TBPS Research Articles

Effects of 1-amino-5-bromouracil on the benzodiazepine-GABA A receptor complex

We investigated the effects of 1-amino-5-bromouracil on the benzodiazepine-γ-aminobutyric acid (GABA) A receptor complex to elucidate its central action. 1-Amino-5-bromouracil neither displaced nor enhanced [ 3H]muscimol, [ 35S] t-butylbi-cyclophosphorothionate (TBPS), or [ 3H]dehydroepiandrosterone sulfate binding to the rat brain synaptosomal membranes. The anesthesia induced by 1-amino-5-bromouracil was potentiated by diazepam, pentobarbital, and muscimol, and was antagonized by picrotoxin but not by bicuculline. 1-Amino-5-bromouracil protected mice from picrotoxin-induced seizure and slightly ameliorated TBPS-induced seizure, but did not antagonize bicuculline-induced seizure. Diazepam antagonized both the bicuculline- and the picrotoxin-induced seizure, and pentobarbital antagonized the picrotoxin- and the TBPS-induced seizure. Our in vivo studies suggest that part of the central action of 1-amino-5-bromouracil is concerned with the benzodiazepine-GABA A receptor complex including the chloride channel.

Actions of agonists and convulsant antagonists on a Drosophila melanogaster GABA receptor (Rdl) homo-oligomer expressed in Xenopus oocytes

Cytoplasmic injection of Xenopus oocytes with cDNA-derived RNA (cRNA) encoding a wild-type Drosophila melanogaster GABA receptor subunit (Rdl) resulted in functional expression of a GABA receptor homo-oligomer. Membrane currents recorded in response to muscimol, trans (TACA)- and cis (CACA)-4-aminocrotonic acid reversed at membrane potentials close to E Cl− and were insensitive to 1.0 × 10 −4 M bicuculline methiodide. An order of potency of GABA ∼ muscimol ∼ TACA > CACA > glycine was observed. Response of the expressed Drosophila Rdl receptor to GABA was reduced by t-butylbicyclophosphorothionate (TBPS), 4- n-propyl-4′-ethynylbicycloorthobenzoate (EBOB), picrotoxinin and the novel insecticide, fipronil (all at 1.0 × 10 −5 M).

The protective effects of stress control may be mediated by increased brain levels of benzodiazepine receptor agonists

Control over stress protects against many of the deleterious effects of stress exposure, but the endogenous mediators responsible for these prophylactic effects have remained elusive. Using behavioral pharmacology, in vitro radioligand binding and neurochemical analyses, we demonstrate that exposure to escapable stress results in brain and behavior changes reminiscent of benzodiazepine administration. The stress control group shows significant protection against picrotoxinin-induced seizures, reductions in [ 35 S]t- butylbicyclophosphorothionate (TBPS) binding and a 3-fold increase of benzodiazepine-like substances in brain in comparison to both yoked-inescapable shock and non-shock controls. These observations suggest that coping behavior leads to the release of endogenous benzodiazepine-like compounds in brain which protect the organism from stress pathology.

Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABAA receptor subtypes

1. [35S]t-butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABA(A) receptors. Here we examined TBPS binding to the cloned receptors made of alpha 1, alpha 3 or alpha 6 in combination with beta 2 or beta 2 and gamma 2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7-dimethoxy-4-methyl-beta-carboline-3-carboxylate (DMCM), 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (5 alpha-THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF-9 insect cells by infecting with recombinant baculoviruses. 2. In alpha beta subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the alpha isoform (alpha 3 beta 2 >> alpha 1 beta 2). In alpha beta gamma subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of gamma 2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (alpha 6 beta 2) to 1 microM (alpha 6 beta 2 gamma 2). 3. Allosteric ligands also altered TBPS binding to sensitive GABA(A) receptor subtypes. For instance,diazepam only in the alpha 1 beta2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes, and 5 alpha-THDOC in all the subtypes enhanced TBPS binding in the absence of GABA, and intensified the inhibitory effect of GABA. Pentobarbitone exhibited only the latter effect in all the subtypes we examined.4. DMCM and Zn, inhibitors of GABA-induced Cl currents in alpha beta gamma and alpha beta subtypes, respectively,produced opposite effects to agonists, decreasing TBPS binding in the absence of GABA and attenuating(or eliminating in the case of Zn) the inhibitory effect of GABA on TBPS binding.5. These results show that GABA binding sites and their conformational coupling with TBPS sites are differentially affected by the alpha isoform (particularly alpha 6 as compared to alpha l or alpha 3) and by quaternary interactions involving the gamma 2 subunit. Moreover, changes in TBPS binding by allosteric ligands include not only direct (allosteric) effects on TBPS sites but also indirect effects via GABA sites, and are consistent with their known subtype selectivity and functionality from previous studies.

Radioautographic Evidence that the GABAA Receptor Antagonist SR 95531 is a Substrate Inhibitor of MAO‐A in the Rat and Human Locus Coeruleus

The locus coeruleus (LC), a major noradrenergic nucleus in the brain, probably has a functional role in the regulation of anxiety states as well as vigilance, attention, learning and memory. LC neurons are under the inhibitory control of gamma-aminobutyric acid (GABA) via ionotropic GABAA receptors. However, to date, little is known of the receptor binding characteristics of these neurons. In the present investigation we therefore examined by receptor radioautography the localization of the binding sites for different components of the GABAA receptor complex in the rat and human LC. Both rat and human LC neurons have a high density of binding sites for the pyridazinyl-GABA derivative [3H]SR 95531 (gabazine, a GABAA receptor antagonist for low affinity GABA recognition sites). However, at the concentrations used, no binding sites in the LC were detectable for the benzodiazepine receptor antagonist [3H]flumazenil, the GABAA receptor agonist (for high affinity sites) [3H]muscimol or the ionophore ligand [35S]t-butyl bicyclophosphorothionate (TBPS). Unexpectedly, the pharmacological specificity of [3H]SR 95531 binding to the LC differed markedly from that to most brain regions (IC50 values for GABA and RU 5135 respectively in the LC were > 10(-2) and 10(-3) M; and, for example, in the dentate gyrus the most labelled structure after the LC, 8 x 10(-7) and 1.8 x 10(-9) M). These differences prompted the further characterization of [3H]SR 95531 binding in the LC, revealing a significant affinity for monoamine oxidase type A (MAO-A), which is highly concentrated in this nucleus. In a competition binding study, a reduction of up to 25% of the [3H]SR 95531 binding was observed with MAO-A but not MAO-B inhibitors, at concentrations which produce maximum but selective enzyme inhibition. Correspondingly, 2 h after the oral administration of supramaximal doses of the MAO-A inhibitors moclobemide and Ro 41-1049 (but not the MAO-B inhibitor lazabemide) the in vitro binding of [3H]SR 95531 was markedly reduced (by 77 and 82% of controls respectively). Moreover, enzyme radioautography with [3H]Ro 41-1049 revealed that SR 95531 has a significant affinity for MAO-A (IC50 values were 10(-5) and 4 x 10(-6) M in the LC and dentate gyrus respectively) but not for MAO-B ([3H]lazabemide binding). Altogether, these findings suggest that the high-affinity binding of [3H]SR 95531 to the LC mainly reflects its affinity for MAO-A, which questions its utility as a selective ligand for low-affinity GABA recognition sites in the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Long-term treatment with abecarnil fails to induce tolerance in mice

The effects of long-term treatment (3 times a day for 4 weeks) with a pharmacologically active dose (0.1 mg/kg i.p.) of the novel anxiolytic, abecarnil, on exploratory behaviour and [ 35S]TBPS ( t-butylbicyclophosphorothionate) binding were compared to those of diazepam (1 mg/kg i.p.) in mice. A challenge dose (0.1 mg/kg) of abrecarnil given 12 h after the last administration of the treatment protocol markedly inhibited exploratory behaviour in animals treated chronically with abecarnil (−62%) or vehicle (−87%). Consistent with this behavioural effect, the same challenge dose of abecarnil significantly reduced [ 35S]TBPS binding to unwashed cerebral cortical membranes from mice treated chronically with abecarnil (−28%) or vehicle (−30%). In contrast, a challenge dose (1 mg/kg) of diazepam failed to affect motor behaviour and [ 35S]TBPS binding in mice chronically exposed to diazepam; in animals chronically treated with vehicle, diazepam markedly inhibited both exploratory behaviour (−55%) and [ 35S]TBPS binding (−21%). These results indicate that long-term treatment with abecarnil failed to induce tolerance to the effect of this drug on γ-aminobutyric acid type A (GABA A) receptor function. Accordingly, [ 35S]TBPS binding was increased (+15–26%) 12 and 48 h after discontinuation of long-term diazepam administration while no such increase in [ 35S]TBPS binding was observed for mice chronically treated with abecarnil. Moreover, whereas a significant decrease (−15%) in [ 35S]TBPS binding was observed 96 h after discontinuation of long-term diazepam treatment, chronic treatment with abecarnil did not modify this parameter. Together, these data indicate that long-term treatment with a pharmacologically effective dose of abecarnil did not induce tolerance or the discontinuation syndrome in mice.

Sesquilignan Haedoxans: Interaction with the GABAAReceptor in Rat Brain

(+)-Haedoxan A, an insecticidal sesquilignan, inhibited the specific binding of the noncompetitive GABA antagonist [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes in concentration-dependent and noncompetitive manners. The data show haedoxan’s interaction with a site coupled to the TBPS-binding site on the GABAA receptor in rat brain, while the physiological significance of the interaction remains to be discovered.

Alterations in t-Butylbicyclophosphorothionate binding in the brains of lidocaine-kindled rats

This autoradiographic study examines regional GABAA receptors in lidocaine-kindled rats. [35S]t-Butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel, was used to radiolabel GABAA complexes. Male Sprague-Dawley rats were injected daily with lidocaine (65mg/kg, i.p.). Seizure activity was evaluated using the Racine Scale (Racine, 1972). The animals displayed a gradual increase in the indices and by day 20 greater than 50% were in stage 4 or 5. Regression of behavior was seen in half of the experimental group and this subgroup was considered 'compensated'. Autoradiographs were analyzed using a computer-based image analysis system. Several regions within the kindled group display a decrease in TBPS binding, including the subiculum, posterior lateral thalamic nuclei, the lateral hippocampus CA1, and the lateral hippocampus CA3. Conversely, within the compensated group these regions display normal or heightened TBPS binding. The data support the theory that alterations in the GABAA receptors are involved in the kindling model of epilepsy.

Decreased forebrain [ 35S]TBPS binding and increased [ 3H]muscimol binding in rats that do not develop stress-induced behavioral depression

Recent evidence suggests that anxiety and its biological concomitants may be involved in the pathophysiology of depression. In the present study, the in vitro radioligand binding of [ 3H]flunitrazepam, [ 3H]muscimol and[ 35S]t-butylbicyclophosphorothionate (TBPS) sites on the benzodiazepine /GABA chloride ionophore receptor complex (BGRC) was examined using the learned helplessness paradigm. Only rats which did not develop the syndrome showed a significant increase in [ 3H]muscimol binding in cerebral cortex and a decrease in [ 35S]TBPS binding in cerebral cortex and hippocampus in comparison to naive controls. For both ligands, this represented a change inB max rather than a change in affinity. Adrenalectomy had no impact on these alterations indicating that critical endogenous factors are not manufactured by the adrenal glands. These findings suggest that the BGRC in the forebrain may be a site mediating the ‘coping’ ability of rats that do not develop the learned helplessness syndrome. The possible involvement of neurosteroids in the effect is discussed.

Swim stress selectively alters the specific binding of a benzodiazepine antagonist in mice

The ability of flurazepam to antagonize the electrical precipitation of tonic hindlimb extension is reduced 24 h after mice are forced to swim for 10 min in cold water (6°C). Presumably, this reduction in flurazepam's antiseizure efficacy reflects an environmental stress-induced modification of the GABA A receptor complex. The current study employed a variety of complementary in vitro approaches to characterize the delayed effects of cold-water swim stress on binding parameters of the GABA A receptor complex that may be associated with flurazepam's reduced antiseizure efficacy. The specific binding of [ 3H]flunitrazepam and the potentiation of this binding by chloride ions did not change after stress in the cerebral cortex, hippocampus, and cerebellum. Moreover, swim stress did not alter the ability of GABA to inhibit the binding of [ 35 S]t- butylbicyclophosphorothionate (TBPS), a ligand that is a specific biochemical marker of the GABA-associated chloride ionophore, to crude membranes prepared from the cerebral cortex and cerebellum. Swim stress was associated with alterations of the specific binding of [ 3H]Ro 15-1788, a benzodiazepine receptor antagonist, to crude hippocampal and cerebellar membranes. The results are considered in the context of new insights derived from molecular cloning studies of the GABA A receptor complex.

Stereospecific actions of the inhalation anesthetic isoflurane at the GABA A receptor complex

The inhalation anesthetic isoflurane stereoselectively modulates ligand binding to the GABAA receptor complex. The (+)-isomer of isoflurane was more potent and efficacious than the (-)-isomer in enhancing [3H]flunitrazepam binding to benzodiazepine receptors. For example, concentration effect curves for Cl- enhancement of [3H]flunitrazepam binding were significantly different (P < 0.001) in the presence of (+)- and (-)-isoflurane (0.44 and 0.88 mM). At the higher anesthetic concentration, they potency of Cl- to increase [3H]flunitrazepam binding was increased 3.2- and 1.45-fold by (+)- and (-)-isoflurane, respectively (P < 0.05). Likewise, concentration-effect curves for (+) isoflurane-enhanced [3H]flunitrazepam binding were significantly different (P < 0.05-P < 0.001) from the (-)-isomer in the presence of 0-200 mM Cl-. Stereoselectivity was not observed with respect to the potencies of these enantiomers as inhibitors of [35S]t-butylbicyclophosphorothionate (TBPS) binding to sites within the Cl- ionophore. In this measure, the isomers had similar potencies (P > 0.05), although at higher concentrations (> 0.1 mM) (+)-isoflurane produced significantly more inhibition than (-)-isoflurane. While the absolute potency differences between isomers were modest (< or = 2-fold) and measure dependent, these effects were manifested at clinically relevant concentrations of isoflurane and are consistent with in vivo studies demonstrating (+)-isoflurane is a more effective anesthetic than the (-)-isomer. This is the first demonstration of an inhalation anesthetic producing a stereoselective perturbation of the GABAA receptor complex.

Region-specific changes of GABA A receptors by tolerance to and dependence upon pentobarbital

An experimental model for inducing tolerance to and dependence upon pentobarbital was characterized. Rats were infused with pentobarbital, 300 μg/10 μ1 per h i.c.v. for 6 or 7 days, through pre-implanted cannulas by osmotic minipumps. Measurement of brain and serum levels showed that pentobarbital remained mainly inside of the brain. Measurements of sleeping time and susceptibility to convulsant-induced seizures indicated a substantial degree of tolerance and dependence (24 h after termination of infusion). Results of GABA A receptor binding assays showed marked regional variations. While [ 35S]t-butylbicyclophosphorothionate (TBPS) binding sites were increased in frontal cortices and striata of pentobarbital-dependent animals, K D was increased in striata of tolerant animals. Dependence upon pentobarbital was correlated with increased [ 3H]flunitrazepam binding sites in all three regions examined. Both high and low affinity [ 3H]muscimol binding sites were increased in dependent animals, but low affinity sites were decreased in frontal cortices of tolerant animals. K Ds of [ 3H]muscimol high affinity sites were increased in cerebellum after animals developed dependence upon pentobarbital. K Ds of [ 3H]muscimol low affinity sites were decreased in striata of pentobarbital tolerant animals. These findings further support the hypothesis that GABA A receptors in discrete areas of the brain have different subunit compositions and are regulated differentially by pharmacological modulators.

Interaction of La 3+ with GABA A receptors in rat cerebrocortical membranes as detected with [ 35S]t-butylbicyclophosphorothionate binding

Lanthanides at micromolar concentrations stimulated binding of [ 35S]t-butylbicyclophosphorothionate (TBPS), a highly sensitive marker for allosteric modulation of the chloride channel of GABA A receptors, to purified rat cerebrocortical synaptosomal membranes in the absence of GABA. This trivalent cation effect appeared to reflect a specific and direct interaction with GABA A receptors, and could not be mimicked by divalent metal ions including Ca 2+, Cd 2+, Co 2+, Sr 2+, Mg 2+ and Mn 2+. On the other hand, Zn 2+, a known allosteric regulator of GABA A receptors, inhibited TBPS binding, but its presence could not prevent stimulation of TBPS binding by La 3+. We further examined the lanthanide binding site by monitoring La 3+-induced changes in TBPS binding. La 3+ reduced the K d for TBPS without affecting B max. Stimulation of TBPS by La 3+ was not additive with the increased TBPS binding induced by other agonists, including GABA (at nanomolar concentrations), neurosteroids or benzodiazepines (in the absence of GABA). GABA at micromolar concentrations inhibited TBPS binding and its inhibitory effect was potentiated by neurosteroids and benzodiazepines, but not by La 3+. Thus, GABA, in the presence of La 3+, inhibited TBPS binding in a monophasic manner with a Hill coefficient of approximately 2 and an IC 50 of 6 μM. In the absence of La 3+, the dose-response profile for GABA became biphasic with an apperance of a stimulatory phase at concentrations of GABA lower than 1 μM. Analysis of the initial stimulation yielded a Hill coefficient of 1 and an EC 50 of 88 nM. These data are consistent with the stimulation of TBPS binding by GABA resulting from the occupation of its high affinity site and its inhibition of TBPS from binding occupation of a low affinity site. It appears that GABA binding at the high affinity site induced the conformational change(s) at the TBPS binding site (which is presumably located near the mouth of the chloride channel as the picrotoxin binding site), which is qualitatively similar to the conformational change induced by La 3+, neurosteroids or benzodiazepines (in the absence of GABA). These similar and nonadditive effects on TBS binding may reflect changes on the multimeric GABA A receptors prior to opening of the chloride channels. Finally, the lanthanide site on GABA A receptors examined here with La 3+ appears to be different from the Zn 2+ site, and from the previously reported monovalent and divalent cation recognition sites (Squires and Saederup, 1982, Mol. Pharmacol. 22, 327).

GABA A receptor function in the γ-hydroxybutyrate model of generalized absence seizures

γ-Hydroxybutyric acid (GHB) produces absence-like seizures when given to animals. One of the distinguishing characteristics of experimental generalized absence seizures is that they are exacerbated by GABA A agonists. Therefore, the hypothesis that GHB-induced absence seizures result from an interaction between GHB and the GABA A receptor complex was tested. The effect of GHB on the function of various components of the GABA A receptor complex in the cortex of the rat, was determined in a series of in vitro experiments. Similar studies were carried out at various times following systemic administration of the prodrug of GHB, γ-butyrolactone (GBL) and changes in the GABA A receptor were correlated with electrographic and behavioral changes. γ-Hydroxybutyric acid had no effect on the binding of [ 3H]muscimol, [ 3H]flunitrazepam and [ 35S] t-butyl-bicyclophosphorothionate (TBPS) or on the uptake of 36Cl − into synaptoneurosomes in the in vitro studies. Nor were changes observed after the administration of GBL before the onesetof GHB-induced absence seizures. However, at the onset of GHB-induced spike wave discharge, there was a significant ( P < 0.04) decrease in the binding of [ 35S]TBPS, associated with a significant decrease in muscimol-stimulated uptake of 36Cl − with no other biochemical change. One minute after onset of GHB-induced absence seizure, a significant ( P < 0.05) increase in the binding of [ 3H]muscimol was noted. Ten minutes later the decrease in muscimol-stimulated uptake of 36Cl − had normalized, while the changes in binding of [ 3H]muscimol and [ 35S]TBPS persisted. Because GABA A function remained unchanged in the in vitro studies, as well as prior to the onset of GHB-induced absence seizures in the in vivo experiments, these studies do not support the hypothesis that GHB interacts directly with the GABA A receptor complex to produce absence-like seizures.

Relative anticonvulsant effects of GABAmimetic and GABA modulatory agents.

Anticonvulsant properties of compounds that enhance GABA-mediated inhibition through modulatory sites on the GABAA receptor [phenobarbital (PB), clonazepam (CZP), alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL)] were compared with anticonvulsant effects of compounds believed to be antagonists at these modulatory sites (Ro15-1788 and alpha-isopropyl-alpha-methyl-gamma-butyrolactone gamma-IMGBL)] and to 4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol (THIP, GABAA receptor agonist), (+/-) baclofen (GABAB receptor agonist), and gamma-vinyl GABA, a compound that increases endogenous GABA. The compounds were tested for their ability to block experimental seizures caused by maximal electroshock, pentylenetetrazol, picrotoxin, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), bicuculline (BIC), aminophylline, strychnine, and t-butyl-bicyclophosphorothionate (TBPS) in mice. CZP blocked all but strychnine seizures. PB was also highly effective, blocking all but TBPS seizures. alpha-EMTBL, representing a new class of experimental anticonvulsant drugs, prevented all seizures except strychnine (STR)- and aminophylline-induced seizures. The antagonists are effective only against one convulsant stimulus. Ro15-1788 and alpha-IMGBL prevented only DMCM- and pentylenetetrazol (PTZ)-induced seizures, respectively. THIP and gamma-vinyl GABA both blocked only BIC and picrotoxin seizures. Baclofen had no anticonvulsant activity. These data demonstrate that compounds that increase neuronal inhibition by potentiating the action of GABA have a broader spectrum of anticonvulsant action than either antagonists or GABAmimetic agents or compounds that increase endogenous GABA.

Sex differences in sensitivity to pentylenetetrazol but not in GABAA receptor binding

Female rats have a higher threshold than males for seizures induced by the convulsant pentylenetetrazol, a GABAA receptor-chloride channel complex blocker. No sex difference was observed for the anticonvulsant activities of ethanol or diazepam to protect against pentylenetetrazol seizures. Ovariectomy reduces the pentylenetetrazol seizure threshold of females to that of males. In contrast, females have a lower threshold than males to electroshock seizures. Pentylenetetrazol receptors were compared in males and females and gonadectomized animals by binding of several radioligands to the GABAA receptor complex. No differences were found for these four groups of animals in the binding of [3H]flunitrazepam to the benzodiazepine sites and [35S]t-butyl bicyclophosphorothionate (35S]TBPS) to the cloride channel/convulsant sites in membrane homogenates, nor for allosteric modulation of binding by GABA, the steroid anesthetic alphaxalone, or the benzodiazepine Ro 5-4864. In tissue section autoradiography, no difference was observed for these same assays nor for the binding of [3H]muscimol in the presence and absence of alphaxalone in several major regions. We conclude that circulating female sex hormones, possibly neurosteroid metabolites of progesterone, known to interact directly with the GABAA receptor complex, are involved in the sex differences in pentylenetetrazol seizure susceptibility.

The GABA A receptor complex in experimental absence seizures in rat: An autoradiographic study

The regional distribution of radioactive ligand binding for different receptors of the γ-aminobutyric acid A (GABA A)-benzodiazepine-picrotoxin chloride channel complex was measured on tissue section by autoradiography in brains taken from a genetic strain of Wistar rats with spontaneous absence-like seizures, the genetic absence epilepsy rats from Strasbourg (GAERS), and a control colony. The ligands employed included [ 3H]muscimol for high affinity GABA agonists sites; [ 3H]SR 95531 for the low-affinity GABA sites; [ 3H]flunitrazepam for the benzodiazepine sites; and [ 35 S]t- butyl bicyclophosphorothionate (TBPS) for the picrotoxin site. There was no significant change between GAERS and control animals in [ 3H]flunitrazepam and [ 35S]TBPS binding. However, there was significantly decreased [ 3H]muscimol and [ 3H]SR 95531 binding in the CA2 region of the hippocampus of the GAERS. This was due to a decrease in B max of both [ 3H]muscimol and [ 3H]SR 95531 binding in the epileptic strain.

Aging related alterations in GABA A receptor subunit mRNA levels in Fischer rats

The influence of aging on the binding of ligands to picrotoxin binding sites as well as steady state levels of mRNA for various α subunits of γ-aminobutyric acid (GABA) receptor complex was investigated in male Fischer F-344 rats. In aged rats, the binding of [ 35 S]t- butyl-bicyclophosphorothionate (TBPS) was significantly reduced. This decrease in TBPS binding derived from a reduced density of binding sites, rather than from affinity changes, in both cerebral cortex and cerebellum. In aged rats, α 1 mRNA level decreased approximately 70% between age 6 months and 24 months in the cerebral cortex ( P < 0.005). In contrast, α 1 mRNA remained unchanged in the cerebellum of old rats. The association of a decrease in picrotoxin binding sites in the cerebral cortex with a decline in α 1 mRNA level in the cerebral cortex and in α 2 mRNA level in the cerebellum is indicated. α 6 mRNa level increased with age in the cerebellum. These findings indicate a selective age related modulation in the stoichiometry of GABA A receptor in aging.

Carbon dioxide inhalation reduces the function of GABA A receptors in the rat brain

The effect of CO 2 inhalation on the function of the GABA A-coupled chloride channel was evaluated in rat brain. This treatment decreased the capability of GABA to stimulate 36Cl − uptake and produced a significant increase of [ 35S]-TBPS ([ 35S]t-butylbicyclophosphorothionate) binding in the cerebral cortex, cerebellum and hippocampus. These results demonstrate that a brief exposure of rats to CO 2 inhalation reduces the function ofthe GABA A-ionophore receptor complex in rat brain.

Characterization of the enantiomeric 3-substituted-1,4-benzodiazepin-2-ones binding to the GABA—BDZ—chloride ionophore receptor complex

Gamma aminobutyric acid (GABA) is an important and abundant inhibitory neurotransmitter in the mammalian central nervous system (CNS). Its interaction with the GABAA receptor subtypes opens anion channels in the membrane through which chloride ions pass down their electrochemical gradient, thus stabilizing the resting level of membrane potential. GABA* receptors, associated with the anion channel, are part of a protein complex containing distinct but interacting recognition sites for convulsants [such as picrotoxin and t-butylbicyclo-phosphorothionate (TBPS)] and depressants [such as benzodiazepines (BDZs) and barbiturates] [ 11. Therefore, the macromolecular make-up and interactions of the BDZ binding sites with the GABA receptor and chloride channel have to be accounted for in models of BDZ receptor function [2-41. Several papers report the structure activity relationships as well as the influence of the stereochemistry of BDZs on their in vitro binding to the CNS receptor [5-111. However, to the best of our knowledge, nothing is known about BDZ receptor function depending on the enantioselective binding of chiral BDZs to their receptor. In the present paper the interaction to the CNS receptor of a series of chiral3-substituted1,4-benzodiazepin-2-ones, resolved as pure enantiomers by preparative chiral HPLC, was studied. The study of the interaction of the BDZ receptor with the GABA receptor and chloride channel was utilized to profile the in vitro differences between enantiomeric BDZs as agonists, antagonists, and inverse agonists. Quantifiable differences in the coupling of the BDZ receptors to GABA receptors can be determined by measuring the GABA-shift in the affinities of BDZs to displace 3H-flunitrazepam [2, 11-131. Thus, the influence of the stereochemistry on the pharmacological profile of the BDZ receptor ligands has been investigated as GABA-ratio determination. Further, the study of possible interaction between BDZ receptor and the chloride channel has been performed by the use of 35S-TBPS (a selective label of chloride anion recognition site). Thus the effect exerted by chiral BDZs on the chloride channel regulation was evaluated by the modulation of 35S-TBPS binding.