Tert-butylbicyclophosphorothionate Research Articles

Positive allosteric γ-aminobutyric acid type A receptor modulation prevents lipotoxicity-induced injury in hepatocytes in vitro.

To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABAA ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury. Allosteric modulation of the GABAA receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABAA channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-κB subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting. Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABAA channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABAA receptor. In HepG2 cells, which expressed the main GABAA receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and NF-κB phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI. HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs.

A Novel Approach for the Identification of Selective Anticonvulsants Based on Differential Molecular Properties for TBPS Displacement and Anticonvulsant Activity: An Integrated QSAR Modelling

AbstractIn order to gain insight into the structural and molecular requirements influencing the anticonvulsant activity against Pentylenetetrazole (PTZ)‐induced seizures and [35S] tert‐Butyl‐bicyclophosphorothionate (TBPS) displacement property (a measure of binding to GABAA receptor), Quantitative Structure–Activity Relationship (QSAR) studies have been performed on a series of congeneric anticonvulsant agents proposed to act by binding to the lactone site of the GABAA receptor. The aim of this work was to identify and analyse the various functionalities, which determine the TBPS displacement property and anticonvulsant activity by correlating with various molecular descriptors. Statistical techniques like Principal Component Analysis (PCA), Partial Least Squares (PLS), Multiple Linear Regression (MLR) and Genetic Function Approximation (GFA) were applied to identify the structural and physicochemical requirements for TBPS displacement property and anticonvulsant activity. The generated equations were statistically validated using leave‐one‐out cross‐validation technique and randomization. The best models were also validated by prediction of activity of compounds, not used for the development of QSAR models. The results from reasonably good QSAR models (statistically validated) clearly indicated that TBPS displacement property and the anticonvulsant activity are defined by different molecular parameters. Based on this finding, a novel approach is proposed, using integrated QSAR modelling, for the identification of potential and selective anticonvulsant agents.

Molecular Determinants of Picrotoxin Inhibition of 5-Hydroxytryptamine Type 3 Receptors

Previously, we reported that the GABA(A) receptor antagonist picrotoxin also antagonizes serotonin (5-HT)3 receptors and that its effects are subunit-dependent. Here, we sought to identify amino acids involved in picrotoxin inhibition of 5-HT3 receptors. Mutation of serine to alanine at the transmembrane domain 2 (TM2) 2' position did not affect picrotoxin (PTX) sensitivity in murine 5-HT3A receptors. However, mutation of the 6' TM2 threonine to phenylalanine dramatically reduced PTX sensitivity. Mutation of 6' asparagine to threonine in the 5-HT3B subunit enhanced PTX sensitivity in heteromeric 5-HT3A/3B receptors. Introduction of serine (native to the human 3B subunit) at the 6' position also increased PTX sensitivity, suggesting a species-specific effect. Mutation of the 7' leucine to threonine in 5-HT3A receptors increased PTX sensitivity roughly 10-fold, comparable with that observed in GABA(A) receptors, and also conferred distinct gating kinetics. The equivalent mutation in the 3B subunit (i.e., 7' valine to threonine) had no impact on PTX sensitivity in 5-HT3A/3B receptors. Interestingly, [3H]ethynylbicycloorthobenzoate ([3H]EBOB), a high-affinity ligand to the convulsant site in GABA(A) receptors, did not exhibit specific binding in 5-HT3A receptors. The structurally related compound, tert-butylbicyclophosphorothionate (TBPS), which potently inhibits GABA(A) receptors, did not inhibit 5-HT3 currents. Our results indicate that the TM2 6' residue is a common determinant of PTX inhibition of both 5-HT3 and GABA(A) receptors and demonstrate a role of the 7' residue in PTX inhibition. However, lack of effects of EBOB and TBPS in 5-HT3A receptors suggests that the functional domains in the two receptors are not equivalent and underscores the complexity of PTX modulation of LGICs.

Allopregnanolone-stimulated GABA-mediated chloride ion flux is inhibited by 3β-hydroxy-5α-pregnan-20-one (isoallopregnanolone)

Altered gamma-aminobutyric acid (GABA)-ergic function is associated with neurological and psychiatric disorders. Certain progesterone metabolite 3α-hydroxy-5α-pregnan-20-one, or allopregnanolone (ALLO), increases the GABA-mediated chloride ion (Cl −) flux through GABA A receptors in a similar fashion as benzodiazepines and barbiturates. We have studied the effect of its 3β diastereomer, 3β-hydroxy-5α-pregnan-20-one, or isoallopregnanolone (ISO), on the Cl − flux and investigated the interaction between ISO and ALLO on GABA-mediated Cl − uptake in cortical homogenates from adult male Wistar rats. We found that ISO from 1 μM to 1 mM does not affect baseline Cl − uptake in rat cortical homogenates. Neither does ISO at dose range of 100 nM to 100 μM interact with 10 μM GABA in the Cl − uptake assay. In addition, ISO at the dose range of 1–30 μM does not affect flunitrazepam and pentobarbital-induced increase of Cl − uptake. We conclude that ISO selectively inhibits the ALLO-induced Cl − uptake with respect to ALLO concentrations. The IC 50 of ISO inhibition on 1 μM ALLO-induced Cl − uptake was calculated to be 12.25 μM. On the other hand, we have studied the effect of 30 μM ISO on ALLO (0.01 nM to 1 μM) induced displacement of tert-butylbicyclophosphorothionate (TBPS) binding. We did not note any interaction between ALLO and ISO on TBPS binding assay. These results indicate that ISO may be useful functional blocker of GABA A receptor potentiating steroid ALLO when used at concentrations that do not affect baseline GABAergic neurotransmission.

Age-related changes of GABA-activated chloride channel properties in brain cortex synaptic plasma membrane: evidence for phospholipase involvement.

Brain aging decreases binding of tert-butylbicyclophosphorothionate (TBPS), a specific ligand for Cl- channels, but has no effect on Cl- influx. Detailed studies on the kinetics of TBPS dissociation allowed the characterization of Cl- channel properties. Aging lowers, exclusively in the presence of GABA agonist, muscimol, the half-life of the fast phase of TBPS dissociation, indicating an opening time of receptor-dependent Cl- channels shorter than that in adult brain. The half-life of the slow phase of TBPS dissociation is significantly lower in aged brain in the presence and absence of muscimol. These results suggest a sustained Cl- current, including also the other channel(s) not connected with GABAA receptor activation. The analysis of biphasic TBPS dissociation demonstrates a lowered number of binding sites resulted in the reduction of the number of Cl- channels in the "open" state. This may explain an observed decrease of TBPS binding in aged brain. One of the possible factors involved in modification of GABAA receptor behavior during aging may be arachidonic acid or diacylglycerol, known to be accumulated in aged brain. The action of these compounds on the Cl- channel, observed in this study, correlates well with the effect of aging.

Modulation Of [35S]-tert-butylbicyclophosphorothionate binding by somatostatin in rat hypothalamus.

1. The present study was designed to assess the effect of the tetradecapeptide somatostatin on the GABA(A) receptor complex in the rat hypothalamus. 2. GABA(A) receptors were labelled with [35S]-tert-butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel, and binding as assessed by in vitro quantitative autoradiography using a computer-assisted image analysis system. 3. Somatostatin inhibited the binding of [35S]-TBPS to the convulsant site of the hypothalamic GABA(A) receptor complex of rat slide-mounted hypothalamic structures in a concentration-dependent manner with an affinity in the micromolar range (10(-6) to 3 x 10(-6) mol/L). Somatostatin appeared to mimic the effects of the neurosteroid 5alpha-pregnane-3alpha ol-one (5alpha3alphaP), GABA and picrotoxin on [35S]-TBPS binding in the rat hypothalamus in all structures examined. Furthermore, GABA or muscimol (a GABA(A) receptor agonist), when added to the incubation medium, enhanced the capacity of somatostatin to inhibit [35S]-TBPS binding, with an IC50 of 10(-7) mol/L. However, incubation with bicuculline (a GABA(A) receptor antagonist) led to the abolition of the inhibitory effect of somatostatin on [35S]-TBPS specific binding in rat hypothalamus. 4. The present results demonstrate the presence of a modulatory effect of somatostatin on the GABA(A) receptor complex in rat hypothalamic structures. Furthermore, the data suggest that somatostatin allosterically modifies [35S]-TBPS binding through a mechanism similar to that of GABA. Taken together, these results provide evidence for the presence of somatostatin- GABA interactions in rat hypothalamus.

Noncompetitive antagonist-binding sites of rat and housefly γ-aminobutyric acid receptors display different enantiospecificities for tert-butyl(isopropyl)bicyclophosphorothionate

The enantiomers of 4- tert-butyl-3-isopropyl-2,6,7-trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfide (TBIPPS) were prepared in nine steps from diethyl tert-butylmalonate, and their abilities to compete with [ 3H]1-(4-ethynylphenyl)-4- n-propyl-2,6,7-trioxabicyclo[2.2.2]octane (EBOB), a noncompetitive antagonist of ionotropic γ-aminobutyric acid (GABA) receptors, at their binding site were investigated using rat brain and housefly head membranes. The ( S)-(−)-isomer of TBIPPS (IC 50=398 nM) was more potent than was the ( R)-(+)-isomer of TBIPPS (IC 50=1220 nM) in rat receptors, while the potencies of ( S)-TBIPPS (IC 50=104 nM) and ( R)-TBIPPS (IC 50=94.4 nM) in housefly receptors were almost the same. The different enantiospecificities of rat and housefly receptors indicate that the three-dimensional structure of the binding site might be different between these receptors. In a region of the rat binding site there might be a steric bulk that interacts less favorably with ( R)-TBIPPS than with ( S)-TBIPPS, while in the corresponding region of the housefly binding site there might not be such a steric bulk that leads to specificity for these compounds.

A simple polar deacetylated caloporoside derivative is a positive modulator of the GABA A chloride channel complex in cortical mammalian neurones

Synthesis of octyl- O-β- d-mannopyranoside, a caloporoside analogue was achieved by the activation of 2,3,4,6- tetra- O-benzyl-1- O-1′,3′2′-dioxaphosphacyclohexane-α,β- d-mannopyranosyl-2-oxide with TMSOTf (Trimethyl silyl triflate) and subsequent debenzylation. At 100 μM the molecule significantly and reversibly increased the magnitude of GABA A currents evoked in cultured rat pyramidal neurones whilst concomitantly reducing the incidence of spontaneous synaptic activity. These results contradict earlier proposals that such molecules bind to the TBPS ( tert-Butylbicyclophosphorothionate) site to block the chloride channel.

Identification of amino acid residues of GABA(A) receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site.

A chimeric GABA(A) receptor subunit was constructed that contained the beta3 sequence from the N-terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the alpha1 subunit. On co-expression with alpha1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high-affinity [3H]muscimol but no high-affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the beta3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the beta3 subunit into this chimera was sufficient to reconstitute the specific high-affinity [35S]TBPS binding site in receptors composed of the chimera and alpha1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the beta3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure-function relationship of GABA(A) receptors.

Effects of the chlorotriazine herbicide, cyanazine, on GABA A receptors in cortical tissue from rat brain

Chlorotriazine herbicides disrupt luteinizing hormone (LH) release in female rats following in vivo exposure. Although the mechanism of action is unknown, significant evidence suggests that inhibition of LH release by chlorotriazines may be mediated by effects in the central nervous system. GABA A receptors are important for neuronal regulation of gonadotropin releasing hormone and LH release. The ability of chlorotriazine herbicides to interact with GABA A receptors was examined by measuring their effects on [ 3H]muscimol, [ 3H]Ro15-4513 and [ 35S] tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes. Cyanazine (1–400 μM) inhibited [ 3H]Ro15-4513 binding with an IC 50 of approximately 105 μM ( n=4). Atrazine (1–400 μM) also inhibited [ 3H]Ro15-4513 binding, but was less potent than cyanazine (IC 50=305 μM). However, the chlorotriazine metabolites diaminochlorotriazine, 2-amino-4-chloro-6-ethylamino- s-triazine and 2-amino-4-chloro-6-isopropylamino- s-triazine were without significant effect on [ 3H]Ro15-4513 binding. Cyanazine and the other chlorotriazines were without effect on [ 3H]muscimol or [ 35S]TBPS binding. To examine whether cyanazine altered GABA A receptor function, GABA-stimulated 36Cl − flux into synaptoneurosomes was examined. Cyanazine (50–100 μM) alone did not significantly decrease GABA-stimulated 36Cl − flux. Diazepam (10 μM) and pentobarbital (100 μM) potentiated GABA-stimulated 36Cl − flux to 126 and 166% of control, respectively. At concentrations of 50 and 100 μM, cyanazine decreased potentiation by diazepam to 112 and 97% of control, respectively, and decreased potentiation by pentobarbital to 158 and 137% of control ( n=6). Interestingly, at lower concentrations (5 μM), cyanazine shifted the EC 50 for GABA-stimulated 36Cl − flux into synaptoneurosomes from 28.9 to 19.4 μM, respectively ( n=5). These results suggest that cyanazine modulates benzodiazepine, but not the muscimol (GABA receptor site) or TBPS (Cl − channel), binding sites on GABA A receptors. Furthermore, at low concentrations, cyanazine may slightly enhance function of GABA A receptors, but at higher concentrations, cyanazine antagonizes GABA A receptor function and in particular antagonizes the positive modulatory effects of diazepam and pentobarbital.

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C3 length and the 4-alkyl groups of C4 length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC50 = 45.2 nM), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC50 = 62.2 nM). Their receptor selectivities (rat IC50/housefly IC50) were 52 and 0.038, respectively. The insecticidal activity (LD50) of 20 active analogues was well correlated with their potency (IC50) in inhibiting [3H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABAA receptors. © 1999 Society of Chemical Industry

Ionotropic GABA receptor from lobster olfactory projection neurons.

This study reports an ionotropic GABA (gamma-aminobutyric acid) receptor in projection neurons acutely dissociated from the olfactory lobe of the brain of the spiny lobster and analyzed by whole cell and cell-free patch-clamp recording. GABA evokes a macroscopic current in the cells that is linear from -100 to + 100 mV, reverses at the imposed chloride equilibrium potential, has a permeability sequence of Cl- > acetate > bicarbonate > phosphate > propionate and SCN- > Br- > I- > Cl- > F-, and is reversibly blocked by the Cl channel blocker picrotoxin but not tert-butylbicyclophosphorothionate (TBPS). The current is bicuculline insensitive and activated by muscimol, isoguvacine, cis-4-aminocrotonic acid (CACA), and trans-aminocrotonic acid (TACA), as well as by the GABA(C)-receptor antagonists 4,5,6,7-tetrahydroisoxazolo [5,4,-c]pyridin-3-ol (THIP), 3-amino-1-propanesulfonic acid (3-APS), and imidazole-4-acetic acid (I-4AA), but not the GABA(B)-receptor agonists baclofen and 3-aminopropylphosphonic acid (3-APA). Agonist potency for the receptor is TACA > muscimol > GABA > I-4AA > isoguvacine > 3-APS > CACA > THIP. Unitary chloride currents in cell-free, outside-out patches from the cells share enough of these pharmacological properties to indicate that the channel underlies the macroscopic current. The receptor mediates an inhibitory current in the cells in vivo. The receptor is similar, if not identical, to one from neurons cultured from the thoracic ganglia of the clawed lobster. The more extensive pharmacological characterization of the receptor reported here indicates that this lobster CNS receptor is pharmacologically distinct from previously characterized ionotropic GABA receptors.

Anesthetic activity of novel water-soluble 2 beta-morpholinyl steroids and their modulatory effects at GABAA receptors.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

Evidence that GABAA receptor subunit mRNA expression during development is regulated by GABAA receptor stimulation.

The expression of six mRNA species (alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. alpha 3, alpha 5, and beta 3 subunit expression declined, alpha 2 expression increased, whereas beta 2 and gamma 2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert-butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the alpha 3, alpha 5, or beta 3 subunit mRNA expression declined and prevented the increase in alpha 2 subunit mRNA expression. As well, allopregnanolone down-regulated beta 2 subunit mRNA expression. TBPS, on the other hand, up-regulated alpha 3, alpha 5, beta 2, and beta 3 subunit mRNA expression. It also down-regulated the expression of alpha 2 subunit mRNA. Both allopregnanolone and TBPS had no effect on gamma 2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.

Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells.

1. Radioligand binding and patch-clamp techniques were used to study the actions of gamma-aminobutyric acid (GABA) and the general anaesthetics propofol (2,6-diisopropylphenol), pentobarbitone and 5 alpha-pregnan-3 alpha-ol-20-one on rat alpha 1 and beta 3 GABAA receptor subunits, expressed either alone or in combination. 2. Membranes from HEK293 cells after transfection with alpha 1 cDNA did not bind significant levels of [35S]-tert-butyl bicyclophosphorothionate ([35S]-TBPS) (< 0.03 pmol mg-1 protein). GABA (100 microM) applied to whole-cells transfected with alpha 1 cDNA and clamped at -60 mV, also failed to activate discernible currents. 3. The membranes of cells expressing beta 3 cDNAs bound [35S]-TBPS (approximately 1 pmol mg-1 protein). However, the binding was not influenced by GABA (10 nM-100 microM). Neither GABA (100 microM) nor picrotoxin (10 microM) affected currents recorded from cells expressing beta 3 cDNA, suggesting that beta 3 subunits do not form functional GABAA receptors or spontaneously active ion channels. 4. GABA (10 nM-100 microM) modulated [35S]-TBPS binding to the membranes of cells transfected with both alpha 1 and beta 3 cDNAs. GABA (0.1 microM-1 mM) also dose-dependently activated inward currents with an EC50 of 9 microM recorded from cells transfected with alpha 1 and beta 3 cDNAs, clamped at -60 mV. 5. Propofol (10 nM-100 microM), pentobarbitone (10 nM-100 microM) and 5 alpha-pregnan-3 alpha-ol-20-one (1 nM-30 microM) modulated [35S]-TBPS binding to the membranes of cells expressing either alpha 1 beta 3 or beta 3 receptors. Propofol (100 microM), pentobarbitone (1 mM) and 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) also activated currents recorded from cells expressing alpha 1 beta 3 receptors. 6. Propofol (1 microM-1 mM) and pentobarbitone (1 mM) both activated currents recorded from cells expressing beta 3 homomers. In contrast, application of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) failed to activate detectable currents. 7. Propofol (100 microM)-activated currents recorded from cells expressing either alpha 1 beta 3 or beta 3 receptors reversed at the Cl- equilibrium potential and were inhibited to 34 +/- 13% and 39 +/- 10% of control, respectively, by picrotoxin (10 microM). 5 alpha-Pregnan-3 alpha-ol-20-one (100 nM) enhanced propofol (100 microM)-evoked currents mediated by alpha 1 beta 3 receptors to 1101 +/- 299% of control. In contrast, even at high concentration 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) caused only a modest facilitation (to 128 +/- 12% of control) of propofol (100 microM)-evoked currents mediated by beta 3 homomers. 8. Propofol (3-100 microM) activated alpha 1 beta 3 and beta 3 receptors in a concentration-dependent manner. For both receptor combinations, higher concentrations of propofol (300 microM and 1 mM) caused a decline in current amplitude. This inhibition of receptor function reversed rapidly during washout resulting in a "surge' current on cessation of propofol (300 microM and 1 mM) application. Surge currents were also evident following pentobarbitone (1 mM) application to cells expressing either receptor combination. By contrast, this phenomenon was not apparent following applications of 5 alpha-pregnan-3 alpha-ol-20-one (10 microM) to cells expressing alpha 1 beta 3 receptors. 9. These observations demonstrate that rat beta 3 subunits form homomeric receptors that are not spontaneously active, are insensitive to GABA and can be activated by some general anaesthetics. Taken together, these data also suggest similar sites on GABAA receptors for propofol and barbiturates, and a separate site for the anaesthetic steroids.

Actions of picrodendrin antagonists on dieldrin-sensitive and -resistant Drosophila GABA receptors.

1. A series of terpenoid compounds, recently isolated from Picrodendron baccatum, share a picrotoxane skeleton with picrotoxinin, an antagonist of ionotropic GABA receptors. Referred to as picrodendrins, they inhibit the binding of [35S]-tert-butylbicyclophosphorothionate (TBPS) to rat GABAA receptors. Hitherto, their effects on GABA receptors have not been investigated electrophysiologically. Under two-electrode voltage-clamp, the actions of picrodendrins and related terpenoids have been assayed on homooligomeric GABA receptors formed by the expression of a Drosophila GABA receptor subunit (RDLac) in Xenopus oocytes. 2. All the terpenoids tested, dose-dependently antagonized currents induced by 30 microM (EC50) GABA. 3. Tutin and its analogues (dihydrotutin and isohyenanchin) differ in the structure of their axial C4 substituents. Of these compounds, tutin, which bears an isopropenyl group at this carbon atom, was the most potent antagonist of RDLac homo-oligomers, whereas isohyenanchin, which bears a hydroxyisopropyl group, was the least potent antagonist tested. 4. Picrodendrins differ mainly in the structure of their C9 substituents. The IC50s of picrodendrins ranged from 17 +/- 1.3 nM (picrodendrin-Q) to 1006 +/- 1.3 nM (picrodendrin-O). As such, the most potent picrodendrins (Q, A and B) were approximately equipotent with picrotoxinin as antagonists of RDLac homo-oligomers. 5. Certain picrodendrin compounds effected a use-dependent blockade of RDLac homo-oligomers. Such a biphasic block was not observed with tutin analogues. 6. Picrotoxin-resistant RDLacA3025 homo-oligomers, which have a single amino acid substitution (A302S) in the 2nd transmembrane region, were markedly less sensitive to picrodendrin-O than the wild-type, dieldrin-sensitive, homo-oligomers. 7. The relative potency of tutin analogues demonstrates that the structure-activity relationship of the C4 substituent of picrotoxane-based compounds is conserved in vertebrates and insects. However, the relative order of potency of picrodendrins on RDLac homo-oligomers is distinctly different from that observed in previous radioligand binding studies performed on vertebrate GABAA receptors. As picrodendrin compounds differ in the structure of their C9 substituents, these data suggest that the optimal convulsant pharmacophores of vertebrate GABAA receptors and RDLac homo-oligomers differ with respect to this substituent.

Enhancement by GABA of the association rate of picrotoxin and tert‐butylbicyclophosphorothionate to the rat cloned α1β2γ2 GABAA receptor subtype

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.

Effects of 5-HT 3 receptor antagonists on binding and function of mouse and human GABA A receptors

Both 5-HT 3 receptor antagonists and benzodiazepine receptor ligands have effects on anxiety, and alter the behavioral action of ethanol. For these reasons, we tested the ability of several 5-HT 3 receptor antagonists to inhibit the ligand binding and function of the γ-aminobutyric acid A/benzodiazepine receptor Cl − channel complex of mouse brain membranes. MDL 72222 (1-a-H-3-a-5-aH-optropan-3yl-3,5-dichlorobenzoate) and LY 278584 (1-methyl- N-(8-methyl-8-azabicyclo[3.2.1.] oct-3-yl)-1H- indazole-3-carboxamide) inhibited [ 3H]flunitrazepam binding with K i values of approximately 20 μM; ICS 205–930 (3α-tropanyl-1H-indole-3-carboxylic acid ester) was more potent with a K i of 0.8 μM. ICS 205–930 (50 μM) had no effect on [ 3H]muscimol binding. ICS 205–930, MDL 72222, and LY 278584 all inhibited the binding of [ 35S]TBPS (tert-butylbicyclophosphorothionate) with K i values of approximately 10 μM and reduced muscimol-dependent 36Cl − flux into mouse cortical microsacs by 30–45% at a concentration of 10 μM. ICS 205–930, MDL 72222, and LY 278584 (at micromolar concentrations) reduced GABA-gated chloride currents studied in Xenopus oocytes expressing human α 1 β 1 γ 2S GABA A receptor subunits. ICS 205–930 differed from the other two 5-HT 3 receptor antagonists in that it induced a biphasic effect on GABA-gated currents: at concentrations from 0.1 to 5 μM it potentiated GABA responses, whereas at higher concentrations (50–100 μM) it produced inhibition. The stimulatory action induced by ICS 205–930 was due to interaction at the benzodiazepine recognition site because expression of the γ 2 subunit was required and Ro 15–1788 (1 μM) completely prevented the potentiation caused by ICS 205–930. Thus, several 5-HT 3 receptor antagonists inhibit benzodiazepine binding and affect GABA A receptor function. These actions are most pronounced for ICS 205–930 and likely involve direct affects on the GABA/benzodiazepine complex rather than interactions with 5-HT 3 receptors.

Pharmacology of novel GABA receptors found on rod horizontal cells of the white perch retina

A novel type of GABA receptor is present on rod-driven (H4) horizontal cells of the white perch retina (Qian and Dowling, 1993a). These receptors have been tentatively termed GA-BAC receptors. In this study, the pharmacological properties of these receptors were further investigated by applying several conformationally restricted GABAA receptor agonists, GABAA antagonists, and a GABAB agonist to the H4 horizontal cells. GABA analogs locked in a partially folded conformation had a variety of effects. Isonipecotic acid had no effect on these receptors, whereas isoguvacine activated them but with low potency (EC50 = 137 microM). THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) acted as a competitive antagonist on these receptors with an inhibition constant of 82.5 microM. P4S (piperidine-4-sulfonic acid) activated the receptors at high concentrations (> 1 mM), but at lower concentrations it was a competitive antagonist with an inhibition constant of 80.9 microM. 14AA (imidazole-4-acetic acid), a GABA analog with an extended conformation, potently inhibited the GABA responses on H4 horizontal cells with an inhibition constant of 1.67 microM. Muscimol, which can assume both partially folded and extended conformations, acted as a mixed agonist-antagonist. The GABA responses on H4 horizontal cells were resistant to several competitive GABAA receptor antagonists including bicuculline, hydrastine, and SR-95531, but they were very sensitive to picrotoxin (IC50 = 237 nM). The inhibition by picrotoxin was both competitive and noncompetitive in nature. On the other hand, TBPS (tert-butyl-bicyclophosphorothionate), another GABAA receptor channel blocker, had minimal effects on these receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and structure-activity relationships of a series of insecticidal dioxatricyclododecenes acting as the noncompetitive antagonist of GABAA receptors

Chlorinated analogues of 5-substituted 2,3:8,7-endo-4,6-dioxatricyclo[7.2.1.0 2,8 ]dodec-10-ene (DTD) and 2,3:8,7-endo-4,6-dioxa-5-thiatricyclo[7.1.1.0 2,8 ]dodec-10-ene 5-oxide were prepared. With -CH 2 -, -CH(Me)-, and -S(O)- at the 5 position, the presence of chlorine atoms in the norbornene moiety was important in exerting toxic effects to houseflies, while with -CH(n-Pr)- and -CH(Ph-CN-4)- the introduction of chlorine atoms resulted in a loss of toxic activity. The effect of the presence of chlorine atoms on the potency in inhibiting [ 35 S]tert-butylbicyclophosphorothionate (TBPS) binding to rat brain membranes was found to be correlated to their insecticidal activite