T-butylbicyclophosphorothionate Binding Sites Research Articles

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).

35S]t-butylbicyclophosphorothionate binding sites in susceptible and cyclodiene-resistant houseflies.

4-aminobutyric acid (GABA)-gated chloride ion channels are important molecular targets for a number of polychlorocycloalkane compounds including cyclodiene insecticides. Previous radioligand binding studies have indicated that cyclodiene insecticides are potent inhibitors of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to housefly thorax and abdomen membranes. In the present study, a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica) showed resistance to a number of cyclodiene insecticides. Specific, saturable [35S]TBPS binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of [35S]TBPS binding data from CSMA and CYW membranes revealed no significant differences between the two strains in either the affinity (Kd) or the density (Bmax) of specific, saturable binding sites. There were no differences in the comparative effectiveness of a range of polychlorocycloalkanes, including cyclodiene insecticides, as inhibitors of specific [35S]TBPS binding to CSMA and CYW thorax and abdomen membranes. Therefore, if an alteration in target site is a mechanism for resistance to cyclodienes in the CYW strain, it is not readily measurable using [35S]TBPS.

GABA A antagonists reveal binding sites for [ 35S]TBPS in cerebellar granular cell layer

Autoradiography revealed picrotoxin-sensitive [ 35S]TBPS([ 35S]t-butylbicyclophosphorothionate) binding sites in the cerebellar granule cell layer only in the presence of GABA A receptor antagonists. However, under the same conditions, the binding was decreased in other brain regions, including the cerebellar molecular cell layer. Thus, the convulsant binding sites at GABA A receptors in the granule cell layer are highly sensitive to down-regulation by GABA, which may be due to a unique combination of receptor subunits.

Prenatal benzodiazepine administration. II. Lorazepam exposure is associated with decreases in [ 35S]TBPS binding but not benzodiazepine binding

Prenatal benzodiazepine exposure has been associated with neurobehavioral alterations in humans and animals. To determine effects of prenatal benzodiazepine exposure on binding at the benzodiazepine and t-butylbicyclophosphorothionate (TBPS) sites on the GABA A receptor in mature offspring, we treated mice with lorazepam, 2 mg/kg/day, during days 13–20 of gestation. Binding was assessed at 6 weeks of age. There were no differences among controls, vehicle- or lorazepam-exposed mice in benzodiazepine receptor binding determined in vivo or in vitro. However, receptor density for [ 35S]TBPS binding sites was decreased in lorazepam-exposed offspring compared to the other groups. These data are consistent with prior neurochemical results indicating decreased TBPS binding and GABA A receptor function in several systems.

Inactivation of [ 35S]t-butylbicyclophosphorothionate binding sites by the arginine reagent, 2,3-butanedione

The “cage” convulsant [ 35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) binds with high affinity to specific sites “at” or “near” a γ-amino-butyric acid (GABA)-gated chloride channel according to current hypothesis. We now report that pretreatment of membranes with 2,3-butanedione, an arginine-specific reagent, causes a dose- and time-dependent decrease in the number of [ 35S]TBPS binding sites. No decrease occurs when membranes are pretreated with 2,3-butanedione in the presence of picrotoxinin. Binding of [ 35S]TBPS to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.

Asymmetrical activation of GABA-gated chloride channels in cerebral cortex

A pronounced left/right asymmetry of GABA-gated chloride channels was observed in rat cerebral cortex. This asymmetry was manifest as a higher apparent affinity and density of binding sites for the “cage” convulsant [ 35S]t-butylbicyclophosphorothionate (TBPS) in the right compared to left cortex. Asymmetries in [ 35S]TBPS binding were not observed in other brain areas, and were restricted to the occipital and entorhinal/pyriform areas of cerebral cortex. Evaluation of other components of the benzodiazepine/GABA receptor chloride ionophore complex in cerebral cortex suggests that this asymmetry is not present in either benzodiazepine receptors or that population of GABA receptors linked to benzodiazepine receptors. Brief restraint-stress significantly increased both the apparent affinity and number of [ 35S]TBPS binding sites in cortex, but the left/right asymmetry was no longer apparent. These findings indicate a time-dependent, asymmetric response of cortical GABA-gated chloride channels to stress rather than an anatomical hemispheric difference, and suggest that GABA-gated chloride channels may be involved in a differential processing of stressful stimuli by the cerebral hemispheres.

Antidepressants and metabolites that block GABA A receptors coupled to 35S- t-butylbicyclophosphorothionate binding sites in rat brain

Twenty-three clinically effective antidepressants and 9 metabolites of antidepressants (together, about 50% of all antidepressants tested) were found to, fully or partially, reverse the inhibitory action of 1 μM GABA on 35S-TBPS binding. Amoxapine, the most potent of the complete reversers, is a strong convulsant in overdosage while mianserin, the most potent of the partial reversers and a proconvulsant, almost never produces convulsions in humans, even after massive overdosage. Convulsant side effects, a pervasive property of antidepressants, can probably be attributed to GABA antagonism, perhaps at a subset of GABA A receptors. Further, the structures of the antidepressants, as well as the criteria used to select them for clinical trials, are diverse, raising the possibility that GABA antagonism, perhaps at another subset of GABA A receptors, could be also involved in clinical antidepressant action. We speculate that selective blockade of excessive GABAergic inhibition of reward systems may contribute to the clinical effects of many antidepressants, in some cases via active metabolites.

Alteration of the t-butylbicyclophosphorothionate binding site as a mechanism of vertebrate cyclodiene insecticide resistance

Insecticide-resistant populations of mosquitofish ( Gambusia affinis) have developed a resistance to cyclodiene and cyclodiene-type insecticides which bind to picrotoxinin binding sites of the γ-aminobutyric acid receptor-ionophore complex in the central nervous system. Scatchard analysis of [ 35S]t-butylbicyclophosphorothionate binding data from wild populations of resistant and susceptible mosquitofish suggests a reduction in binding affinity in resistant fish. Reduction in binding capacity correlates with endrin LC 50 values in laboratory-reared endrin-resistant mosquitofish. These studies indicate that vertebrate endrin-resistance involves a genetically based alteration in central nervous system target sites which is the result of environmental selection pressures from toxic insecticide residues.

Toxicity of chlorinated bornane (toxaphene) residues isolated from Great Lakes lake trout (Salvelinus namaycush).

Lake trout samples from the Great Lakes contain residues of a complex pattern of chlorinated bornanes similar to the insecticide toxaphene. These residues, although structurally similar to toxaphene, are composed of a different profile of compounds than the analytical standard. Chlorinated bornane residues were isolated from tissues of Lake Michigan and Siskiwit Lake (Isle Royale) lake trout using a variety of purification techniques. Toxicity studies were conducted to determine the toxicological properties of the isolated residues in comparison to both technical material and the procedural standard. Static 24-hr acute bioassays with mosquito larvae(Aedes egypti) demonstrated that the residues were as toxic as the toxaphene standard. Additionally, experiments performed on the picrotoxinin receptor of the GABA (γ-aminobutyric acid)-chloride ionophore complex of the central nervous system revealed that the residues were very potent at the [35S]-t-butylbicyclophosphorothionate binding site. Therefore, both non-specific and specific measures of toxicologic potency show that environmentally derived residues of toxaphene retain significant biologic activity. There is no trend for decreasing toxicity of toxaphene residues for the period of 1982–85. Also, the residues found in samples from Siskiwit Lake were as toxic as those found in Lake Michigan.

The chloride channel blocking agent, t-butyl bicyclophosphorothionate, binds to the γ-aminobutyric acid-benzodiazepine, but not to the glycine receptor in rodents

The inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA) both activate transmembrane chloride channels of similar physical characteristics. A common ion channel component has therefore been postulated for both the glycine and GABA receptor proteins. Different convulsant drugs as picrotoxin and t-butyl bicyclophosphorothionate (TBPS) have been reported as channel-blocking ligands of the GABA receptor. Here, we show that the distribution of [35S]TBPS binding sites parallels the binding of the GABA receptor ligand [3H]flunitrazepam, but not that of the glycine receptor antagonist [3H]strychnine. Binding was examined in membrane fractions from different regions of the rat CNS and of the mutant mouse spastic, an animal deficient in glycine receptors. Also, affinity purification of the glycine receptor on aminostrychnine-agarose resulted in almost complete removal of [35S]TPBS binding sites from the receptor preparation. It is concluded that TBPS selectively binds to the GABA, but not glycine, receptor chloride channel complex.

Environmentally-induced modification of the benzodiazepine/GABA receptor coupled chloride ionophore.

The benzodiazepine/GABA receptor coupled chloride ionophore was examined in brain membranes of rats maintained in either a conventional animal facility or a "protected" (low-stress) environment. Following a 10 min ambient temperature swim, animals maintained in both environments had qualitatively similar increases in the number (Bmax) of [35S]t-butylbicyclophosphorothionate (TBPS) binding sites, the apparent affinity of this radioligand, and the efficacy and potency of Cl- to enhance [3H]flunitrazepam binding. Nonetheless, the Bmax of [35S]TBPS and efficacy of Cl- to enhance [3H]flunitrazepam binding were significantly lower in animals housed in the protected environment compared to those maintained in a conventional facility both before and after swim stress. Furthermore, in rats housed in a protected environment, sequential removal of animals from a common cage (cohort removal), produced a very rapid increase (less than or equal to 15 s) in Cl(-)-enhanced [3H]flunitrazepam binding in cortical and hippocampal but not cerebellar membranes. Cohort removal also produced a sequential increase in the number of [35S]TBPS binding sites and apparent affinity of this radioligand in cerebral cortical membranes. The effects of cohort removal were not observed in animals subjected to ambient temperature swim or if animals were removed from different cages. Changes in the benzodiazepine/GABA receptor coupled chloride ionophore produced by cohort removal from a common cage preceded any statistically significant changes in circulating levels of alpha-MSH, beta-endorphin, ACTH or corticosterone. These findings suggest that the benzodiazepine/GABA receptor chloride ionophore complex (supramolecular complex) is under both tonic and acute regulation by the environment, and may subserve a physiologically relevant function in the response to stressful or anxiety producing stimuli.

Effects of insecticides and GABAergic agents on a house fly [ 35S] t-butylbicyclophosphorothionate binding site

Specific binding of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) to a house fly thorax-plus-abdomen membrane preparation at 20°C is characterized by apparent K d and B max values of 0.21 μ M and 2.5 pmol/mg protein, respectively, an association half-time of 13 min at 2 n M, and a biphasic dissociation curve showing half-times of 15 and 35 min. Specific binding is reduced at 37°C apparently due to instability of the receptor-ligand complex and at 0°C as the result of very slow association. [ 35S]TBPS binding is diminished by detergents, stimulated by GABA at low ligand concentration, and inhibited by picrotoxinin and certain barbiturates, benzodiazepines, bicyclophosphorus compounds, and polychlorocycloalkane insecticides. The potency of TBPS and three related phosphorothionates in displacing [ 35S]TBPS parallels their toxicity on injection into house flies; the corresponding bicyclophosphates are less active in both assays. Cyclodienes of low toxicity are generally poor inhibitors of radioligand binding. α-Endosulfan and syn-12-hydroxyendrin are more potent than their β and anti isomers, respectively, both as inhibitors of TBPS binding and as toxicants. Analysis of Scatchard plots indicates that picrotoxinin and heptachlor epoxide are non-competitive inhibitors of [ 35S]TBPS binding. The [ 35S]TBPS binding site of the house fly membrane preparation differs from that extensively studied in mammalian brain with respect to their responses to many insecticides and GABAergic agents.

O-221 - Neurochemical studies on γ-aminobutyric acid(GABA) receptor.(XV) Pharmacological properties of purified GABAA/benzodiazepine receptor complex from bovine cerebral cortex and its co-purification with [35S]t-butylbicyclophosphorothionate binding site by benzodiazepine affinity column chromatography

O-221 - Neurochemical studies on γ-aminobutyric acid(GABA) receptor.(XV) Pharmacological properties of purified GABAA/benzodiazepine receptor complex from bovine cerebral cortex and its co-purification with [35S]t-butylbicyclophosphorothionate binding site by benzodiazepine affinity column chromatography

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-β-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-β-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 [ 35H]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.

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 prepration obtained on treatment with the zwitterionic detergent CHAPS. In the membranes, GABA at 0.3−1 μM 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 μM both the apparent density and affinity are significantly decreased. On treatment with CHAPS the solubilized preparation and the corresponding pallet fraction become less sensitive to GABA which even at 5 μM 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 α-cyanopyrethroid cypermethrin and the barbiturate 5-(1′,3′-dimenthylbutyl)-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.

Convulsant potencies of tetrazoles are highly correlated with actions on GABA/benzodiazepine/picrotoxin receptor complexes in brain

A series of tetrazole convulsants were examined for their potencies in displacing [ 35S]-t-butylbicyclophosphorothionate (TBPS) from the picrotoxin site on the benzodiazepine-GABA-chloride ionophore receptor complex. All of the tetrazole derivatives tested inhibited [ 35S]-TBPS binding from rat forebrain membranes, and except for one (undecamethylenetetrazole), had Hill coefficients near unity. Similar to other chemically unrelated convulsants the inhibition of [ 35S]-TBPS binding by the various tetrazole derivatives was unaffected by the addition of the bicucculine-like GABA antagonist, R 5135. To ascertain whether the inhibition of specific [ 35S]-TBPS binding by the tetrazole derivatives was related to their convulsant properties, we compared their in vitro potencies in displacing [ 35S]-TBPS binding with their minimum convulsant potencies in mice. A very good correlation was observed (r=0.96, p<0.001) between their relative affinities for the [ 35S]-TBPS binding site and their convulsant potencies, indicating that pentamethylenetetrazol and related tetrazoles may produced their convulsant and anxiogenic actions via the GABA-benzodiazepine-chloride ionophore receptor complex.

Differential interactions of GABA agonists, depressant and convulsant drugs with [ 35S]-t-butylbicyclophosphorothionate binding sites in cortex and cerebellum

Effects of three GABA agonists, four GABA antagonists and convulsants (picrotoxinin, α-dihydropicrotoxinin [DHP], pentamethylenetetrazole [PTZ] and isopropylbicyclophosphate ester) and three depressant drugs (pentobarbital (+)etomidate and etazolate) were investigated on [ 35S]t-butylbicyclophosphorothionate (TBPT) in cortex and cerebellum. All the convulsants tested were equipotent in inhibiting [ 35S]TBPT binding in cortex and cerebellum. Convulsants like picrotoxinin inhibited [ 35S]-TBPT binding competitively in both cortex and cerebellum. In contrast, γ-aminobutyric acid (GABA) agonists (muscimol, GABA and 4,5,6,7-tetrahydroisoxazol[5,4- C]pyridine-3-ol[THIP]), and depressants like etazolate, (+)etomidate ande more potent inhibitors of [ 35S]TBPT binding in cerebellum than in cortex. GABA inhibition of [ 35S]TBPT binding appears to be mediated through a low-affinity site. GABA and pentobarbital inhibited [ 35S]TBPT binding in cortex and cerebellum noncompetitively. Depressants like pentobarbital appear to interact with the TBPT sites allosterically. These results suggest that depressant and convulsant drugs that modulate GABAergic transmission interact differently with the TBPT binding sites in cortex and cerebellum.

35S]-t-butylbicyclophosphorothionate binding sites are constituents of the gamma-aminobutyric acid benzodiazepine receptor complex

t- Butylbicyclophosphorothionate ( TBPS ), a derivative of potent GABA antagonistic cage convulsants, has recently been introduced ( Squires , R. F., J.E. Casida , M. Richardson, and E. Saederup (1983) Mol. Pharmacol. 13:326-336) as ligand for a GABA-A receptor-linked drug receptor. Using conventionally prepared washed membrane fractions from rat cerebral cortex, we have confirmed that in the presence of 200 mM NaBr [35S] TBPS binds to a high affinity population of binding sites (Kd 26 +/- 5 nM) and that muscimol inhibits [35S] TBPS binding (IC50 0.32 microM) allosterically. In 200 mM NaCl the apparent affinity of [35S] TBPS binding sites is lower (Kd 60 +/- 5 nM), and muscimol has biphasic effects with stimulation at low concentrations of muscimol (EC50 0.023 microM) followed by inhibition at high concentrations (IC50 0.72 microM). Both base line [35S] TBPS binding (in 200 mM NaCl) and muscimol inhibition of [35S] TBPS binding (in 200 mM NaBr) are bidirectionally modulated by the occupancy of benzodiazepine receptors with its ligands. Benzodiazepine receptor agonists, regardless of their structure, enhance and inverse benzodiazepine receptor agonists inhibit base line [35S] TBPS binding and muscimol inhibition of [35S] TBPS binding. Fourteen ligands for benzodiazepine receptors display a similar in vitro profile as benzodiazepine receptor agonists or inverse benzodiazepine receptor agonists on [35S] TBPS binding as their anti- or proconvulsive effects in vivo suggest (Jensen, L. H., E. N. Petersen, and C. Braestrup (1983) Life Sci. 33: 393-399). That [35S] TBPS binding sites are constituents of a GABA benzodiazepine receptor complex is also suggested by a number of membrane pretreatments.(ABSTRACT TRUNCATED AT 250 WORDS)