Barbiturates allosterically inhibit GABA antagonist and benzodiazepine inverse agonist binding

Barbiturates and the related depressant drugs, etazolate and etomidate, inhibited both the binding of [3H]bicuculline methochloride (BMC) to gamma-aminobutyric acid (GABA) receptor sites and the binding of [3H] beta-carboline-3-carboxylic acid methyl ester (beta CCM) to benzodiazepine receptor sites in mammalian brain. These concentration-dependent effects were chemically specific and stereospecific in a manner correlating with the activity of barbiturates to enhance GABA responses in neurons and to enhance GABA and benzodiazepine receptor agonist binding in vitro. The barbiturate inhibition of [3H]BMC binding involved a decrease in affinity which at high concentrations of barbiturates results in an effective complete loss of detectable binding. The maximal inhibition of [3H] beta CCM binding involved a more modest decrease in affinity (increase in KD from 1.35 to 1.85 nM). The barbiturate inhibitions of both ligands could be reversed by picrotoxin, suggesting an indirect action at previously defined picrotoxin/barbiturate modulatory sites on the GABA-benzodiazepine receptor/chloride ion channel complex.     

Enhancement of the in vivo binding of [3H]flunitrazepam by the atypical neuroleptic, clozapine

Modulation of the benzodiazepine receptor/GABA receptor/chloride ionophore complex in vivo involves a number of intricate regulatory interactions between the three components of the receptor complex. One way to assess these potential interactions involves the in vivo labelling of the benzodiazepine receptor with [3H]flunitrazepam. In these studies, we used this approach to demonstrate that the neuroleptic, clozapine, increases [3H]flunitrazepam binding in mouse brain in a bicuculline-reversible manner. This potentiation of benzodiazepine binding was not antagonized by picrotoxin and was found to result from a slower dissociation of [3H]flunitrazepam from the benzodiazepine receptor. These data suggest that clozapine acts to increase [3H]flunitrazepam binding via a GABAergic mechanism, independent of the chloride channel.     

Properties of a high affinity binding site for [3H]avermectin B1a

The specific high affinity binding of [3H]avermectin B1a was investigated in membranes from several rat brain regions. Binding occurred rapidly, was reversible and partially dependent on the presence of chloride ions in the incubation medium. Specific high affinity binding of [3H]avermectin B1a was partially inhibited by GABA receptor agonists and this effect was blocked by GABA receptor antagonists. Pentobarbital and etazolate inhibited, and picrotoxin, picrotoxinin and IPTBO stimulated high affinity binding of [3H]avermectin B1a. All these effects were influenced by the presence of chloride ions in the incubation medium. The results indicate that the high affinity binding site of [3H]avermectin B1a is associated with the GABA-benzodiazepine receptor-chloride ion channel complex.     

Action of pyrazolopyridines as modulators of [3H]flunitrazepam binding to the gaba/benzodiazepine receptor complex of the cerebellum

The pyrazolopyridines etazolate (SQ 20009) and cartazolate (SQ 65396) have strong modulatory effects on the GABA/benzodiazepine receptor complex of rate cerebellum. Thus, etazolate and cartazolate directly stimulate [3H]flunitrazepam binding (with EC50 values of 1.2 microM and 0.3 microM respectively) by increasing the apparent affinity of [3H]flunitrazepam for its binding sites. Stimulation of [3H]flunitrazepam binding by pyrazolopyridines is dependent on the presence of certain anions like chloride, bromide, iodide, nitrite, nitrate but not fluoride, acetate, formate or sulfate. If is inhibited by bicuculline-methiodide, and by the "chloride channel drugs' picrotoxinin and IPTBO. isoTHAZ, a GABA analogue with GABA antagonist properties in vivo, fails to inhibit binding stimulated by etazolate but antagonizes [3H]flunitrazepam binding stimulated by GABA. The pyrazolopyridines have also indirect effects on benzodiazepine receptor binding since they enhance the apparent sensitivity of those GABA recognition sites which are coupled to benzodiazepine binding sites. Thus, in the presence of 10 microM etazolate, GABA and muscimol enhance [3H]flunitrazepam binding, with EC50 values of 109 nM and 12 nM respectively. This sensitization effect is partially dependent on the presence of chloride ions. The pyrazolopyridines facilitate also the stimulation of benzodiazepine receptor binding by beta-alanine and taurine and by the rigid and flattened GABA analogues THIP and piperidine-4-sulfonic acid. Taken together, these results suggest that the pyrazolopyridines modulate [3H]flunitrazepam binding by acting at a site closely related to GABA receptor-regulated chloride ion channels.     

Effect of chronic treatment of ethanol on benzodiazepine and picrotoxin sites on the GABA receptor complex in regions of the brain of the rat

Ethanol has been shown to enhance gamma-aminobutyric acid (GABA)ergic transmission. In this study an examination was made of the effect of chronic treatment with ethanol and its withdrawal at 24 h on the binding of [3H]flunitrazepam and [35S]t-butylbicyclophosphorothionate (TBPS) to brain regions in rat. Rats were rendered tolerant to, and dependent on, ethanol by an intragastric intubation method. The affinity (KD) or the binding capacity (Bmax) of [3H]flunitrazepam or [35S]TBPS was not altered by chronic treatment with ethanol or during withdrawal from ethanol. Neither the enhancing effect of GABA on the binding of [3H]flunitrazepam nor its inhibitory effect on the binding of [35S]TBPS were affected by chronic treatment with ethanol or its withdrawal at 24 h. These results suggest that the sensitivity of benzodiazepine and picrotoxin sites on the oligomeric GABA receptor complex is not affected during tolerance to, or withdrawal from ethanol. It is suggested that the effects of ethanol on GABAergic transmission may be produced at the level of coupled chloride ion channels.     

Anion-dependent modulation of [3H]muscimol binding and of GABA-stimulated [3H]flunitrazepam binding by picrotoxin and related CNS convulsants

Picrotoxin, isopropylbicyclophosphate (IPTBO) and related CNS-convulsants have allosteric effects on the binding of ligands to the GABA/benzodiazepine receptor complex. When binding experiments were performed at 23 degrees C and at 35 degrees C, these drugs inhibited [3H]muscimol binding and muscimol- or GABA-stimulated [3H]flunitrazepam binding, respectively. Both effects required the presence of C1-, Br-, I- but not of F- or SO4(2-). Picrotoxin and IPTBO could only partially inhibit [3H]muscimol binding. In contrast other GABA antagonists and convulsants like bicuculline, 3 alpha-hydroxy-16-imino-5 beta-17-aza-androstan-11-one (R 5135), strychnine and d-tubocurarine interferred completely with [3H]muscimol binding, also in the absence of those ions mentioned above which were essential for the effects of picrotoxin. Our results support the notion that drugs like picrotoxin and IPTBO which interfere with the GABA receptor effector system, may lead to an allosteric perturbation of GABA-recognition sites.     

Enhancement of benzodiazepine binding by a diaryltriazine,LY81067

Ly81067, a diaryltriazine, represents a new class of compounds to enhance [³H]flunitrazepam binding to membranes of rat cerebral cortex. The enhancement induced by LY81067 exceeds that induced by γ-aminobutyric acid (GABA) and is partially abolished by the GABA antagonists bicuculline and picrotoxin. Scatchard analysis on the saturable binding of [³H]flunitrazepam reveals that LY81067 at 10 μM increases mainly the affinity for the [³H]ligand, due to a reduced rate of dissociation of the receptor-bound [³H]flunitrazepam. A dependence on chloride anions was demonstrated in the enhancement of [³H]flunitrazepam binding by LY81067. These findings suggest that the diaryltriazine LY81067 enhances [³H]flunitrazepam binding by exerting its effect at or near the picrotoxinsensitive anion recognition sites of the GABA/benzodiazepine receptor complex.     

Solubilization and characterization of benzodiazepine receptors in frontal cortex of rabbit brain

[3H]Flunitrazepam binding to benzodiazepine receptors solubilized by the detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate (CHAPS) was saturable and showed non-linear Scatchard plot with KD1 0.31 nmol/L and KD2 6.7 nmol/L. The affinities of soluble receptors to benzodiazepine were consistent with P2 membrane. One radioactive zone was found by SDS-PAGE after photoaffinity labelling of soluble membrane and the apparent molecular weight of 55,000 was reported. [3H]Flunitrazepam binding to soluble receptors was enhanced by GABA, NaCl or KCl and barbiturates, but inhibited by bicuculline and picrotoxinin. The enhancement of GABA on [3H]flunitrazepam binding was amplified by NaCl or KCl and antagonized by bicuculline and picrotoxinin. These results suggest that the benzodiazepine receptors solubilized by CHAPS have their pharmacological properties and are still associated with GABA receptors and chloride channel.     

GABA induces down-regulation of the benzodiazepine-GABA receptor complex in the rat cultured neurons

Cultured neurons from embryonic rat brain display central type benzodiazepine receptors characterized by high-affinity binding of [3H]flunitrazepam which is allosterically enhanced in the presence of gamma-aminobutyric acid (GABA). A 48 h treatment of the cultured neurons with 1 microM diazepam, 0.1 microM clonazepam or 0.1 microM beta-carboline ester derivatives did not change either Bmax or KD values of the [3H]flunitrazepam specific binding. A 48 h incubation in the presence of GABA (1 mM) or muscimol (0.1 mM) induced a 30% decrease of the Bmax value of [3H]flunitrazepam specific binding without change of the KD value. The down-regulation was dependent on GABA concentrations and temperature, and was partially inhibited by bicuculline but not by the benzodiazepine antagonist Ro 15-1788. The other subunits of the benzodiazepine-GABA-chloride channel receptor complex also seemed to be down-regulated by GABA since there was a decrease of the specific binding of [3H]muscimol and [35S]t-butylbicyclophosphorothionate (TBPS) to the GABAA and chloride channel sites respectively. The GABA-induced down-regulation of the GABA-benzodiazepine receptor seems to be selective since the specific binding of ligands to other receptors was not affected. Our results suggests that activation of the low-affinity GABA subunit which is involved in cellular electrophysiological responses, induced the receptor down-regulation.     

Modulation of the chloride ionophore by benzodiazepine receptor ligands: influence of gamma-aminobutyric acid and ligand efficacy

t-Butylbicyclophosphorothionate (TBPS) produces dose-dependent enhancement of [3H]propyl beta-carboline-3-carboxylate ([3H]PCC, 40 pM) binding to the benzodiazepine1 (BZ1) receptor subtype in hippocampus. Furthermore, TBPS enhancement of [3H]PCC binding was antagonized by micromolar concentrations of gamma-aminobutyric acid (GABA) in a way reversible by bicuculline. BZ receptor ligands that are "GABA positive" (i.e., enhance GABA neurotransmission) allosterically inhibited [35S]TBPS binding, whereas "GABA-negative" ligands (i.e., inhibit GABA neurotransmission) produced the opposite effect. The efficacy of the ligands as modulators of [35S]TBPS binding was consistent with their reported in vivo pharmacology. The effects of positive and negative ligands on [35S]TBPS binding were modulated by micromolar concentrations of GABA. Examination of the kinetics of [35S]TBPS binding suggested the presence of slowly and rapidly dissociating components. The GABA-positive clonazepam stabilized the rapidly dissociating component of [35S]TBPS binding, whereas methyl beta-carboline-3-carboxylate had a similar effect on the slowly dissociating component. It is speculated that the slowly dissociating component of [35S]TBPS binding is associated with a closed chloride channel, whereas the opposite is proposed for the rapidly dissociating component. The differential effects of GABA-positive versus GABA-negative ligands on [35S]TBPS binding and the modulatory effect of GABA provide further evidence to suggest that [35S]TBPS labels a site near the chloride ionophore linked to the GABA-BZ receptor complex.     

Enhancement of benzodiazepine and GABA binding by the novel anxiolytic, tracazolate

Tracazolate (ICI 136,753) 4-butylamine-1-ethyl-6-methyl-1H-pyrazolo[3,4]pyridine-5-carboxylic acid ethyl ester is a non-benzodiazepine with anxiolytic-like activity in animal models. In contrast to the benzodiazepines, it enhances [3H]flunitrazepam binding in rat synaptic membrane fragments. The enhancement is potential by chloride ion and is due to an increase in affinity of the receptor. The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) is additive with that of tracazolate; however, the GABA antagonist bicuculline blocks the enhancement by both compounds. Tracazolate enhances [3H]GABA binding to frozen and thawed Triton X-100-treated membrane fragments. The enhancement is due to an increase in the number of sites and potentiated by chloride. Benzodiazepines also enhanced GABA binding but the effect was due to an apparent change in affinity and not potentiated by chloride. The rank order to chlorodiazepoxide, diazepam and flunitrazepam for enhancement of GABA binding and displacement of [3H]flunitrazepam binding were the same. The enhancement of [3H]GABA binding by flunitrazepam and tracazolate were additive. Possible interactions between these various receptors are discussed.     

The modulation by chlormethiazole of the GABAA-receptor complex in rat brain.

1. The interactions of chlormethiazole with gamma-aminobutyric acid (GABA) synthesis and release, and with ligand binding to sites associated with the GABAA-receptor complex and the GABAB-receptor have been studied in the rat. The GABAA-receptor was studied using [3H]-muscimol, [3H]-flunitrazepam was used to label the benzodiazepine modulatory site, and [35S]-butyl-bicyclophosphorothionate ([35S]-TBPS) to label the chloride channel. 2. Chlormethiazole had no effect on GABA synthesis in the cortex, hippocampus and striatum or on GABA release from cortical slices in vitro. Chlormethiazole did not displace [3H]-baclofen binding to the GABAB-receptor. 3. Chlormethiazole (IC50 = 140 microM) and pentobarbitone (IC50 = 95 microM) both inhibited [35S]-TBPS binding by increasing the rate of [35S]-TBPS dissociation. In addition, chlormethiazole caused an apparent decrease in the affinity of [35S]-TBPS binding. 4. Chlormethiazole enhanced the binding of [3H]-muscimol but had no effect on [3H]-flunitrazepam binding. In contrast, the sedative barbiturate pentobarbitone enhanced both [3H]-muscimol and [3H]-flunitrazepam binding. 5. It is concluded that the sedative and anticonvulsant effects of chlormethiazole are probably mediated through an action at the GABAA-receptor. However, chlormethiazole does not interact with the GABAA-receptor complex in an identical manner to the sedative barbiturate pentobarbitone.     

Enhancement of GABA binding by the benzodiazepine partial agonist CGS9896

Compounds have been reported that act on the benzodiazepine receptor as full agonists (diazepam and CL218872), full antagonists (CGS8216 and RO15-1788), on partial agonists (CGS9896). We examined the effect of these compounds on [3H]GABA binding to membrane fragments from rat brain. Incubations were performed at 37 degrees C in a buffer containing EGTA to reduce free calcium ion levels. Centrifugation was then used to separate bound from free [3H]GABA. Diazepam caused a 20-45% enhancement of [3H]GABA binding and this effect was inhibited by 5 mM CaCl2. The magnitude of the enhancement of [3H]GABA by CL218872 was similar to that of diazepam. In contrast, the benzodiazepine antagonists, RO15-1788 and CGS8216 caused little enhancement of [3H]GABA binding. Finally, the partial agonist CGS9896 was distinguishable from both the benzodiazepine antagonists and full agonists by an intermediate level of enhancement of [3H]GABA binding. The extent of enhancement of [3H]GABA binding appears to be predictive of the pharmacological efficacy of compounds acting at the benzodiazepine receptor.     

Interaction of pitrazepin with the GABA/benzodiazepine receptor complex and with glycine receptors

Pitrazepin (3-(piperazinyl-1)-9H-dibenz(c,f)triazolo(4,5-a)azepin) is a new GABAA receptor antagonist reported to antagonize electrophysiological effects of GABA. We have investigated in some detail the interaction of pitrazepin with the GABA/benzodiazepine receptor chloride channel complex. Pitrazepin was found to be a competitive inhibitor of the GABAA receptor which is coupled to [3H]diazepam and [35S]TBPS binding sites; the KI value obtained by Schild analyses was 80 nM. Although pitrazepin interacted weakly with BZ receptors the compound did not affect the chloride gating mechanism (labelled with [35S]TBPS or [3H]avermectin B1a). Further, pitrazepin was a non-selective GABA antagonist since glycine receptors, labelled with [3H]strychnine, were affected at low concentrations (the KI values in rat brain-stem were 71-110 nM).     

Gamma-hydroxybutyric acid binding sites in rat and human brain synaptosomal membranes

The binding of gamma-hydroxy[2,3-3H]butyric acid (GHB) was characterized in rat and human brain synaptosomal membranes. Binding was shown to be saturable, pH dependent, and linear with protein concentration. There was a distinct regional distribution of binding sites in both rat and human brain, with the hippocampus being the richest and the cerebellum the poorest, in density of [3H]GHB binding sites. Competition and saturation experiments revealed two different population of binding sites, a high-affinity site with a KD1 of 580 nM and a B max1 of 1.8 pmoles/mg protein and a low-affinity site with a KD2 of 2.3 microM and a B max2 of 11.3 pmoles/mg protein. [3H]GHB binding was not inhibited by gamma-aminobutyric acid (GABA), GABA receptor agonists, opiate antagonists or anticonvulsant drugs. These data suggest that GHB may play a role as a neurotransmitter or neuromodulator in brain independent of GABA.     

Radiation inactivation of brain [35S]t-butylbicyclophosphorothionate binding sites reveals complicated molecular arrangements of the GABA/benzodiazepine receptor chloride channel complex

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a bicyclic cage convulsant, binds to the anion gating mechanism of the GABA/benzodiazepine receptor chloride channel complex. Using a carefully calibrated radiation inactivation technique, the molecular weight of [35S]TBPS binding complexes from frozen rat cerebral cortex was estimated to be 137,000 daltons. The GABA agonist muscimol reduced [35S]TBPS binding to 0-10% of the control value, in a way which is independent of the radiation dose. This shows that the GABA receptor (Mw = 55,000 daltons) is included in the 137,000-dalton [35S]-TBPS binding complex; the [35S]TBPS binding protein alone accounts for 137,000-55,000 = 82,000 daltons. The pyrazolopyridazine etazolate (SQ 20.009) and etomidate in appropriate concentrations both reduced specific binding of [35S]TBPS. The ability of SQ 20.009 and etomidate to reduce [35S]TBPS binding was greatly reduced by exposure to low radiation doses, suggesting that SQ 20.009 and etomidate reduce [35S]TBPS binding by an allosteric mechanism requiring a molecular structure of 450,000-500,000 daltons. Benzodiazepine agonists (ethyl 4-methoxymethyl-6-benzyloxy-beta-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) enhance and reduce [35S]TBPS binding, respectively, in repeatedly frozen and washed membrane preparations. The effects of ZK 93423 and DMCM on [35S]TBPS binding disappeared upon exposure of membranes to low radiation doses. This suggests that the benzodiazepine receptor site interacts allosterically with the [35S]TBPS binding site, requiring a molecular complex of at least c. 400,000 daltons. The [35S]TBPS site alone in these latter conditions of membrane preparation (repeatedly frozen/washed) revealed a molecular weight of 221,000 daltons (TBPS-site + GABA receptor + unknown structures). The number of binding sites for [35S]TBPS (145 pmol/g tissue) was only slightly higher than for [3H]flunitrazepam (130 pmol/g tissue) in cerebral cortex. These results are all consonant with the conclusion that the GABA/BZ receptor chloride channel complex is composed of highly integrated multimeric subunits, tentatively accounted for by a tetramic complex of molecular weight 548,000 daltons.     

Modulation of [3H]flunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine

The influence of phosphatidylserine on ligand binding to the benzodiazepine/GABA receptor complex was assessed in rat cerebellar synaptic membranes and in a detergent-solubilized membrane preparation. Intact synaptic membranes or membranes solubilized with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate) were incubated with a range of concentrations of phosphatidylserine for 2 h at 4 degrees C, prior to use in radioligand binding assays. Phosphatidylserine, an endogenous membrane phospholipid, facilitated the site-specific binding of [3H]flunitrazepam to synaptic membranes and CHAPS-solubilized preparations. In addition, phosphatidylserine inhibited the facilitation of [3H]flunitrazepam binding induced by either cartazolate or gamma-aminobutyric acid (GABA). Although the maximum effect (38% facilitation of [3H]flunitrazepam binding; greater than 90% inhibition of the cartazolate action) was produced using 130 microM phosphatidylserine, a significant enhancement of [3H]flunitrazepam binding could be observed upon preincubation of synaptic membranes with concentrations of phosphatidylserine as low as 5 microM. These results suggest that endogenous phosphatidylserine may play a role in the regulation of benzodiazepine/GABA receptor function, possibly through modulation of the mechanisms which functionally link the various components of this complex receptor system.     

Proconflict effect of GABA receptor complex antagonists. Reversal by diazepam

The effect of drugs which down-regulate the function of GABA at the level of the GABA/benzodiazepine receptor complex was studied on the conflict test in the rat. The GABA receptor antagonist, bicuculline, and the blockers of the GABA-receptor-coupled chloride channel, picrotoxin and pentylenetetrazol, produced a dose-dependent proconflict effect. This effect occurred at dose levels which failed to affect unpunished behaviour. The most effective compounds were bicuculline and picrotoxin. The proconflict effect of these drugs was prevented by diazepam but not by the specific benzodiazepine antagonist, Ro15-1788. The data indicate that a diminished GABAergic activity at different subunits of the GABA receptor complex resulted in an enhancement of punishment-suppressed behaviour in rats.     

Increased benzodiazepine receptor number elicited in vitro by a novel purine, EMD 28422

EMD 28422 (N6-[2-(4-chlorophenyl)-bicyclo-2.2.2.octyl-(3)]-adenosine) was demonstrated to increase the number of binding sites for [3H]diazepam (Bmax) in vitro without an accompanying increase in receptor affinity (KD). The increase in receptor number was observed in both crude synaptosomal preparations (P2) and thrice-washed membrane preparations with and without the addition of 50 microM GABA. Furthermore, this effect appeared to be independent of the concentration of chloride ion, since the increases in Bmax were observed in both Tris-HCl and Tris-maleate buffer. The effects of EMD 28422 were stereospecifically antagonized by the GABA antagonist bicuculline, despite the lack of effect of EMD 28422 on [3H]muscimol binding at concentrations which markedly increased benzodiazepine receptor number. Neither EMD 39011 nor adenosine, the two parent moieties of EMD 28422, increased [3H]diazepam binding at concentrations of up to 1 mM. The increases in benzodiazepine receptor number observed with EMD 28422 in vitro suggests that this compound induces a conformational change in the benzodiazepine receptor which may cause the dissociation of an endogenous noncompetitive inhibitor of [3H]diazepam binding from the membrane, thus 'unmasking' binding sites. The stereospecific antagonism of this effect by bicuculline and the apparent inability of GABA to alter the action of EMD 28422 suggests the presence of a novel type or different functional state of GABA receptor which may play a permissive role in the rapid modulation of benzodiazepine receptor number in vitro.     

Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors.

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.