TBPS Binding Sites Research Articles

Multiple actions of fenamates and other nonsteroidal anti-inflammatory drugs on GABAA receptors

The nonsteroidal anti-inflammatory drug (NSAID) niflumic acid, a fenamate in structure, has many molecular targets, one of them being specific subtypes of the main inhibitory ligand-gated anion channel, the GABAA receptor. Here, we report on the effects of other fenamates and other classes of NSAIDs on brain picrotoxinin-sensitive GABAA receptors, using an autoradiographic assay with [35S]TBPS as a ligand on mouse brain sections. We found that the other fenamates studied (flufenamic acid, meclofenamic acid, mefenamic acid and tolfenamic acid) affected the autoradiographic signal at low micromolar concentrations in a facilitatory-like allosteric fashion, i.e., without having affinity to the [35S]TBPS binding site. Unlike niflumic acid that shows clear preference for inhibiting cerebellar granule cell layer GABAA receptors, the other fenamates showed little brain regional selectivity, indicating that their actions are not receptor-subtype selective. Of the non-fenamate NSAIDs studied at 100 μM concentration, diclofenac induced the greatest inhibition of the binding, which is not surprising as it has close structural similarity with the potent fenamate meclofenamic acid. Using two-electrode voltage-clamp assays on Xenopus oocytes, the effect of niflumic acid was found to be dependent on the β subunit variant and the presence of γ2 subunit in rat recombinant α1β and α1βγ2 GABAA receptors, with the β1 allowing the niflumic acid inhibition and β3 the stimulation of the receptor-mediated currents. In summary, the fenamate NSAIDs constitute an interesting class of compounds that could be used for development of potent GABAA receptor allosteric agonists with other targets to moderate inflammation, pain and associated anxiety/depression.

Phenobarbitone enhances [35S]TBPS binding to extensively washed rat cortical membranes.

Pentobarbitone and phenobarbitone enhance [35S]TBPS binding to extensively washed rat cortical membranes with ED50 values of 15 and 120 microM, respectively, indicative of an interaction with the chloride ionophore part of the GABA-receptor different from the TBPS binding site, but allosterically coupled to this site.

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

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. 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. 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. 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.

Autoradiographic imaging of altered synaptic αβγ2 and extrasynaptic αβ GABA A receptors in a genetic mouse model of anxiety

To image the possible alterations in brain regional GABA A receptor subtype properties in a genetic animal model of human anxiety, mice heterozygous for the deletion of GABA A receptor γ2 subunit (γ2 +/−) were studied using ligand autoradiographic assays on brain cryostat sections. The [ 35 S ]TBPS binding assay was designed to reveal impaired GABA and channel site coupling shown to be more prominent in recombinant α1/6β3 than in α1/2β3γ2 or β2 subunit-containing GABA A receptors expressed in HEK 293 cells. Increased GABA-insensitive [ 35 S ]TBPS binding in the γ2 +/− mouse brains was evident in the cerebral cortex and in subcortical regions, the alterations being regionally similar to the loss of γ2 subnunit-dependent benzodiazepine (BZ) sites as revealed by [ 3 H ]Ro 15-4513 autoradiography. As the γ2 subunit protein is needed for synaptic clustering of GABA A receptors, these results indicate that the extrasynaptic αβ3 receptors can be visualized in vitro as atypical GABA-insensitive [ 35 S ]TBPS binding sites. The results suggest that GABA Aergic synaptic inhibition is widely decreased in the brains of anxiety-prone γ2 +/− mice, while extrasynaptic GABA A receptors are increased. These autoradiographic imaging findings further demonstrate the need to develop GABA A receptor subtype-selective in vivo ligands to aid in assessing the contributions of various subcellular receptor populations in anxious and other patient groups.

Neonatal 6-Hydroxydopamine Treatment Affects GABAA Receptor Subunit Expression in the Frontal Cortex but Not the Hippocampus of Rats during Postnatal Development

The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the maturation of GABA_A receptors in the frontal cortex and hippocampus was studied using 5- to 40-day-old rats. In situ hybridization with antisense oligonucleotide probes was performed for α₁, α₂, α₅, β₂, β₃ and γ₂ subunit mRNAs of the GABA_A receptor. We demonstrated that neonatal treatment with 6-OHDA temporarily delays the postnatal transcription of the α₁ and γ₂ subunits in the rat prefrontal cortex, as assessed by in situ hybridization histochemistry. The effect was selective for these subunits (the α₂, α₅, β₂, and β₃ subunit mRNAs remained unchanged) and for this region (the mRNA levels in the hippocampus were not changed). The reduction in mRNA levels at early postnatal stages (postnatal day 5, PD5, and PD10) also affected the subunit protein levels, as shown by immunohistochemistry for the α₁ subunit, and the formation of GABA_A receptor-associated picrotoxinin-insensitive TBPS binding sites, as shown by autoradiography. Our findings indicate that without a noradrenergic influence, the maturation of GABAergic interneurons in the frontal cortex is transiently delayed (from PD5 to PD40). However, it is possible that this transient reduction of the expression of certain GABA subunits – caused by depletion of noradrenergic innervation – cannot cause a lasting alteration to the GABAergic function in the prefrontal cortex.

A novel recognition site for somatostatin-14 on the GABA(A) receptor complex.

Functional interactions between gamma-aminobutyric acid (GABA) and somatostatin are suggested by the presence of synaptic contacts between GABA and somatostatin neurons, colocalisation of GABA and somatostatin and reciprocal modulation of somatostatin and GABA release. Nevertheless, a direct interaction of somatostatin with the GABA(A) receptor complex has not yet been investigated. A quantitative autoradiographic technique was used to determine the ability of somatostatin to interact with the [35S]t-butylbicyclophosphothionate [35S]TBPS binding sites of the GABA(A) receptor complex: somatostatin inhibited [35S]TBPS binding with IC50 values in the micromolar range in all brain regions studied. These results demonstrate for the first time a direct interaction between somatostatin and the GABA(A) receptor complex.

Quantitative Autoradiography on [(35)S]TBPS Binding Sites of Gamma- Aminobutyric Acid(A) Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred forHigh- and Low-Alcohol Preference.

It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA(A) receptors are involved in alcohol preference in HAD rats.

Effect of Psychological Stress on [ 35S]TBPS Binding in Rat Brain

This study was designed to determine whether psychological stress alters the function of the GABAergic synapse, examined as biochemical changes of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) binding, in unwashed membranes of rat cerebral cortex. Psychological stress increased the number of [ 35S]TBPS binding sites by 22%. This enhancement was very similar to that after acute foot shock (24%). Psychological stress was induced very rapidly, because only 1 day after previous foot shock exposure, [ 35S]TBPS binding was increased by 23%. Diazepam [3 mg/kg intraperitoneally (subcutaneously)] and ipsapirone (5 mg/kg subcutaneously), injected 30 min before psychological stress, antagonized the enhancement of [ 35S]TBPS binding. This result suggests that psychological stress is a good animal model for investigating the various biochemical changes related to stress, avoiding the physical components associated with most of the normally used stressors and mimicking only emotional state alterations.

Clozapine and some other antipsychotic drugs may preferentially block the same subset of GABA(A) receptors.

Selective blockade of a subset of GABA(A) receptors may be involved in the antipsychotic effects of Clozapine and several other antipsychotic drugs. Seven antipsychotic drugs, and 11 drugs classified as antidepressants that only partially reverse the inhibitory effect of 1 microM GABA on [35S]TBPS binding, do not yield additive reversal when tested pairwise with Clozapine, which also only partially reverses the inhibitory effect of GABA. This suggests that all of these antipsychotic/antidepressant drugs may block a common subset of GABA(A) receptors. DMCM and Ro 5-4864 are also partial reversers of GABA's inhibitory effect, but they yield additive reversals when tested pairwise with the antipsychotic/antidepressant drugs, and also with each other, suggesting that DMCM, Ro 5-4864, and the antipsychotic drugs define three heterogeneous subsets of GABA(A) receptors, with variable overlap, depending on the drug. Several potent ligands for benzodiazepine binding sites can block the GABA inhibitory effects of DMCM and Ro 5-4864, but with different patterns: the ligands generally blocked DMCM less potently, but more completely than Ro 5-4864. Ro 5-4864 was not blocked by Flumazenil or CGS-8216, ligands that potently blocked DMCM. Nine additional antipsychotic/antidepressant drugs, as well as Clozapine, and 7 "classical" GABA(A) receptor blockers, all of which reversed GABA nearly completely, when tested at lower concentrations that only reverse approximately 20-35%, yielded almost complete additivity when tested pairwise with DMCM or Ro 54864. Another convulsant benzodiazepine, KW-1937, a positional isomer of Brotizolam, fully reverses the inhibitory effect of 1 microM GABA. At a lower concentration yielding about 50% reversal, KW-1937 is completely additive with DMCM, but entirely nonadditive with Ro 5-4864. The 50% reversal obtained with KW-1937 was potently blocked by Triazolam, but with a plateau similar to that obtained with Ro 5-4864. The results with KW- 1937 suggest that its 50% reversal largely corresponds to the reversal obtained with Ro 5-4864, and that virtually all of the [35S]TBPS binding sites inhibited by 1 microM GABA are coupled to benzodiazepine binding sites. The fraction of GABA(A) receptors preferentially blocked by all the antipsychotic/antidepressant drugs, roughly 25% of the [35S]TBPS binding sites inhibited with 1 microM GABA, are sensitive to KW-1937, but not to DMCM or to Ro 5-4864.

Quantitative Autoradiography on [^35S]TBPS Binding Sites of Gamma- Aminobutyric AcidA Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred for High- and Low-Alcohol Preference

Quantitative Autoradiography on [^35S]TBPS Binding Sites of Gamma- Aminobutyric AcidA Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred for High- and Low-Alcohol Preference

Kinetics of tert-[35S]butylbicyclophosphorothionate binding in the cerebral cortex of newborn and adult rats: effects of GABA and receptor desensitization.

The effects of GABA on the kinetics of tert-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to the convulsant site of GABAA receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [35S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast (t1/2, 11 min) and slow (t1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 microM GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 microM GABA (dissociation started by adding 40 microM picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 microM picrotoxin plus 2 microM GABA). Taken together, these results suggest that (1) the closed state of GABAA receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.

An autoradiographic study comparing the interactions of 3?-OH-5?-pregnan-20-one, pregnenolone sulfate and pentobarbital with [35S]-TBPS binding sites and their modulation by GABA in different structures of the rat brain

Using quantitative autoradiography, we have studied the distribution of the [35S]-TBPS binding sites of the GABA-A receptor complex in various structures of the rat brain. High densities of binding sites were observed in layer IV of the cerebral cortex, in the globus pallidus, and in the thalamus. Intermediate densities of binding sites were observed in superficial and deep layers of the cerebral cortex, in the dentate gyrus and in the hippocampus. For all of these structures, the interactions of 3 alpha-OH-5 alpha-pregnan-20-one (3 alpha 5 alpha P), pregnenolone sulfate (PS), and pentobarbital with [35S]-TBPS binding, in the presence or the absence of GABA were studied. In the absence of GABA, IC50 values for the inhibition of [35S]-TBPS binding were 10(-6) M for 3 alpha 5 alpha P and 10(-4) M for PS and pentobarbital in all of the brain regions studied. In the presence of GABA (5 x 10(-6) M), IC50 values were decreased by one order of magnitude for 3 alpha 5 alpha P, PS, and pentobarbital in all structures studied except layer IV of the cortex, where the IC50 value for PS was more markedly decreased (up to two orders of magnitude). By contrast, IC50 values for picrotoxin and TBPS to inhibit [35S]-TBPS binding were 10(-7) M and 10(-8) M, respectively, in the presence or absence of GABA.

Age-related levels of GABA/benzodiazepine binding sites in cerebrum of F-344 rats: Effects of exercise

We examined γ-aminobutyric acid (GABA), benzodiazepine and convulsant sites of postsynaptic GABA/benzodiazepine receptors (GBZR) in cerebral membranes of inbred Fischer 344 male rats as a function of age. In aged rats (23 to 24 months), the benzodiazepine binding site as determined by [ 3H]flunitrazepam was 47% and 43% lower than corresponding values in young adult (3 to 4 months) and mature (10 to 12 months) rats, respectively. The decrease was due to the loss of binding density rather than a change in affinity. No statistically significant age-related changes in [ 3H]muscimol binding were observed when 5 nM or 40 nM labeled muscimol were used. GABA produced a dose-dependent stimulation of flunitrazepam binding in all age groups, but the maximum stimulation in aged animals was significantly higher (24%) than in young and mature animals. The [ 35S]TBPS binding site, the convulsant site of GABA/benzodiazepine receptors, was unaffected with age. We also studied the effects of exercise on GBZR binding sites of aged rats. The decline of flunitrazepam binding sites and the high sensitivity of flunitrazepam binding to regulation by GABA in aged animals were reversed by 8 to 10 weeks of endurance exercise. Endurance exercise did not have any significant effect on muscimol or TMPS binding sites. Results suggest that there are aged-related alterations of GBZR binding sites and that these modifications can be reversed by exercise.

Fast Na +-current induced transmitter release from Na +-loaded sympathetic nerves in the absence of functioning Ca 2+-channels

The ontogenesis of the GABA-gated Cl− channel was investigated in the cerebral cortex of the rat by monitoring the binding parameters of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) at intervals after birth (1–90 days). To investigate the influence of the developmental changes in the content of GABA on [35S]TBPS-binding, the assays were carried out in unwashed membranes, in which the concentration of GABA was dependent on its content in vivo, and in repeatedly washed membranes in the presence of defined concentrations of exogenous GABA. At birth, the density (Bmax) of [35S]TBPS-binding sites in unwashed membranes was similar to that found in well-washed membranes. However, in unwashed membranes, the number of [35S]TBPS-binding sites increased by two-fold within 10 days after birth whereas in washed membranes it increased by four-fold during the same period. The higher density of [35S]TBPS-binding sites in washed membranes as compared with the unwashed counterparts persisted throughout development. In unwashed membranes, the apparent Kd for [35S]TBPS-binding increased with age whereas in washed membranes the affinity of [35S]TBPS for its binding sites remained constant throughout development. The binding of [35S]TBPS to the GABA-gated Cl− channel is allosterically modulated by drugs acting on different sites of the GABAA receptor complex. Thus, GABA and diazepam decrease [35S]TBPS-binding whereas the GABAA receptor antagonist, bicuculline, and the inverse agonist for benzodiazepine receptors, 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid methyl ester, increase it. The magnitude of the effects of positive and negative modulators of the GABAA receptor on [35S]TBPS-binding did not change significantly from birth through adulthood, provided the concentration of GABA in the incubation medium was kept constant at 3 μM. These results reflect the early development of the functional interactions between the different components of the GABAA receptor.

Allosteric modulation of [ 35S]TBPS-binding in the cerebral cortex of the rat during postnatal development

The ontogenesis of the GABA-gated Cl − channel was investigated in the cerebral cortex of the rat by monitoring the binding parameters of [ 35 S]t- butylbicyclophosphorothionate ([ 35S]TBPS) at intervals after birth (1–90 days). To investigate the influence of the developmental changes in the content of GABA on [ 35S]TBPS-binding, the assays were carried out in unwashed membranes, in which the concentration of GABA was dependent on its content in vivo, and in repeatedly washed membranes in the presence of defined concentrations of exogenous GABA. At birth, the density ( B max) of [ 35S]TBPS-binding sites in unwashed membranes was similar to that found in well-washed membranes. However, in unwashed membranes, the number of [ 35S]TBPS-binding sites increased by two-fold within 10 days after birth whereas in washed membranes it increased by four-fold during the same period. The higher density of [ 35S]TBPS-binding sites in washed membranes as compared with the unwashed counterparts persisted throughout development. In unwashed membranes, the apparent K d for [ 35S]TBPS-binding increased with age whereas in washed membranes the affinity of [ 35S]TBPS for its binding sites remained constant throughout development. The binding of [ 35S]TBPS to the GABA-gated Cl − channel is allosterically modulated by drugs acting on different sites of the GABA A receptor complex. Thus, GABA and diazepam decrease [ 35S]TBPS-binding whereas the GABA A receptor antagonist, bicuculline, and the inverse agonist for benzodiazepine receptors, 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylic acid methyl ester, increase it. The magnitude of the effects of positive and negative modulators of the GABA A receptor on [ 35S]TBPS-binding did not change significantly from birth through adulthood, provided the concentration of GABA in the incubation medium was kept constant at 3 μM. These results reflect the early development of the functional interactions between the different components of the GABA A receptor.

Steroid modulation of GABA A receptors in an amphibian brain

Steroids can modulate γ-aminobutyric acid (GABA A) receptor function in rat brains, but the physiological relevance of this mechanism is still unclear. To determine whether this phenomenon is widespread among vertebrates, we investigated steroid modulation of GABA A receptors in amphibian brain tissue. Equilibrium binding parameters for t-butylbicyclophosphorothionate ([ 35S]TBPS) and [ 3H]flunitrazepam were similar in Tricha glanulosa and mammalian brains, as was the allosteric regulation of [ 35S]TBPS and [ 3H]flunitrazepam binding in GABA. The rank order and absolute potencies of steroids to inhibit [ 35S]TBPS binding and enhance [ 3H]flunitrazepam binding were also similar in Taricha and rat brains. As in mammalian studies, physiological concentrations of corticosterone had no effect on ligand binding of GABA-stimulated Cl − uptake. In autoradiogrphic studies, 3α-hydroxy-5α-pregnan-20-one inhibited [ 35S]TBPS binding sites in all brain regions examined, whereas corticosterone had no effect on [ 35S]TBPS binding. These studies suggest that the steroid recognition sites on GABA A receptors bave been highly conserved through vertebrate evolution and thus portend physiologically important functions. However, the pharmacological profiles for the GABA A receptor and high-affinity coritcosteroid receptor are apparently different, suggesting there are multiple types of steroid recognition sites on neuronal membranes.

Studies on picrotoxin binding sites of GABA A receptors in rat cortical synaptoneurosomes

Experiments were performed to characterize [ 35S]TBPS binding in rat cortical Synaptoneurosomes, which have vesicular structures containing both pre- and postsynaptic elements. Scatchard analysis revealed a single component of [ 35S]TBPS binding sites with K D and B max values of 76.1 nM and 1.97 pmoles/mg protein, respectively, under physiological conditions. GABA and muscimol inhibited [ 35S]TBPS binding in a concentration-dependent manner. IC 50 values of these GABA A agonists in displacing synaptoneurosomal [ 35S]TBPS binding are comparable to previously reported EC 50 values of the agonist-stimulated 36C1 − uptake in Synaptoneurosomes by these agents. Furthermore, the IC 50 values of these GABA A agonists were better correspondence to those determined by [ 3H]muscimol binding in synaptoneurosomal preparations as reported by Delorey and Brown (3) than those determined in membrane preparations. Although bicuculline increased [ 35S]TBPS binding in a concentration dependent manner in cortical membranes, it did not affect synaptoneurosomal [ 35S]TBPS binding. Benzodiazepine agonists and inverse agonists (0.1 to 10 μM) did not show any effects on the binding in the absence of muscimol. However, benzodiazepine agonists potentiated and inverse agonists antagonized muscimol-induced inhibition of synaptoneurosomal [ 35S]TBPS binding. In addition, an anesthetic steroid, THDOC, and pentobarbital inhibited synaptoneurosomal [ 35S]TBPS binding in a concentration dependent manner. These results suggest that allosteric modulation of [ 35S]TBPS binding by various ligands which interact with GABA A supramolecular complexes remain intact in Synaptoneurosomes. It appears that this preparation is useful for investigating correlation between functional 36Cl − uptake and individual binding studies of each of the GABA A receptor complex.

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.

House Fly Head GABA-Gated Chloride Channel: Four Putative Insecticide Binding Sites Differentiated by [ 3H]EBOB and [ 35S]TBPS

Optimization of 4′-ethynyl-4- n-[ 3H]propylbicycloorthobenzoate ([ 3H]EBOB) and reexamination of [ 35S] t-butylbicyclophosphorothionate ([ 35S]TBPS) as radioligands for the GABA receptor complex of house fly head membranes established their respective binding parameters as follows: specific binding, 75 and 50-60%; apparent K D s, 2 and 145 n M; B maxs, 0.35 and 2.4 pmol/mg protein. Five groups of insecticides, all inhibitory to [ 3H]EBOB binding, were evaluated for potency at the [ 3H]EBOB and [ 35S]TBPS binding sites and competitive or noncompetitive inhibition with [ 3H]-EBOB. They are ( i) polychlorocycloalkanes with emphasis on lindane analogs; ( ii) 1-aryl-trioxabicyclooctanes, 2-aryl-dithiane, aryl-silatrane, and picrotoxinin; ( iii) trioxabicyclooctanes including bicyclophosphorus esters and trithiabicyclooctanes and dithianes with smaller terminal substituents; ( iv) phenylpyrazoles; and ( v) avermectins. Groups i-iv are putative channel blockers and v is a channel activator. Additional observations were made on poisoning signs, temperature coefficient of poisoning, and cross-resistance in a dieldrin-resistant strain. The findings are interpreted in light of our earlier differentiation, from studies with mice, of "Type A" action for compounds in groups i and ii and "Type B" action for the trioxabicyclooctanes in group iii. In house flies, Type A action involves the EBOB site as toxicologically relevant for i and ii, and there are characteristic hyperexcitation signs, a positive temperature coefficient, and cross-resistance with dieldrin. Type B action (for some of the compounds in group iii) presumably involves the TBPS site, although its toxicological relevance is not established, and there are different poisoning signs, a negative temperature coefficient, and no cross-resistance. Mixed Types A and B action is suggested for other compounds in group iii. Phenylpyrazoles ( iv) are related to Type A action except for noncompetitive inhibition of [ 3H]EBOB binding in fly membranes and low potency at the [ 3H]EBOB site in mammals and are therefore designated as Type C. The action of the avermectins ( v), designated Type D, is coupled to the EBOB but not the TBPS site with poisoning signs of sedation and diuresis and no cross-resistance.

The human neuroblastoma cell line, IMR-32 possesses a GABA A receptor lacking the benzodiazepine modulatory site

GABA A receptors were identified in IMR-32 cell membranes by the binding of [ 35 S]t- butyl-bicyclophosphorothionate ([ 35S]TBPS) to the chloride channel. GABA (IC 50 2.2μM), muscimol (IC 50 0.8 μM), picrotoxin (IC 50 1.7 μM), pentobarbitone (IC 50 108 μM), etomidate (IC 50 53 μM), chlormethiazole (IC 50 98 μM) and Ro 5-3663 (IC 50 280 μM) all inhibited [ 35S]TBPS binding. The potency of these drugs at the [ 35S]TBPS binding site in IMR-32 cell membranes did not correlate with their potency on [ 35S]TBPS binding to rat cortical membranes (linear correlation of pIC 50 values, r = 0.75, NS). No specific binding of the benzodiazepine ligands [ 3H]flunitrazepam or [ 3H]Ro 15-4513 to IMR-32 cell membranes was observed. Chloride efflux from IMR-32 cells was studied using the fluorescent dye 6-methoxy- N-(3-sulphopropyl) quinolinium. Chloride efflux was stimulated by GABA and muscimol (0.1–100 μM) but not by the GABA B agonist baclofen (100 μM). In the absence of exogenous GABA chloride efflux was stimulated by chlormethiazole (1–100 μM) in a picrotoxin-sensitive manner. Flurazepam (1–100 μM) both alone and in the presence of GABA had no effect on chloride efflux. It is concluded that IMR-32 cells contain a functional GABA A receptor which differs from that in rat cortex both in its general pharmacology and specifically in the absence of the allosteric modulatory site sensitive to benzodiazepines.