Characterisation of GABA(A) receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone

Progesterone metabolites acting via GABA(A) receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA(A) receptors in fetal, newborn and adult sheep brains using [(35)S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity (K(D)) and density (B(MAX)) in the brainstem were not different in fetal, newborn (1-2 days old) and adult brains. In the hypothalamus K(D) and B(MAX) increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K(D) and B(MAX) increased progressively between 85 days and term ( approximately 147 days gestation), and were then not different from postnatal and adult values. The K(i) values for the GABA(A) receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [(35)S]TBPS binding in the presence of 5 microM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA(A) receptors are present in the fetal brain from at least 85 days gestation; (ii), 3alpha-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA(A) receptor binding parameters. Steroid modulation of the GABA(A) receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.     

Characterization of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain

BACKGROUND AND PURPOSE: The aim of the present study was to determine whether binding of [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) to the convulsant binding site of GABA(A) receptors in human postmortem brain samples can be used as an in vitro index of the functional activation of these receptors. EXPERIMENTAL APPROACH: Postmortem stability of [(35)S]TBPS binding was assessed in rat brain samples harvested at various times after death and the binding properties of [(35)S]TBPS binding (K(D) and B(max)) were determined in human postmortem brain using radioligand binding studies. In addition, the ability of human brain [(35)S]TBPS binding to be allosterically modulated by compounds that bind at recognition sites distinct from the convulsant binding site was measured. KEY RESULTS: Whereas binding of [(3)H]Ro 15-1788 to the benzodiazepine binding site and [(3)H]muscimol to the agonist (GABA) binding site were retained over a 20 h postmortem interval, there was a significant decrease in the affinity and number of [(35)S]TBPS binding sites. Nevertheless, [(35)S]TBPS binding in human brain could be inhibited by TBPS, picrotoxin, loreclezole and pentobarbital and modulated by GABA with potencies comparable to those observed in rats. In addition, the GABA-induced reduction in human brain [(35)S]TBPS binding could be modulated by benzodiazepine site ligands in a manner that reflected their intrinsic efficacies. CONCLUSIONS AND IMPLICATIONS: These results suggest that allosteric coupling between the [(35)S]TBPS, GABA and benzodiazepine binding sites is preserved in postmortem human brain and that [(35)S]TBPS binding in this tissue may be used to study functional characteristics of native human GABA(A) receptors.     

Dependence on gamma-aminobutyric acid of pyrethroid and 4'-chlorodiazepam modulation of the binding of t-[35S]butylbicyclophosphorothionate in piscine brain

Binding sites for t-[35S]butylbicyclophosphorothionate ([35S]TBPS) were detected in well-washed membranes from the brain of trout; gamma-aminobutyric acid (GABA) acted as an uncompetitive inhibitor of the binding of [35S]TBPS, decreasing both the number of binding sites and the affinity of TBPS. Inhibition of the binding of [35S]TBPS by deltamethrin, a Type II pyrethroid, was modulated by GABA; both the affinity and the efficacy of this insecticide increased with incremental concentrations of GABA. Deltamethrin also enhanced the potency of GABA as an inhibitor of the binding of [35S]TBPS. The interaction of 4'-chlorodiazepam (Ro5-4864) with [35S]TBPS was dependent on GABA: in the absence of GABA, Ro5-4864 inhibited up to 40% of the binding; in the presence of 10 microM GABA, Ro5-4864 enhanced binding to a maximum value of 170% of control. However, the same absolute amount of binding was observed with both of these effects at micromolar concentrations of Ro5-4864. Also, Ro5-4864 caused a rightward shift in GABA dose-response curves, increasing the IC50 value for GABA more than 6 fold. These results indicate the reciprocal allosteric interactions between a binding site for pyrethroids, a binding site for Ro5-4864, the GABA recognition moiety and the binding site for TBPS in the brain of trout. The similarity of these findings to previous results in preparations of rodent brain highlight the conservation of the modulation of GABAA receptor function during the evolution of vertebrates.     

Regulation of [35S]t-butylbicyclophosphorothionate binding sites in rat brain by GABA, pyrethroid and barbiturate

GABA regulates the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) within the GABA receptor-ionophore complex by decreasing the rate of radioligand association and increasing the rate of dissociation but in different ways for the EDTA/water-dialyzed rat brain membranes and a solubilized preparation obtained on treatment with the zwitterionic detergent CHAPS. In the membranes, GABA at 0.3-1 microM is a non-competitive inhibitor of [35S]TBPS binding, affecting the density of binding sites but not the affinity of the receptor, while at 5 microM both the apparent density and affinity are significantly decreased. On treatment with CHAPS the solubilized preparation and the corresponding pellet fraction become less sensitive to GABA which even at 5 microM acts only as a non-competitive inhibitor. CHAPS solubilization decreases the sensitivity of the receptor-[35S]TBPS complex to GABA-induced dissociation. GABA at micromolar levels also greatly influences the action of compounds within the TBPS domain, facilitating and modulating displacement of [35S]TBPS from EDTA/water-dialyzed membranes by the alpha-cyanopyrethroid cypermethrin and the barbiturate 5-(1',3'-dimethylbutyl)-5-ethylbarbiturate. Large differences in the Hill numbers indicate that different mechanisms may be involved in GABA modulation of the pyrethroid and barbiturate sites. In contrast, GABA does not effect [35S]TBPS displacement by photoheptachlor epoxide which acts directly at the TBPS binding site.     

Radioligand binding studies of caloporoside and novel congeners with contrasting effects upon [35S] TBPS binding to the mammalian GABA(A) receptor

Caloporoside is a natural active fungal metabolite, which was isolated from Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity. We have previously reported evidence that related beta-linked compounds, lactose and octyl-beta-d-mannoside, bind and functionally modulate rodent GABA(A) receptors, respectively. We have characterized the binding pharmacology of synthetic caloporoside and two further congeners, 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid and octyl-beta-d-glucoside on GABA(A) receptors using a [35S]-t-butylbicyclophosphoorothionate (TBPS) radioligand binding assay. Caloporoside and 2-hydroxy-6-([(16R)-(beta-d-mannopyranosyloxy)heptadecyl]) benzoic acid produced concentration-dependent complete inhibition of specific [35S] TBPS binding with overall apparent IC50 values of 14.7+/-0.1 and 14.2+/-0.1 microM, respectively. In contrast, octyl-beta-d-glucoside elicited a concentration-dependent stimulation of specific [35S] TBPS binding (E(max)=144+/-4%; EC50=39.2+/-22.7 nM). The level of stimulation was similar to that elicited by diazepam (E(max)=147+/-6%; EC50=0.8+/-0.1 nM), and was occluded by GABA (0.3 microM). However, the three test compounds failed to elicit any significant effect (positive or negative) upon [3H] flunitrazepam or [3H] muscimol binding, indicating that they did not bind directly, or allosterically couple, to the benzodiazepine or agonist binding site of the GABA(A) receptor, respectively. The constituent monosaccharide, glucose, and both the closely related congeners octyl-beta-d-glucoside or hexyl-beta-d-glucoside have no significant effect upon [35S] TBPS binding. These data, together, provide strong evidence that a beta-glycosidic linkage and chain length are crucial for the positive modulation of [35S] TBPS binding to the GABA(A) receptor by this novel chemical class.     

Unsulfated and sulfated neurosteroids differentially modulate the binding characteristics of various radioligands of GABA(A) receptors following chronic ethanol administration

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) inhibited the binding of [(3)H]flunitrazepam (2 nM), [(3)H]muscimol (5 nM) and 4 nM [(35)S]t-butylbicyclophosphorothionate [(35)S]TBPS in the rat cerebellum as well as cerebral cortex. DHEAS-induced inhibition of binding of these radioligands (62% to 100%) was more pronounced as compared to that in the case of DHEA (5% to 31%). DHEAS, unlike DHEA, inhibited [(3)H]flunitrazepam binding significantly to a lesser extent in the cerebellum of ethanol-dependent rats as compared to the control group (I(max):82+/-1vs.92+/-2%, p<0.005). However, DHEA, unlike DHEAS, inhibited [(35)S]TBPS binding to a greater extent in the ethanol-dependent rat cerebellum as compared to the control group (I(max):31+/-2vs.19+/-2%, p<0.005). Furthermore, DHEA was more potent in inhibiting [(35)S]TBPS binding in the cerebellum (IC(50):55+/-5 vs. 74+/-7 microM, p<0.05) and cerebral cortex (IC(50):26+/-4vs.64+/-9 microM, p<0.05) of ethanol-dependent rats as compared to the control group. These observations indicate that unsulfated and sulfated androstane-steroids modulate the GABA(A) receptors in the control as well as the ethanol-dependent rats differentially, and also suggest that the androstane-steroid binding sites associated with the GABA(A) receptors play an important role during ethanol dependence.     

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 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.     

Pyrethroid insecticides and radioligand displacement from the GABA receptor chloride ionophore complex

Radioligand binding displacement studies were conducted to determine the effects of Type I and II pyrethroids on [3H]flunitrazepam (FLU), [3H]muscimol (MUS), and [35S]t-butylbicyclophosphorothionate (TBPS) binding. Competition experiments with [3H]FLU and [3H]MUS indicate a lack of competition for binding by the pyrethroids. Type I pyrethroids failed to compete for the binding of [35S]TBPS at concentrations as high as 50 microM. Type II pyrethroids inhibited [35S]TBPS binding to rat brain synaptosomes with Ki values ranging from 5-10 microM. The data presented here suggest that the interaction of Type II pyrethroids with the gamma-aminobutyric acid (GABA) receptor-ionophore complex is restricted to a site near the TBPS/picrotoxinin binding site.     

Highly water-soluble derivatives of the anesthetic agent propofol: in vitro and in vivo evaluation of cyclic amino acid esters.

Cyclic amino acid esters of propofol were synthesized in an attempt to develop new water-soluble anesthetic agents. Their solubility and stability in aqueous solution, and their ability to release propofol in vitro under physiological conditions were determined. L-Proline (6a) and racemic nipecotic acid (6c) esters were found to be highly soluble in water. Sufficiently stable at physiological pH (half-lives >6 h), the alpha-amino acid esters, 6a and 6b, were found to be quantitatively hydrolyzed in plasma and liver esterase solutions within a few minutes, showing prodrug behavior. The in vitro activity of the esters, determined either by the [(35)S]tert-butylbicyclophosphorothionate ([(35)S]TBPS) binding assay or electrophysiological measurements of the action at cloned human receptors, proved to be a mechanism involving allosteric modulation of GABA(A) receptors. Indeed, L-proline (6a), and racemic pipecolinate (6b) and nipecotate (6c), like propofol, reduced [(35)S]TBPS binding, whereas isonipecotate (6d) showed bicuculline-like behavior, increasing [(35)S]TBPS binding. A nonlinear relation between GABA(A) receptor binding affinity and lipophilicity, as assessed by reversed-phase high-performance liquid chromatography, emerged as a trend. The in vivo anticonvulsant and anesthetic activities of prolinate 6a, intraperitoneally administered in water solution, showed that is a water-soluble propofol prodrug candidate for developing formulations useful for parenteral administration.     

Coupling between agonist and chloride ionophore sites of the GABA(A) receptor: agonist/antagonist efficacy of 4-PIOL

Eight gamma-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA(A) receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and beta-alanine on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant alpha1beta2gamma2 (widespread major subtype) and alpha6beta2gamma2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.     

Correlation between gamma-aminobutyric acidA receptor ligand-induced changes in t-butylbicyclophosphoro[35S]thionate binding and 36Cl- uptake in rat cerebrocortical membranes.

We have explored the functional significance of various drug-induced changes in t-[35S]butylbycyclophosporothionate (TBPS) binding to gamma-aminobutyric acidA (GABAA) receptors by comparing them with the actions of the drugs on GABA-induced 36Cl- uptake in rat cerebrocortical membrane preparations. In the presence of micromolar concentrations of GABA, various benzodiazepine receptor agonists, 3 alpha-21-dihydroxy-5 alpha-pregnan-20-one, and pentobarbital inhibited [35S]TBPS binding, whereas ethyl-beta-carboline-3carboxylate (beta-CCE), an inverse agonist, stimulated it, in general agreement with earlier reports [Mol. Pharmacol. 23:326-336 (1983); Mol. Pharmacol. 30:218-225 (1986)]. The drug-induced changes in [35S]TBPS binding, after normalization with respect to the corresponding action of diazepam, were closely related to the relative ability of the drugs to affect 36Cl- uptake, with a correlation coefficient of 0.98 and a slope of 0.85. Upon abolishment of GABA action by the use of bicuculline, however, all the tested drugs stimulated [35S]TBPS binding to various degrees, and their relative changes displayed a lower correlation coefficient of 0.69, with a slope of 2. In particular, the effects of the anesthetic steroid and pentobarbital on [35S]TBPS binding were markedly altered by GABA, which at 2 microM increased not only their maximal effects, but also their half-maximal concentrations severalfold. On the other hand, GABA did not significantly affect these parameters for diazepam under our experimental conditions. Also, the GABA-independent changes in [35S]TBPS binding produced by various benzodiazepine receptor agonists matched reasonably well the actions of the drugs on 36Cl- uptake, with a correlation coefficient of 0.85 and a slope of 1.0. These data suggest more pronounced functional coupling of the GABA sites with those for the steroid and the barbiturate, as compared with the benzodiazepine site. It appears that the degree of [35S]TBPS binding in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptors.     

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.     

Loreclezole and La3+ differentiate cerebellar granule cell GABA(A) receptor subtypes

The effects of loreclezole and La3+ on native cerebellar GABA(A) receptors were compared between GABA(A) receptor alpha6 subunit-deficient (alpha6-/-) and wildtype mouse lines, produced through homologous recombination, using t-[35S]butylbicyclophosphorothionate ([35S]TBPS) autoradiography in brain sections. In the alpha6 subunit-deficient mice, the GABA receptor antagonistic ability of La3+ was abolished in the cerebellar granule cell layer, consistent with its opposite actions on alpha6- and of alpha1 subunit-containing receptors. La3+ significantly potentiated the action of GABA in the molecular layer of the alpha6-/- mice, but not in that of the wildtype mice. The potentiation of agonistic GABA inhibition of [35S]TBPS binding by loreclezole in alpha6-/- granule cells was reduced, suggesting an emergence of low-affinity GABA(A) receptors. The present results thus identified two ligands that may be useful in studying functional roles of cerebellar alpha1 and alpha6 subunit-containing GABA(A) receptor subtypes.     

Comparison of the effects of zaleplon,zolpidem, and triazolam at various GABA(A) receptor subtypes

The pyrazolopyrimidine zaleplon is a hypnotic agent that acts at the benzodiazepine recognition site of GABA(A) receptors. Zaleplon, like the hypnotic agent zolpidem but unlike classical benzodiazepines, exhibits preferential affinity for type I benzodiazepine (BZ(1)/omega(1)) receptors in binding assays. The modulatory action of zaleplon at GABA(A) receptors has now been compared with those of zolpidem and the triazolobenzodiazepine triazolam. Zaleplon potentiated GABA-evoked Cl(-) currents in Xenopus oocytes expressing human GABA(A) receptor subunits with a potency that was higher at alpha1beta2gamma2 receptors than at alpha2- or alpha3-containing receptors. Zolpidem, but not triazolam, also exhibited selectivity for alpha1-containing receptors. However, the potency of zaleplon at these various receptors was one-third to one-half that of zolpidem. Zaleplon and zolpidem also differed in their actions at receptors containing the alpha5 or gamma3 subunit. Zaleplon, zolpidem, and triazolam exhibited similar patterns of efficacy among the different receptor subtypes. The affinities of zaleplon for [(3)H]flunitrazepam or t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding sites in rat brain membranes were lower than those of zolpidem or triazolam. Furthermore, zaleplon, unlike zolpidem, exhibited virtually no affinity for the peripheral type of benzodiazepine receptor.     

Barbiturate recognition site on the GABA/benzodiazepine receptor complex is distinct from the picrotoxinin/TBPS recognition site

The cage convulsant [35S]tert-butylbicyclophosphorothionate [( 35S]TBPS) labels a presumed sedative-convulsant receptor complex. The relative potencies of barbiturates in competing for [35S]TBPS binding parallels their potencies in enhancing benzodiazepine receptor binding. Barbiturates inhibit [35S]TBPS binding in a complex, mixed competitive fashion, leading to a decrease in both the apparent affinity of TBPS for its sites and the apparent number of TBPS sites. All of the barbiturates examined markedly accelerate the dissociation of [35S]TBPS from its recognition sites, while picrotoxinin does not affect the dissociation. These results suggest that the barbiturate and picrotoxinin/TBPS recognition sites are distinct but allosterically linked.     

Ontogenesis and properties of the convulsant recognition site(s) of the gamma-aminobutyric acid (GABA) receptor complex in chicken embryo

Ontogenesis of the convulsant (picrotoxinin or t-butylbicyclophosphorothionate (TBPS] site(s) was defined in chicken embryonic brain with the radioligands [35S]TBPS and [3H]t-butylbicycloorthobenzoate [( 3H]TBOB). Binding of the radioligands is detectable from day 6 of incubation. The increase in binding after day 14 of incubation is due to an increase in the number of binding sites. The pharmacological properties of the embryonic recognition site(s) do not undergo significant changes during hatching based on the affinity of embryonic and chick brain membranes for [3H]TBOB; the rate of association and dissociation for TBOB; non-competitive inhibition of [3H]TBOB binding and modulation of 1R, alpha S-cis-cypermethrin interaction with the recognition site(s) by GABA; and inhibition of radioligand binding by endrin, picrotoxinin and TBPS. Early development and in vitro susceptibility of the recognition site to convulsive toxicants makes the embryo a possible target for a variety of drugs and environmental toxicants acting at this site.     

A 17beta-derivative of allopregnanolone is a neurosteroid antagonist at a cerebellar subpopulation of GABA A receptors with nanomolar affinity

BACKGROUND AND PURPOSE: High-affinity, subtype-selective antagonists of the neurosteroid binding sites of GABA(A) receptors are not available. We have characterized an allopregnanolone derivative as an antagonist of cerebellar GABA(A) receptors with nanomolar affinity. EXPERIMENTAL APPROACH: Receptor binding and electrophysiological methods were used for the allosteric modulation of cerebellar GABA(A) receptors by an allopregnanolone derivative, (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3alpha-ol (HBAO). GABA(A) receptors of rat cerebellar membranes were labelled with the chloride channel blocker [(3)H]ethynylbicycloorthobenzoate (EBOB). The ionophore function of GABA(A) receptors was studied by whole-cell patch clamp electrophysiology in cultured rat cerebellar granule and cortical cells. KEY RESULTS: Partial displacement of cerebellar [(3)H]EBOB binding by nanomolar HBAO was attenuated by 0.1 mM furosemide, an antagonist of alpha(6) and beta(2-3) subunit-containing GABA(A) receptors. Displacement curves of HBAO were reshaped by 30 nM GABA and shifted to the right. However, the micromolar potency of full displacement by allopregnanolone was not affected by 0.1 mM furosemide or 30 nM GABA. The nanomolar, but not the micromolar phase of displacement of [(3)H]EBOB binding by GABA was attenuated by 100 nM HBAO. Submicromolar HBAO did not affect [(3)H]EBOB binding to cortical and hippocampal GABA(A) receptors. HBAO up to 1 microM did not affect chloride currents elicited by 0.3-10 microM GABA, while it abolished potentiation by 1 microM allopregnanolone with nanomolar potency in cerebellar but not in cortical cells. Furosemide attenuated cerebellar inhibition by 100 nM HBAO. CONCLUSIONS AND IMPLICATIONS: HBAO is a selective antagonist of allopregnanolone, a major endogenous positive modulator via neurosteroid sites of cerebellar (probably alpha(6)beta(2-3)delta) GABA(A) receptors.     

Functional correlation between allopregnanolone and [35S]-TBPS binding in the brain of rats exposed to isoniazid, pentylenetetrazol or stress.

1. The relation between changes in the cerebral cortical concentration of allopregnanolone and gamma-aminobutyric acid (GABA) type A receptor function after intracerebroventricular injection of this neurosteroid was investigated in male rats. 2. Intracerebroventricular administration of allopregnanolone (1.25 to 15 micrograms) produced a maximal increase (100 fold at the highest dose) in cortical allopregnanolone concentration within 5 min; the concentration remained significantly increased at 15 and 30 min, before returning to control values by 60 min. 3. The same treatment induced a rapid and dose-dependent decrease in the binding of t-[35S]-butylbicyclophosphorothionate ([35S]-TBPS) to cerebral cortical membranes measured ex vivo, an effect mimicked by the benzodiazepine midazolam but not by the 3 beta-hydroxyepimer of allopregnanolone. The time course of changes in [35S]-TBPS binding paralleled that of brain allopregnanolone concentration. 4. In a dose-dependent manner, allopregnanolone both delayed the onset of convulsions and inhibited the increase in [35S]-TBPS binding to cortical membranes induced by isoniazid. The potency of allopregnanolone in inhibiting [35S]-TBPS binding in isoniazid-treated rats was approximately four times that in control animals. 5. The ability of allopregnanolone to decrease [35S]-TBPS binding in isoniazid-treated rats also correlated with its anticonvulsant activity against pentylenetetrazol-induced seizures as well as its inhibitory effect on the increase in [35S]-TBPS binding induced by foot shock. 6. The results indicate that the in vivo administration of allopregnanolone enhances the function of GABAA receptors in rat cerebral cortex and antagonizes the inhibitory action of stress and drugs that reduce GABAergic transmission.     

Anxiolytic cyclopyrrolone drugs allosterically modulate the binding of [35S]t-butylbicyclophosphorothionate to the benzodiazepine/gamma-aminobutyric acid-A receptor/chloride anionophore complex

The influence of a number of anxiolytic cyclopyrrolone drugs, which include zopiclone and suriclone, on the binding of [35S]t-butylbicyclophosphorothionate (TBPS), to benzodiazepine/gamma-aminobutyric acid-A receptor/chloride anionophore complexes has been characterized in rat brain. Suriclone and its metabolites RP35,489 and RP46,166 are the most potent (IC50 approximately 3nM) inhibitors of [35S]TBPS binding thus far described, about an order of magnitude more potent than TBPS itself. The pattern of inhibition of [35S] TBPS binding by suriclone is distinctive; at approximately 10 nM there is approximately 50% inhibition of [35S]TBPS binding and inhibition "plateaus" at this level until suriclone concentrations exceed 1 microM. RP35,489 and RP46,166 display patterns of inhibition similar to suriclone. In saturation studies of [35S]TBPS binding, suriclone reduces the Bmax of [35S]TBPS-binding sites, with little or no effect on KD. Muscimol also displays a noncompetitive pattern of inhibition of [35S]TBPS binding, whereas inhibition by picrotoxinin appears competitive. [35S]TBPS dissociation is multiphasic and similar whether initiated by 10 microM TBPS or 10 microM picrotoxinin. By contrast, dissociation of [35S]TBPS is much faster (and nearly monophasic) when initiated by 10 microM TBPS/100 nM suriclone, 10 microM TBPS/1 microM muscimol, or 10 microM TBPS/1 mM pentobarbital. These results suggest that suriclone influences [35S]TBPS binding allosterically, at sites distinct from the TBPS/picrotoxinin recognition site. Inhibition of [35S]TBPS binding by suriclone varies regionally with a "plateau" at approximately 20% inhibition in the cerebellum, approximately 50% in the cerebral cortex, hippocampus and brain stem, and approximately 65% in the striatum and midbrain; by contrast, inhibition of [35S] TBPS by picrotoxinin, muscimol, and pentobarbital shows little regional variation. The inhibition of [35S]TBPS binding by suriclone is reversed by bicuculline [ED50 approximately 1 microM] in several brain regions examined. Bicuculline alone has little or no influence on [35S]TBPS binding in the cerebral cortex, hippocampus, and cerebellum, but produces a dose-dependent enhancement of [35S]TBPS binding in the striatum, midbrain, and hypothalamus. Regional differences in the effects of suriclone and bicuculline on [35S]TBPS recognition sites suggest possible heterogeneity in the coupling of cyclopyrrolone and bicuculline recognition sites to [35S]TBPS recognition sites in rat brain.