding sites on rat brain synaptosomes, with a potency intermediate to other benzodiazepines (BDZs), and Hill coefficients near 1 in different brain regions. Incubation at 37 degrees C reduced premazepam's affinity for BDZ receptors to a lower extent than other benzodiazepines and had no effect on the Hill coefficient. The IC50 of PRZ on 3H-RO 15-1788 and 3H-FLU binding was markedly reduced by GABA in rat cortex, like those of reference classical BDZs, but was GABA-independent in the cerebellum. The IC50 of the BDZ antagonist, RO 15-1788 was unaffected by GABA in both brain areas. The possibility that PRZ behaves as a partial agonist in the cortex and as an antagonist in the cerebellum is discussed.     

Independent benzodiazepine and beta-carboline binding sites in the brain of aggressive and anxious-defensive mice

Distribution of specific 3H-flunitrazepam and 3H-beta-carboline-3-carboxylate binding sites in the brain regions of aggressive and timid-defensive mice was investigated before and after subchronic injection of diazepam (5 mg/kg). The absence of differences between the affinity and concentration of 3H-flunitrazepam binding sites in diencephalon and brain cortex in aggressive and defensive mice may be explained by general benzodiazepine receptor reaction on isolation and agonistic interaction stress. Significant predominance of 3H-beta-carboline-3-carboxylate binding sites in the brain cortex, as compared to the concentration of 3H-flunitrazepam binding sites suggests the presence of specific binding sites for beta-carbolines, which have specific distribution in the brain.     

Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.     

Monensin inhibits the binding of 3H-flunitrazepam to and reveals the intracellular passage of GABAA/benzodiazepine receptor.

Effects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H-flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 microM monensin for 3 h blocked the binding of 3H-flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 microM monensin, as detected by assaying the incorporation of 3H-leucine and 3H-galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down-regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Selective affinity of the benzodiazepines quazepam and 2-oxo-quazepam for BZ1 binding site and demonstration of 3H-2-oxo-quazepam as a BZ1 selective radioligand

Quazepam and 2-oxo-quazepam are novel benzodiazepines containing a trifluoroethyl substituent on the ring nitrogen at position #1. Detailed competition binding experiments (25 to 30 concs.) at 4 degrees C were undertaken with these compounds versus 3H-flunitrazepam using synaptic membranes from rat cortex or cerebellum. Unlike other benzodiazepines, both quazepam and 2-oxo-quazepam distinguished two populations of 3H-flunitrazepam binding sites in rat cortex which were present in roughly equal proportions and for which the compounds displayed a greater than 20-fold difference in affinity. In cerebellum, no such discrimination of sites was noted for 2-oxo-quazepam, but quazepam did distinguish a small, low affinity (15% of total) population of sites. 3H-2-oxo-quazepam was prepared and used in competition studies to substantiate the conclusion that these compounds discriminate two populations of benzodiazepine sites in rat cortex. This new radioligand was shown to specifically label BZ binding sites with high affinity in a saturable manner. The competition experiments were then conducted using 3H-2-oxo-quazepam at a radioligand concentration sufficiently low (0.5 nM) to ensure that only the higher affinity binding sites which 2-oxo-quazepam discriminates would be occupied. Competition experiments in both cortex and cerebellum under these conditions indicated single site binding for unlabelled quazepam and 2-oxo-quazepam in every instance. This suggests that 3H-2-oxo-quazepam should be a useful new tool for selectively labeling and studying the BZ1 population of benzodiazepine binding sites.     

GABA-benzodiazepine interactions: physiological, pharmacological and developmental aspects

Many of the pharmacological actions of the benzodiazepines can be attributed to their actions on gamma-aminobutyric acid (GABA) systms in the brain. Electrophysiological studies on dorsal raphe neurons indicate that the benzodiazepines act postsynaptically to potentiate GABAergic inhibition in this midbrain nucleus. Direct binding studies have shown that both in vitro and in vivo binding of [3H]diazepam to a specific high affinity benzodiazepine binding site in cerebral cortical tissue are enhanced by the direct in vitro addition of GABA and GABA agonists or by pretreatment of animals with GABA analogs and agents that elevate GABA levels in brain. Ontogenic development of [3H]diazepam binding in brain parallels the development of the sodium-independent [3H]GABA binding. The ability of GABA to enhance benzodiazepine binding is present throughout development and inversely related to age. These data suggest that there is a functionally significant interaction between the benzodiazepines and GABA throughout development and at maturity. A model is proposed to relate these interactions to conformational changes in a benzodiazepine/GABA/Cl- ionophore complex.     

Structural and functional characterization of the gamma 1 subunit of GABAA/benzodiazepine receptors.

The GABAA receptor gamma 1 subunit of human, rat and bovine origin was molecularly cloned and compared with the gamma 2 subunit in structure and function. Both gamma subunit variants share 74% sequence similarity and are prominently synthesized in often distinct areas of the central nervous system as documented by in situ hybridization. When co-expressed with alpha and beta subunits in Xenopus oocytes and mammalian cells, the gamma variants mediate the potentiation of GABA evoked currents by benzodiazepines and help generate high-affinity binding sites for these drugs. However, these sites show disparate pharmacological properties which, for receptors assembled from alpha 1, beta 1 and gamma 1 subunits, are characterized by the conspicuous loss in affinity for neutral antagonists (e.g. flumazenil) and negative modulators (e.g. DMCM). These findings reveal a pronounced effect of gamma subunit variants on GABAA/benzodiazepine receptor pharmacology.     

Pyritinol inhibition of the GABA and benzodiazepine receptor

In 100 and 200 mumol/l concentration, pyritinol inhibited GABA binding to the GABA receptors of brain synaptosomal membranes. GABA receptors from the cerebral cortex, diencephalon and striatum were inhibited to approximately the same degree; those from the cerebellum and spinal cord were inhibited more. Both high and low affinity receptors were inhibited. Pyritinol did not greatly affect the number of binding sites (Bmax), but reduced the affinity (raised the dissociation constant KD) of both receptors. The benzodiazepine receptor, which is connected with the postsynaptic GABAA receptor, was also inhibited by pyritinol. The character of inhibition was the same as for GABA receptors, i.e. there was no change in the number of binding sites, but there was a decrease in their affinity. It is assumed that the similarity of the effect on GABA and benzodiazepine receptors is associated with their occurrence on one, or on two relatively firmly interconnected, protein molecules. Depression of the affinities of GABA and the associated benzodiazepine receptor, together with inhibition of GABA synthesis, in the presence of pyritinol indicate that diminished activity of the GABA system in the brain might be related to the activating effect of pyritinol.     

Effect of Chronic Ethanol Treatment on the γ-Aminobutyric Acid-Mediated Enhancement of [3H]Flunitrazepam Binding in Rat Cortex and Hippocampus

The mechanism by which ethanol affects the gamma-aminobutyric acid (GABA)/benzodiazepine complex is not clear. It is known that ethanol enhances the Cl- influx mediated by the GABAA receptor complex, and although chronic ethanol administration does not change the KD or Bmax for [3H]flunitrazepam binding, some reports have suggested that it could modify the modulation of benzodiazepine binding produced by GABA. In the present work, we studied the effect of chronic ethanol treatment on the modulation by GABA of [3H]flunitrazepam binding, using light microscopic autoradiography. This technique allows the measurement of densities of benzodiazepine receptors in different brain areas, the visual cortex and hippocampus, which appear to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. We found enhancement of benzodiazepine binding by GABA at concentrations of greater than 10(-6) M for the various cortical and hippocampal areas studied from both control and ethanol-treated animals; this enhancement peaked at 10(-4) M GABA but decreased at 10(-3) M GABA. We found a clear effect of ethanol treatment on the modulatory properties of GABAA receptor, in both cortex and hippocampus, although only in cortex were the differences statistically significant between control and ethanol-treated animals.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Two novel GABAA receptor subunits exist in distinct neuronal subpopulations

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling.     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Daily rhythms of benzodiazepine receptor numbers in frontal lobe and cerebellum of the rat

Behavioural, biochemical and neurophysiological evidence suggests that gamma-aminobutyric acid (GABA) may play an important role in the neural control of circadian rhythms. Central receptors for benzodiazepines are functionally coupled to GABA receptors and appear to mediate behavioural effects of exogenous benzodiazepines. The binding of 3H-flunitrazepam to synaptic plasma membranes prepared from various regions of rat brain was examined at 6-hour intervals over a 36-hour period. Prominent daily rhythms in receptor number (Bmax) were observed in the frontal lobe and the cerebellum but not in the temporoparietal regions, hypothalamus or medulla/pons. Binding was highest during periods of sleep/low activity with a significant decrease occurring just prior to waking. These results suggest that daily fluctuations in benzodiazepine receptor numbers may be related to the temporal control of sleep/wake and muscle activity cycles.     

Inhibition of neuronal membrane GABAB receptor binding by GABA structural analogues

1. A number of compounds structurally related to GABA were tested as inhibitors of baclofen-sensitive GABAB receptor binding to membranes from mouse brain. 2. In addition to two known inhibitors--baclofen and 5-aminovaleric acid--two analogues were shown to possess inhibitory activity. These compounds were 4-aminobutyryl-DL-alanine hydrobromide (IC50 = 3 microM) and trans-2-(aminomethyl)cyclopropane carboxylic acid (IC50 = 90 microM). 3. Both drugs also exhibited affinity for GABAA binding sites. 4. Further experiments are needed to establish if these analogues exert agonist or antagonist action at the GABAB receptor.     

Solubilization of the benzodiazepine receptor from rat brain

A macromolecular moiety having high binding affinity for benzodiazepines was solubilized from a rat brain synaptosomal fraction by extraction with a combination of sodium deoxycholate and potassium chloride. This solubilized fraction exhibits pharmacological specificity for benzodiazepines. Specific ³H-flunitrazepam binding to the solubilized fraction was saturable with the apparent dissociation constant Kd = 1.8 ± 0.3 nM. The binding is sensitive to proteolytic enzymes. The binding is increased by α-aminobutyric acid and decreased by inosine and hypoxanthine. These results are similar to those obtained with intact membranes, suggesting that this moiety may be the benzodiazepine receptor. The molecular weight was estimated to be approximately 200, 000 by sucrose density gradient centrifugation.     

A Gaba/Benzodiazepine Receptor Complex from Bovine Brain: Purification,Reconstitution and Immunological Characterization

Abstract

A GABA/benzodiazepine receptor complex was purified from bovine cerebral cortex. The receptor fraction displayed binding sites for benzodiazepines as well as high and low affinity binding sites for GABA which are characteristics of the membrane-bound receptor. Two monoclonal antibodies of which one was directed against the 50 kd and the other against the 55 kd subunit were used for immunoprecipitation studies. Both of them were shown to quantitatively precipitate the entire receptor population. These results indicate that the binding sites for benzodiazepines and GABA (high and low affinity sites) reside on the same receptor complex containing a mixture of 50 kd and 55 kd subunits. Reconstitution of the receptor in phospholipid vesicles was achieved.     

Uniform regulation of central and peripheral benzodiazepine binding sites by GABA receptor agonists in rats

Central and peripheral benzodiazepine binding sites were studied in vitro after the administration of GABAA and GABAB agonists. Cerebral cortex and kidney homogenates were used in this study. Muscimol (1.5 mg/kg, intraperitoneally) pretreatment significantly increased the affinity of benzodiazepine binding sites not only in the cerebral cortex but also in the kidneys. Similar changes were obtained with (-) and (+) baclofen (5 mg/kg, intraperitoneally), with the only exception that (-) baclofen in addition to changes in the affinity caused a marked decrease in the number of binding sites in both structures studied. The mechanism of action of GABA agonists on peripheral benzodiazepine binding sites is discussed.