TBOB Binding Research Articles

Clarified Açaí (Euterpe oleracea) Juice as an Anticonvulsant Agent: In Vitro Mechanistic Study of GABAergic Targets.

Seizures affect about 50 million people around the world. Approximately 30% of seizures are refractory to the current pharmacological arsenal, so, the pursuit of new therapeutic alternatives is essential. Clarified Euterpe oleracea (EO) juice showed anticonvulsant properties similar to diazepam in an in vivo model with pentylenetetrazol, a GABAA receptor blocker. This study investigated the effects of EO on the main GABAergic targets for anticonvulsant drugs, analyzing the effect on the GABA receptor's benzodiazepine and picrotoxinin binding sites and the GABA uptake. Primary cultures of cortical neurons and astrocytes were treated with EO (0–25%) for up to 90 min. [3H]Flunitrazepam and [3H]TBOB binding, [3H]GABA uptake, cell viability, and morphology were assayed. Nonlethal concentrations of EO increased agonist binding and decreased antagonist binding in cortical neurons. Low concentrations significantly inhibited GABA uptake, especially in astrocytes, suggesting an accumulation of endogenous GABA in the synaptic cleft. The results demonstrate, for the first time, that EO can improve GABAergic neurotransmission via interactions with GABAA receptor and modulation of GABA uptake. Understanding these molecular mechanisms will help in the treatment of seizures and epilepsy, especially in developing countries where geographic isolation and low purchasing power are the main barriers to access to adequate treatment.

In vitro effects of ethanol and gabapentin treatment

Background: The anticonvulsant drug gabapentin, a structural analogue of GABA, showed beneficial effects in the treatment of alcoholism and its consequences. Although the mechanisms of action of gabapentin are not fully understood, studies suggested that gabapentin’s neuroprotective effects could be achieved via GABA-A receptors. The aim of this study was to investigate the potential protective action of gabapentin on the well- known neurotoxic effects of chronic alcohol consumption and withdrawal. Methods: The dose– response relationship and the time course of ethanol and gabapentin treatment were established on human embryonic kidney (HEK) 293 cells, either non-transfected or stably expressing α1β2γ2S GABA-A receptors. A trypan blue exclusion assay and MTT test were performed to assess cell viability. Membrane preparations of stably transfected HEK 293 cells treated with 100 mM ethanol in combination with 1 µM gabapentin for 96 h were used in [3H]flunitrazepam and [3H]TBOB binding studies to determine the number and affinity of benzodiazepine and convulsant binding sites, and their allosteric interactions with GABA binding sites. 100 µM bicuculline, a GABA-A receptor antagonist, was used in other to counteract the effects of gabapentin. The levels of mRNA encoding the α1, β2 and γ2S receptor subunits in stably transfected HEK 293 cells following ethanol and gabapentin treatment were determined by semiquantitative RT-PCR analysis. Results: Treatment with ethanol at concentrations higher than 100 mM for 96 h reduced the number of HEK 293 cells, non- transfected or transfected with α1β2γ2S GABA-A receptors. The cytotoxic effect of ethanol was counteracted by co-administration of 1 µM gabapentin in transfected, but not in non- transfected cells. Exposure to 100 mM ethanol induced an increase in the number of binding sites for benzodiazepines and convulsants, while this effect was reversed by simultaneous exposure to 1 µM gabapentin, 100 µM bicuculline or their combination. Administration of ethanol, as well as bicuculline, induced functional allosteric uncoupling of the GABA and the benzodiazepine binding site, which was prevented by simultaneous gabapentin treatment. The mRNA expression levels of α1, β2 and γ2S GABAA receptor subunits were not influenced by the exposure to either ethanol or gabapentin, but only by their co-administration. Discussion: Although our results support the hypothesis of an involvement of GABA-A receptors in the actions of gabapentin, further research is needed to elucidate the mechanisms of gabapentin’s protective effects against ethanol-induced cytotoxicity.

Is GABA neurotransmission enhanced in auditory thalamus relative to inferior colliculus?

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central auditory system. Sensory thalamic structures show high levels of non-desensitizing extrasynaptic GABAA receptors (GABAARs) and a reduction in the redundancy of coded information. The present study compared the inhibitory potency of GABA acting at GABAARs between the inferior colliculus (IC) and the medial geniculate body (MGB) using quantitative in vivo, in vitro, and ex vivo experimental approaches. In vivo single unit studies compared the ability of half maximal inhibitory concentrations of GABA to inhibit sound-evoked temporal responses, and found that GABA was two to three times (P < 0.01) more potent at suppressing MGB single unit responses than IC unit responses. In vitro whole cell patch-clamp slice recordings were used to demonstrate that gaboxadol, a δ-subunit selective GABAAR agonist, was significantly more potent at evoking tonic inhibitory currents from MGB neurons than IC neurons (P < 0.01). These electrophysiological findings were supported by an in vitro receptor binding assay which used the picrotoxin analog [(3)H]TBOB to assess binding in the GABAAR chloride channel. MGB GABAARs had significantly greater total open chloride channel capacity relative to GABAARs in IC (P < 0.05) as shown by increased total [(3)H]TBOB binding. Finally, a comparative ex vivo measurement compared endogenous GABA levels and suggested a trend towards higher GABA concentrations in MGB than in IC. Collectively, these studies suggest that, per unit GABA, high affinity extrasynaptic and synaptic GABAARs confer a significant inhibitory GABAAR advantage to MGB neurons relative to IC neurons. This increased GABA sensitivity likely underpins the vital filtering role of auditory thalamus.

Quantitative structure–activity relationships of monoterpenoid binding activities to the housefly GABA receptor

Monoterpenoids are a large group of plant secondary metabolites. Many of these naturally occurring compounds have shown good insecticidal potency on pest insects. Previous studies in this laboratory have indicated that some monoterpenoids have positive modulatory effects on insect GABA receptors. In this study, the key properties of monoterpenoids involved in monoterpenoid binding activity at the housefly GABA receptor were determined by developing quantitative structure-activity relationship (QSAR) models, and the relationship between the toxicities of these monoterpenoids and their GABA receptor binding activities was evaluated. Two QSAR models were determined for nine monoterpenoids showing significant effects on [³H]-TBOB binding and for nine p-menthane analogs with at least one oxygen atom attached to the ring. The Mulliken charges on certain carbon atoms, the log P value and the total energy showed significant relationships with binding activities to the housefly GABA receptor in these two QSAR models. From the QSAR models, some chemical and structural parameters, including the electronic properties, hydrophobicity and stability of monoterpenoid molecules, were suggested to be strongly involved in binding activities to the housefly GABA receptor. These findings will help to understand the mode of action of these natural insecticides, and provide guidance to predict more monoterpenoid insecticides.

Modulation of Recombinant GABAA Receptors by Neurosteroid Dehydroepiandrosterone Sulfate

Aim: To investigate whether long-term exposure to the neurosteroid dehydroepiandrosterone sulfate (DHEAS) induces adaptive changes of GABA_A receptors related to the development of tolerance and dependence. Methods: We compared the parameters of [³H]DHEAS binding and the effects of DHEAS on [³H]flunitrazepam binding in the membranes of HEK 293 cells, nontransfected or stably transfected with recombinant α₁β₂γ_2S GABA_A receptors. In HEK 293 cells expressing α₁β₂γ_2S GABA_A receptors, we investigated the effects of long-term DHEAS treatment on the [³H]flunitrazepam and [³H]t-butylbicycloorthobenzoate ([³H]TBOB) binding and on their modulation with GABA. Results: DHEAS behaves as an allosteric antagonist of the recombinant α₁β₂γ_2S GABA_A receptors expressed in HEK 293 cells. Exposure of cells to 100 µmol/l DHEAS for 48 h did not change the number or affinity of benzodiazepine and convulsive binding sites. Long-term DHEAS treatment failed to affect functional allosteric interactions between GABA_A receptor binding sites, as evidenced by an unchanged ability of GABA to stimulate or to inhibit [³H]flunitrazepam and [³H]TBOB binding, respectively. Conclusion: The findings that prolonged DHEAS treatment does not produce changes in GABA_A receptor expression and functional coupling, assumed to underlie the development of tolerance and dependence, might have importance in the long-term therapy necessary for the observed beneficial effects of DHEAS.

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: Importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA A-receptor ligand [ 3H]TBOB to rat forebrain and cerebellar membrane preparations with IC 50 (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [ 3H]flunitrazepam and [ 3H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [ 3H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [ 3H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC 50 values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.

Effects of monoterpenoid insecticides on [ 3H]-TBOB binding in house fly GABA receptor and 36Cl − uptake in American cockroach ventral nerve cord

Monoterpenoids and their derivatives from plant essential oils showed good insecticidal activities in previous studies, but the mechanisms of their action as natural insecticides are not known yet. In the present work, we evaluated the pharmacological action of five monoterpenoids (α-terpineol, carvacrol, linalool, pulegone, and thymol) on native insect GABA receptors from house flies and American cockroaches using radiotracer methods. In the [ 3H]-TBOB binding assay, carvacrol, pulegone, and thymol all enhanced the [ 3H]-TBOB binding to membrane preparation of house fly heads with EC 50 values of 48 μM, 432 μM, and 6 mM, respectively. Moreover, these three monoterpenoids at concentrations of 500 μM and 1 mM also significantly increased the 36Cl − uptake induced by GABA in membrane microsacs prepared from American cockroach ventral nerve cords. These results revealed that carvacrol, pulegone, and thymol are all positive allosteric modulators at insect GABA receptors. The other two monoterpenoids that were tested, α-terpineol and linalool, showed little or no effect in both the [ 3H]-TBOB binding and 36Cl − uptake assays.

Chronic treatment with flumazenil enhances binding sites for convulsants at recombinant α 1β 2γ 2S GABA A receptors

GABA A receptors mediate most of the fast inhibitory neurotransmission in the brain. Prolonged occupancy of these receptors by ligands leads to regulatory changes often resulting in reduction of receptor function. The mechanism of these changes is still unknown. In this study, stably transfected human embryonic kidney (HEK) 293 cells were used as a model to study the effects of prolonged flumazenil (antagonist of benzodiazepine binding sites at GABA A receptors) exposure on the recombinant α 1β 2γ 2S GABA A receptors, the most common type of GABA A receptors found in the brain. Exposure (48 h) of HEK 293 cells stably expressing recombinant α 1β 2γ 2S GABA A receptors to flumazenil (1 or 5 μM) in the presence of GABA (1 μM), enhanced the maximum number ( B max) without affecting the affinity ( K d) of [ 3H]TBOB labeled binding sites for convulsants. Diazepam (1 nM–1 mM) in the presence of GABA (1 μM) modulated [ 3H]TBOB binding to control and flumazenil pretreated cells according to a two-site model. No significant differences between the groups were observed in either the potency or efficacy of diazepam to modulate [ 3H]TBOB binding, as evidenced by a lack of significant changes between their IC 50 and I max values. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant α 1β 2γ 2S GABA A receptors to flumazenil up-regulates the binding sites for convulsants, but it does not appear to affect the functional coupling between these sites and benzodiazepine binding sites. Along with our recent data, these results suggest that chronic treatment with flumazenil enhances the number of GABA A receptors.

Chronic exposure of cells expressing recombinant GABA A 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 GABA A receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant α 1β 2γ 2S GABA A receptors to flumazenil (1 or 5 μM) in the presence of GABA (1 μM) enhanced the maximum number (B max) of [ 3H]flunitrazepam binding sites without affecting their affinity (K d). The flumazenil-induced enhancement in B max was not counteracted by diazepam (1 μM). 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 (EC 50) or efficacy (E max) 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 IC 50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant α 1β 2γ 2S GABA A 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.

Synergy between retigabine and GABA in modulating the convulsant site of the GABA A receptor complex

The molecular mechanism underlying the activity of the novel antiepileptic drug retigabine is not yet fully understood. The aim of this study was to investigate whether retigabine interacts directly with the GABA A receptor complex (γ-aminobutyric acid). Receptor-binding assays were conducted using rat brain membranes. [ 3H]- t-Butyl-bicyclo-orthobenzoate ([ 3H]TBOB) was used as a tracer ligand. We determined the effects of GABA and retigabine in the presence of several concentrations of GABA on the binding of [ 3H]TBOB. GABA inhibited [ 3H]TBOB binding with an EC 50 of 4.8 μM. In the absence of GABA, retigabine inhibited [ 3H]TBOB with an EC 50 of 124 μM and an EC 50 of 42 μM in the presence of 2.5 μM GABA. Isobolic analysis revealed that retigabine acts in synergy with GABA in displacing [ 3H]TBOB. This synergy could be quantified by a molecular model in which GABA and retigabine both allosterically displace [ 3H]TBOB, and retigabine allosterically enhances the binding of GABA and vice versa with a factor of 4. In summary, we found that retigabine does indeed interact with a site on the GABA A receptor complex, and this site is positively allosterically coupled with the GABA site. This GABA-positive effect may well contribute to the clinical anticonvulsive effects of retigabine.

Influence of oxygenated fuel additives and their metabolites on the binding of a convulsant ligand of the γ-aminobutyric acid A (GABA A) receptor in rat brain membrane preparations

As a foundation for evaluating potential mechanisms of the neurological effects (e.g. headache, nausea, dizziness) of some octane boosters, we studied the γ-aminobutyric acid A (GABA A) receptor in a series of binding assays in membranes from rat brain. The GABA A receptor was probed using the radioligand [ 3H] t-butylbicycloorthobenzoate ([ 3H]TBOB) which binds to the convulsant recognition site of the receptor. The results demonstrated that the short-chain t-ethers and their t-alcohol metabolites inhibit binding at the convulsant site of the GABA A receptor. The potency of the inhibition tended to correlate with carbon chain length. For agents having an equal number of carbon atoms, potency of inhibition of [ 3H]TBOB binding was greater in magnitude for the alcohols than for the ethers. The descending rank order of potency for the ethers and alcohols were as follows, t-amyl alcohol (TAA); t-amyl-methyl ether (TAME); ethyl- t-butyl ether (ETBE)> t-butyl alcohol (TBA)>methyl- t-butyl ether (MTBE)>ethanol. In additional saturation binding assays, MTBE reduced apparent density of convulsant binding ( B max).

Formula omitted]t-butylbicycloorthobenzoate binding to recombinant α 1β 2γ 2s GABA A receptor

The interaction of several selected compounds with the binding of the cage convulsant t- [ 3 H] butylbicycloorthobenzoate ( [ 3 H] TBOB) to membranes isolated from human embryonic kidney (HEK) 293 cells stably transfected with α 1β 2γ 2s subtype of GABA A receptors was studied. Scatchard analysis of binding data revealed the existence of a single type of binding site for [ 3 H] TBOB with a K d of 47.06±4.06 nM and a B max value of 6.72±0.52 pmol/mg protein. GABA, thiopental, TBOB, picrotoxin and the neurosteroid dehydroepiandrosterone sulfate displaced concentration—dependently the binding of [ 3 H] TBOB to this recombinant receptor. Dehydroepiandrosterone sulfate reversed the 5 μM GABA-induced inhibition of specific [ 3 H] TBOB binding. It is concluded that membranes isolated from HEK 293 cells stably transfected with α 1β 2γ 2s subunits exhibit specific high-affinity [ 3 H] TBOB binding. The potency of drugs to inhibit [ 3 H] TBOB binding mainly corresponded to that observed for the inhibition of the binding of cage convulsants to the native receptors or to transiently transfected HEK 293 cells.

Diazepam biphasically modulates [3H]TBOB binding to the convulsant site of the GABAA receptor complex.

Interactions of GABA, bicuculline methochloride and diazepam with [3H]TBOB binding to rat brain membranes were evaluated in vitro. GABA displaced [3H]TBOB binding with and IC50 of 4 microM and a slope factor near unity. The competitive GABA antagonist bicuculline methochloride shifted the displacement curve of GABA parallelly to the right, indicating that the interaction of GABA with [3H]TBOB binding is of an allosteric nature. In the presence of GABA, diazepam displaced the binding of [3H]TBOB according to a two-site model: a high affinity site with an IC50 of about 50 nM and a lower affinity site with an IC50 of about 30 microM. Bicuculline methochloride abolished the nanomolar displacement by diazepam and increased the micromolar IC50 value. These results indicate that the interaction of the high affinity diazepam site with the [3H]TBOB binding site is totally GABA dependent and that the low affinity effect of diazepam on [3H]TBOB binding is at least partially GABA dependent. It is likely that the low affinity potency of diazepam to displace [3H]TBOB binding has physiological relevance.

Zinc Inhibition of t‐[3H]Butylbicycloorthobenzoate Binding to the GABAA Receptor Complex

The effect of Zn2+ on t-[3H]butylbicycloorthobenzoate ([3H]TBOB) binding to the GABAA receptor complex was studied autoradiographically in rat brain. Zn2+ inhibited [3H]TBOB binding in a dose-dependent manner at physiological concentrations. Saturation analysis revealed noncompetitive inhibition in various brain regions. The inhibitory effect of Zn2+ had regional heterogeneity; regions showing the greatest inhibition of [3H]TBOB binding were cortical laminae I-III, most areas of hippocampus, striatum, septum, and cerebellar cortex. Regions with relatively less inhibition of [3H]TBOB binding included cortical laminae V-VI, thalamus, superior colliculus, inferior colliculus, and central gray matter. The effect of Zn2+ and those of other GABAA ligands, such as benzodiazepines, bicuculline, isoguvacine, and picrotoxin, on [3H]TBOB binding seemed to be additive. Ni2+, Cd2+, and Cu2+ also inhibited [3H]TBOB binding with a regional heterogeneity similar to that produced by Zn2+. These results are consistent with Zn2+ acting at the previously detected recognition site on the GABAA receptor complex, distinct from the picrotoxin, GABA, and benzodiazepine sites. The regional heterogeneity of the Zn2+ effect may reflect differential regional distribution of GABAA receptor subtypes among brain regions. Other divalent cations probably act at the Zn2+ binding site.

Photoactivatable 2-(4'-azidotetrafluorophenyl)-5-tert-butyl-1,3-dithiane-bis- sulfone and related compounds as candidate irreversible probes for the GABA-gated chloride channels.

Syntheses of 2-substituted photoactivatable derivatives of 5-tert-butyl-1,3-dithiane and their oxidized bis-sulfone derivatives are described with the aim of developing original photoaffinity probes for the GABA-gated chloride channel. Diazocyclohexadienone as well as fluorinated arylazido derivatives were chosen as photosensitive moieties. The dithiane molecules were synthesized by condensation of 2-tert-butylpropane-1,3-dithiol with the appropriate substituted benzaldehydes. The diazocyclohexadienonyl derivatives were synthesized either by diazotization of the corresponding dithiane arylamine precursors (compounds 1, 2, 17, and 18) or by diazotization of the bis-sulfone dithiane arylamine (compounds 19 and 20). The reversible binding properties of the photosensitive probes were established on bovine cortex P2 membranes by displacement of [3H]-tert-butylbicycloorthobenzoate. While the bromo-substituted diazocyclohexadienoyl dithiane derivatives (compounds 1 and 2) exhibited a Ki of about 2-4 microM 2-(4'-azidotetrafluorophenyl)-5-tert-butyl-1,3-dithiane-bis-sulfon e (compound 3) gave a Ki of 0.2 microM. On irradiation, probe 3 produced a 25% irreversible loss of TBOB binding sites in brain membranes. Moreover, this loss was fully protectable by TBOB, demonstrating the specificity of the photochemical inactivation by compound 3 for the convulsant site of the GABAA receptor.

Spirosultam LY219048: A new chemical class of insecticide acting upon the GABA receptor/chloride ionophore complex

AbstractThis study assessed the toxicity and mode of action of a new experimental insecticide, LY219048 in insects and mammals. LY219048 produced rapid convulsions in mice and had LD50 values of 0.7 mg kg−1 and 4 mg kg−1 after intracerebral and intraperitoneal injection, respectively. In initial screens against insects, LY219048 showed low activity against the German cockroach (Blatella germanica L.). Lethality from dietary exposure required one to two weeks, even at concentrations as high as 10000 mg kg−1 (LC50 = 485 mg kg−1). In contrast, it had an LC50 value of 8.3 mg kg−1 against insecticide‐susceptible Drosophila melanogaster (Meig.) when synergized with piperonyl butoxide. Significant resistance to LY219048 (&gt; 12‐fold) was detected in a cyclodiene‐resistant strain of D. melanogaster possessing an altered target site resistance mechanism. This finding suggested that LY219048 blocked the 4‐aminobutyric acid (GABA)‐gated chloride channel in a manner similar to that of the cyclodienes. In physiological studies in larval D. melanogaster central neurons, LY219048 antagonized the reduction of firing caused by 1 mM GABA. Dose‐response experiments showed that the ED50 for blocking inhibition under these conditions was c. 1 μ. Studies of 36CI uptake into bovine brain synaptosomes found that LY219048 was a potent antagonist. At 10 μ it completely blocked chloride flux stimulated by 50 μM GABA. LY219048 competitively displaced [3H]TBOB binding from bovine brain membranes, with an IC50 of 42 nM, which was comparable to values determined for TBPS (35 nM) and picrotoxinin (267 nM). There was little or no displacement (&lt;25%) of [3H]flunitrazepam or [3H]muscimol binding by 10 μM LY219048. Taken together, these results provide strong evidence that this new chemical class of insecticide manifests its acute toxicity by blocking the GABA‐gated chloride channel.

The GABA A receptor complex in relation to epilepsy. Reversal of [ 3H]TBOB inhibition: a prediction of proconvulsive properties?

[ 3H]-t- Butylbicycloorthobenzoate ([ 3H]TBOB) , a convulsant, is known to label a binding site on the GABA A receptor complex. Bicuculline methochloride (bicuculline MCl), folic acid, pentazocine, naloxone, ethyl-β-carboline-3-carboxylate (βCCE) and Ro 5–4864 have (pro)convulsive properties in vivo. In the present study, we determined the extent to which these compounds modify the binding of [ 3H]TBOB in the presence of IC 50 amounts of GABA (5 μM) or diazepam (50 μM). We found that the GABA antagonist bicuculline MCl reversed the inhibitory effect of GABA on [ 3H]TBOB binding completely, as was expected. Folic acid, pentazocine and naloxone also reversed the inhibitory effect of GABA on [ 3H]TBOB binding. This finding is compatible with the view that the proconvulsive effects of these compounds can be credited to a reduction of GABAergic action at the GABA A receptor complex. We suggest that the reversal of GABA's inhibition of [ 3H]TBOB binding is a sufficient (but not a necessary) condition to predict proconvulsive (side) effects of drugs. βCCE and Ro 5–4864 modified [ 3H]TBOB binding in the presence of GABA in a biphasic fashion. A unique relation between βCCE, Ro –4864 and the GABA A complex might exist. Bicuculline MCl reversed the inhibitory effect of diazepam on [ 3H]TBOB binding only partly. βCCE did not reverse the inhibitory effect of diazepam on [ 3H]TBOB binding, neither did Ro 5–4864. The presence of a GABA-independent interaction between a low affinity benzodiazepine recognition site and the TBOB site is proposed.

Dihydrogenfated ergot compounds bind with to GABA A receptor-associated Cl − ionophore

The binding of t-[ 3H]butylbicycloorthobenzoate ([ 3H]TBOB) to crude synaptosomal membranes of the mouse brain (cerebrum minus cortex) in the presence of dihydroergotoxine, dihydroergosine, dihydroergotamine and γ-aminobutyric acid (GABA) was studied in vitro. [ 3H]TBOB binding was inhibited by all drugs used. The rank order of potency was dihydroergotoxine > GABA > dihydroergosine > dihydroergotamine. This suggests that dihydrogenated ergot compounds, especially dihydroergotoxine, possess appreciable binding activity (comparable to that of benzodiazepines and barbiturates) at the GABA A receptor-associated Cl −1 ionophore.

Gamma‐Aminobutyric Acid‐Activated Chloride Channels in Rats Selectively Bred for Differential Acute Sensitivity to Alcohol

Effects of various sedative hypnotic agents on GABA-mediated chloride flux were evaluated in whole brain membrane vesicles (microsacs) prepared from rats selectively bred for high (HAS) and low sensitivity (LAS) to an acute hypnotic dose of alcohol. The HAS rats were more sensitive to the effects of pentobarbital, phenobarbital, flunitrazepam, and ethanol on GABA-mediated chloride flux compared with the LAS rats. No differences between the lines in GABA-stimulated chloride flux were observed. Modulation of 1-[3H]-phenyl-4-butyl-2,6,7-trioxabicyclo(2.2.2)octane ([3H]-TBOB) and [3H]-diazepam binding also was measured. The lines did not differ in inhibition of [3H]-TBOB binding by pentobarbital, phenobarbital, muscimol or picrotoxin. Although the lines displayed almost identical KD and Bmax for [3H]-diazepam binding, the GABA agonist, muscimol, was a more potent stimulator of [3H]-diazepam binding in membranes prepared from HAS rats than from LAS rats. These findings are discussed in light of previous work using other selected lines.

Photoaffinity ligands for the [3H]TBOB binding site of the GABAA receptor.

Two types of 1-substituted-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octanes are examined as candidate photoaffinity ligands to irreversibly inhibit the [3H]TBOB binding site of the GABAA receptor complex present in bovine brain membranes. The 1-substituted-(4-bromo-6-diazocyclohexa -1,4-dien-3-onyl) derivative (IC50 1.1 microM) dissociates completely in the dark, but on irradiation produces over 40% photoirreversible inhibition of [3H]TBOB binding. This photoinactivation can be partially (64%) protected using a potent and completely dissociating TBOB analog. The 1-substituted-(4-azidophenyl) derivative (IC50 149 nM) also dissociates completely in the dark and at 2.2 microM gives 20% photoirreversible inhibition which is completely protectable. The corresponding 1-(4-azido-3,5-dichlorophenyl) analog and its 4-(1-methylpropyl) isomer are 16- and 8-fold more potent inhibitors, respectively, than the azidophenyl derivative but these dichloro probes are not examined further because they do not dissociate in the dark by an apparently non-covalent mechanism. Thus, the bromodiazocyclohexadienonyl and azidophenyl compounds photoirreversibly and therefore covalently modify the [3H]TBOB binding site of the GABAA receptor complex.