Polychlorocycloalkane insecticide-induced convulsions in mice in relation to disruption of the GABA-regulated chloride ionophore

The toxicity to mice of intraperitoneally-administered polychlorocycloalkane (PCCA) insecticides is generally correlated with their potency as in vitro inhibitors of the brain specific [35S]t-butylbicyclophosphorothionate [( 35S]TBPS) binding site with correction for metabolic activation and detoxification. These findings from our earlier studies are extended here to in vivo investigations relating convulsant action to inhibition of the TBPS binding site in poisoned mice. Radioligand binding assays involved brain P2 membranes washed three times with 1 mM EDTA to remove endogenous gamma-aminobutyric acid (GABA) or other modulator(s) which otherwise serves as a noncompetitive inhibitor of [35S]TBPS binding at the GABA-regulated chloride ionophore. Examination of lindane, technical toxaphene, toxaphene toxicant A, and 10 polychlorocyclodiene insecticides revealed 62 +/- 4% binding site inhibition 30 min after their LD50 doses with 32 +/- 3% inhibition at one-half and 6 +/- 3% inhibition at one-quarter of their LD50 doses. This correlation between binding site inhibition and convulsant action is also evident in dose- and time-dependency studies with endosulfan sulfate. The brain P2 membranes of treated mice contain the parent compound with each of the PCCAs plus activation products of some of the cyclodienes, i.e. endosulfan sulfate from alpha- and beta-endosulfan and 12-ketoendrin from isodrin and endrin. The finding that the brains of treated mice contain sufficient PCCA or its activation products to achieve a magnitude of [35S]TBPS binding site inhibition correlated with the severity of the poisoning signs supports the hypothesis that the acute toxicity of PCCA insecticides to mammals is due to disruption of the GABA-regulated chloride ionophore.     

Chlorocyclohexane insecticides and male medfly attractants: similar stereospecificity for neuroactivity and interactions with a housefly [35S]t-butylbicyclophosphorothionate binding site

[35S]t-Butylbicyclophosphorothionate [( 35S]TBPS) undergoes saturable specific binding to a membrane preparation from housefly thoraxes and abdomens with apparent Kd and Bmax values at equilibrium of 0.17 microM and 2.2 pmol/mg protein at 20 degrees C. Lindane is more potent than three other isomers of hexachlorocyclohexane as a toxicant for houseflies and in displacing [35S]TBPS from this housefly membrane preparation. This correlation of similar stereospecificity for neuroactivity and interaction with the TBPS receptor extends to the Mediterranean fruit fly male attractant trimedlure and its components and analogs. The relative attractancy of t-butyl cis-4-chloro-trans-2-methylcyclohexanecarboxylate and of three less active isomers of this trans-chlorocyclohexane-carboxylate parallel their potency in the [35S]TBPS binding assay. With both trimedlure and the related cyclohexene attractant siglure the trans-isomers are more potent than the cis-isomers as attractants and in displacing [35S]TBPS. Scatchard analyses indicate that lindane binds at the same site as [35S]TBPS. The hexachlorocyclohexane isomers and trimedlure components are much more potent inhibitors with membrane preparations from houseflies than from rat brain. The housefly TBPS receptor possibly serves as a model for the primary target sites, thereby suggesting that both the insecticide and the attractant may interact with some component of the putative GABAergic nervous or neuromuscular system.     

t-[35S]Butylbicyclophosphorothionate Binding Sites in Invertebrate Tissues

Specific high affinity binding of the cage convulsant t-[35S]butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S]TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22 degrees C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4 degrees C temperature and 150-min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD = 50 nM, Bmax = 250 fmol/mg protein in housefly; KD = 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1 = 8 X 10(6) M-1 min-1), but dissociation was described better by two rate constants (k-1 = 0.28 min-1 and 0.042 min-1; calculated KD values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50 = 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1-2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclodiene resistance at the insect GABA receptor/chloride channel complex confers broad cross resistance to convulsants and experimental phenylpyrazole insecticides

This study investigated the pharmacological profile of cyclodiene resistance in Drosophila melanogaster and the mode of action of a phenylpyrazole insecticide, JKU 0422. Toxicological studies were performed with a sucrose bait assay containing the synergist piperonyl butoxide. The Maryland strain of D. melanogaster was resistant to dieldrin, lindane, picrotoxinin, TBPS, p-CN-TBOB, and JKU 0422. In contrast, this strain was susceptible to cypermethrin and the avermectins MK-243, abamectin, and abamectin 8,9-oxide. Neurophysiological studies showed that both TBPS and JKU 0422 reversed the inhibitory action of GABA in central nerve preparations from susceptible D. melanogaster. However, the response to these compounds was attenuated in nerve preparations from the resistant Maryland strain, which indicated that the resistance was expressed at the level of the nerve. Topical toxicity bioassays with JKU 0422 on susceptible (CSMA) and cyclodiene-resistant (LPP) strains of German cockroach revealed a resistance ratio of 553-fold for this compound. These studies demonstrate that cyclodiene resistance in D. melanogaster confers broad cross resistance toward compounds thought to block the GABA-gated chloride channel in a manner similar to the cyclodienes. Moreover, the cross resistance extends to JKU 0422, and resistance to this compound is also present in a strain of cyclodiene-resistant German cockroach. These toxicological results, along with the neurophysiological studies, confirm that JKU 0422 has a mode of action that is similar to the cyclodienes and TBPS. These findings suggest that the introduction and use of new chloride channel antagonists as insecticides should be managed carefully in order to prevent the rapid development of resistance in the field. © 1994 Wiley-Liss, Inc.     

Actions of Insecticides on the Insect Gaba Receptor Complex

Abstract

The actions of insecticides on the insect γ-aminobutyric acid (GABA) receptor were investigated using [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) binding and voltage-clamp techniques. Specific binding of [³⁵S]TBPS to a membrane homogenate derived from the brain of Locusta migratoria locusts is characterised by a Kd value of 79.3 ± 2.9 nM and a B_max value of 1770 ± 40 fmol/mg protein. [³⁵S]TBPS binding is inhibited by mM concentrations of barbiturates and benzodiazepines. In contrast dieldrin, ivermectin, lindane, picrotoxin and TBPS are inhibitors of [³⁵S]TBPS binding at the nanomolar range. Bicuculline, baclofen and pyrethroid insecticides have no effect on [³⁵S]TBPS binding. These results are similar to those obtained in electrophysiological studies of the current elicited by GABA in both Locusta and Periplaneta americana central neurones. Noise analysis of the effects of lindane, TBPS, dieldrin and picrotoxin on the cockroach GABA responses reveals that these compounds decrease the variance of the GABA-induced current but have no effect on its mean open time. All these compounds, with the exception of dieldrin, significantly decrease the conductance of GABA-evoked single current.     

Polychlorinated Convulsant Insecticides Potentiate the Protective Effect of NaCl Against Heat Inactivation of [3H]Flunitrazepam Binding Sites

Six polychlorinated convulsant insecticides that potently inhibit t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes also potentiate the protective effect of NaCl (200 mM) against heat inactivation of [3H]flunitrazepam binding sites on the same membranes. Similar effects were obtained with all "cage" convulsants tested. The rank order of potencies as heat protection potentiators was similar to the rank order of potencies as inhibitors of [35S]TBPS binding (alpha-endosulfan greater than endrin greater than dieldrin greater than toxaphene greater than lindane). alpha-Endosulfan and endrin are more potent in both respects than any previously reported picrotoxin-like (cage) convulsant, but are much less toxic to mammals. The greatly reduced toxicities of alpha-endosulfan and endrin in mammals may reflect partial gamma-aminobutyric acid (GABA)-neutral properties of these compounds. Time courses of heat inactivation of [3H]flunitrazepam binding sites in the presence of 200 mM NaCl plus saturating concentrations of endrin or picrotoxin revealed monophasic components constituting about 88% of the binding sites, suggesting that virtually all [3H]flunitrazepam binding sites are coupled to picrotoxin binding sites in the GABA/benzodiazepine/picrotoxin receptor complex. Protection against heat inactivation constitutes a useful tool for characterizing the various allosterically linked binding sites in neurotransmitter receptor complexes.     

Similar properties of [35S]t-butylbicyclophosphorothionate receptor and coupled components of the GABA receptor-ionophore complex in brains of human, cow, rat, chicken and fish

No significant differences are evident in the specific binding characteristics of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to EDTA/water-dialyzed P2 membranes of human, cow, rat, chicken and fish brain. This species similarity includes dissociation constants of 61-77 nM at 37 degrees C, maximum receptor densities of 3-7 pmol/mg protein, and sensitivity to inhibition or displacement by gamma-aminobutyric acid (GABA), two cage convulsants (picrotoxinin and t-butylbicycloorthobenzoate) and the insecticide [1R,cis, alpha S]-cypermethrin, indicating a constancy during vertebrate evolution of the [35S]TBPS binding site and its coupling with other components of the GABA receptor-ionophore complex. As a possible exception, chicken and fish brain membranes appear to be less sensitive than the others to the insecticide alpha-endosulfan. Human and rat preparations are also essentially identical relative to the inhibition of radioligand binding by two GABA mimetics (muscimol and 3-amino-propanesulfonic acid), six other cage convulsants (including examples of three classes of polychlorocycloalkane insecticides), a potent anthelmintic agent (Ivermectin), dimethylbutylbarbiturate, the convulsant benzodiazepine Ro 5-3663, and ethanol. The findings to date with [35S]TBPS and the GABA receptor-ionophore complex in rat brain membranes are therefore generally applicable to human preparations. Cow brain is an appropriate source for large scale preparations in receptor purification studies since it is essentially identical to human and rat preparations in all parameters examined. Species differences in sensitivity to the toxic effects of the convulsants and polychlorocycloalkane insecticides considered are apparently not attributable to receptor site specificity.     

t-[35S]Butylbicyclophosphorothionate Binding under Equilibrium and Nonequilibrium Conditions: Differential Effects of Barbiturates and γ-Aminobutyric Acid in the Long-Sleep and Short-Sleep Selected Mouse Lines

Significant differences were demonstrated between the long-sleep (LS) and short-sleep (SS) selected mouse lines in the abilities of barbiturates and gamma-aminobutyric acid (GABA) to inhibit t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) binding to well-washed cerebral cortical membranes. Thus, using phenobarbital to initiate the dissociation of [35S]TBPS, the extent of inhibition was significantly greater in LS mice (but not SS mice) than would be predicted using equilibrium conditions. Pentobarbital had the opposite effect, causing [35S]TBPS to dissociate to a greater extent in SS than LS membranes. [35S]TBPS binding was dissociated from LS and SS membranes by GABA to a greater and lesser extent, respectively, than would be predicted from equilibrium studies. Because no line differences in the potencies of these drugs to inhibit [35S]TBPS binding were found using equilibrium conditions, these results indicate that the association rates of barbiturates and GABA may be different between these lines. These findings are consistent with neurochemical studies indicating differences in the benzodiazepine/GABA receptor-chloride channel complex in these selected lines and may explain their differential sensitivities to certain agents acting through this supramolecular complex.     

Blocking actions of heptachlor at an insect central nervous system GABA receptor.

The actions of the polychlorocycloalkane insecticide heptachlor, and its epoxide metabolite, were examined on GABA receptors in insects and vertebrates. Electrophysiological experiments on the cell body of the cockroach (Periplaneta americana) fast coxal depressor motor neuron (Df), and GABA-activated 36Cl- uptake experiments on microsacs prepared from cockroach ventral nerve cords showed that both heptachlor and heptachlor epoxide blocked functional GABA receptors. The block appeared to be non-competitive and was voltage-independent over the membrane potential range -75 mV to -110 mV. There was no significant difference between the potencies of heptachlor and heptachlor epoxide in the functional assays for insect GABA receptors. Both compounds inhibited [35S]-t-butylbicyclophosphorothionate [( 35S]TBPS) binding in insects and vertebrates. The findings provide further evidence for block of an insect GABA receptor/Cl- channel by the cyclodiene class of polychlorocycloalkanes, and reveal differences in the insecticide-[35S]TBPS binding site interactions of insects and vertebrates.     

Solubilization and Detergent Effects on Interactions of Some Drugs and Insecticides with the t-Butylbicyclophosphorothionate Binding Site Within the γ-Aminobutyric Acid Receptor-Ionophore Complex

Specific binding of [35S]t-butylbicyclophosphorothionate (TBPS) to rat brain membranes (RBM) is enhanced nine-fold by EDTA/water dialysis and 1.3- to 4.2-fold by 50 nM ketosteroid R 5135, or 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or related piperazine-N-alkanesulfonate buffers, or extensive washing with NaCl/Na phosphate or Na phosphate/citrate solution. About one-fifth of the [35S]TBPS binding capacity appears in the soluble fraction whereas the rest remains in particulate form on treatment of the EDTA/water-dialyzed RBM with 20 mM CHAPS. Similar KD values (64-86 nM) are obtained for the original EDTA/water-dialyzed membranes and the CHAPS-treated and/or -solubilized preparations. The Bmax of the EDTA-treated RBM is reduced five-fold on solubilization with CHAPS. The potency for displacement of [35S]TBPS changes in the presence of CHAPS or on CHAPS solubilization: gamma-aminobutyric acid (GABA) and muscimol inhibit specific [35S]TBPS binding more strongly in the absence than in the presence of CHAPS: TBPS, picrotoxinin, and photoheptachlor epoxide are almost equally active with RBM, RBM + CHAPS, and RBM solubilized with CHAPS. Levels of (1R, alpha S)-cis-cypermethrin and dimethylbutylbarbiturate which are inhibitory with RBM are moderately stimulatory after TBPS receptor solubilization. Thus CHAPS defines three regions of the GABA receptor-ionophore complex, i.e., the GABA and benzodiazepine receptors, the TBPS/picrotoxinin/polychlorocycloalkane receptor(s), and the sites at which the alpha-cyano pyrethroid and the barbiturate interact with TBPS binding.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Effects of Iodide and Chloride on Allosteric Interactions of the GABAA Receptor

t-[35S]Butylbicyclophosphorothionate [( 35S]TBPS) has been shown to bind to the GABAA receptor complex. The binding is modulated allosterically by drugs that interact at components of the receptor complex. The present studies were designed to evaluate the influence of ionic environment and state of equilibrium on the allosteric modification of [35S]TBPS binding. In both I- and Cl- under nonequilibrium conditions, diazepam, gamma-aminobutyric acid (GABA), and pentobarbital (PB) stimulate and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) inhibits [35S]TBPS binding. In addition, there is an inhibitory component to the effect of GABA and PB at higher drug concentrations. These effects are blocked by the appropriate antagonists for each drug. In Cl-, the stimulation of [35S]TBPS binding by drugs disappears at equilibrium, whereas the inhibition by GABA and PB persists. The inhibitory effect of DMCM in Cl- also disappears at equilibrium. When assayed in I- at equilibrium, however, DMCM stimulates [35S]TBPS binding. In addition, bicuculline, which is without effect under nonequilibrium conditions in either Cl- or I-, stimulates [35S]TBPS binding in I- at equilibrium. The persistent effects of DMCM, bicuculline, and GABA in I- are accompanied by alterations in the affinity of [35S]TBPS for its receptor. In addition, the stimulation of [35S]TBPS binding by GABA is associated with a decreased number of [35S]TBPS binding sites. These data demonstrate that receptor complex interactions with anions influence the responsiveness to drug binding.     

The General Anesthetic Propofol Enhances the Function of γ-Aminobutyric Acid-Coupled Chloride Channel in the Rat Cerebral Cortex

The effect of the general anesthetic propofol on t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to unwashed membrane preparations from rat cerebral cortex was studied and compared to that of other general anesthetics (pentobarbital, alphaxalone) which are known to enhance GABAergic transmission. Propofol produced a concentration-dependent complete inhibition of [35S]TBPS binding, an effect similar to that induced by pentobarbital and alphaxalone, although these agents differ markedly in potency (alphaxalone greater than propofol greater than pentobarbital). The concomitant addition of propofol either with alphaxalone or pentobarbital produced an additive inhibition of [35S]TBPS binding, suggesting separate sites of action or different mechanisms of these drugs. Moreover, although bicuculline (0.1 microM) completely antagonized the propofol-induced inhibition of [35S]TBPS binding, the effect of this anesthetic was not due to a direct interaction with the gamma-aminobutyric acidA (GABAA) recognition site. In fact, propofol, like alphaxalone and pentobarbital, markedly enhanced [3H]GABA binding in the rat cerebral cortex. Finally, propofol was able to enhance [3H]GABA binding in membranes previously incubated with the specific chloride channel blocker picrotoxin. Taken together these data strongly suggest that propofol, like other anesthetics and positive modulators of GABAergic transmission, might exert its pharmacological effects by enhancing the function of the GABA-activated chloride channel.     

γ-Aminobutyric Acid Receptor Ionophore Complexes: Differential Effects of Deltamethrin, Dichlorodiphenyltrichloroethane, and Some Novel Insecticides in a Rat Brain Membrane Preparation

The binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), a gamma-aminobutyric acid (GABA)-activated chloride ionophore ligand; [3H]diazepam, a benzodiazepine agonist; and [3H]muscimol, a GABA receptor probe, were used to assess the effects at 100 microM of deltamethrin, dichlorodiphenyltrichloroethane (DDT), and three experimental insecticides--a DDT-pyrethroid hybrid, GH414 (cycloprothrin), and two DDT-analogues, GH266 and GH149 (EDO), on GABA receptor ionophore complexes in a rat brain membrane preparation. GH266 and GH149 were found to inhibit a greater percentage of [35S]TBPS binding than the same concentration of deltamethrin or DDT, although GH414 had little effect. GH266 and GH149 enhanced [3H]diazepam binding by nearly 200%, in contrast to the inhibitory effects of deltamethrin, DDT, and GH414. GH266 and GH149 also caused a dramatic enhancement of [3H]muscimol binding, 367 and 236% of control, respectively, whereas DDT and deltamethrin caused only a moderate enhancement. The effects of the insecticides on binding affinity and density were examined for each of the ligands. The results show a differential interaction of the insecticides on the various ligand binding sites.     

Binding Characteristics of t-[35S]Butylbicyclophosphorothionate in Discrete Brain Regions of Rats Made Tolerant to and Dependent on Pentobarbital

The effects of acute and continuous pentobarbital administration by pellet implantation on binding characteristics of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) in discrete regions of rat brains were examined. Acute administration of pentobarbital (60 mg/kg, s.c.) affected neither the KD nor the Bmax values of [35S]TBPS binding in any of the regions studied. The cerebella of pentobarbital-tolerant rats had an increased density of [35S]TBPS binding sites with no change in their apparent affinity. There were no significant changes in the binding characteristics in the frontal cortex (FC), the striatum (ST), and the substantia nigra (SN) of these animals. Twenty-four hours after removal of the pentobarbital pellets, a significant decrease in the latency of onset of first twitch response induced by pentylenetetrazol (PTZ) (50 mg/kg, i.p.) was observed. In addition, the density of [35S]TBPS binding sites was significantly increased in the FC, the SN, and the cerebellum but not in the ST. In all brain regions studied, placebo pellet implantation and pentobarbital tolerance and dependence caused no changes in the apparent affinity of [35S]TBPS binding or the IC50 of pentobarbital for the inhibition of [35S]TBPS binding. These results suggest that [35S]TBPS binding was significantly increased following the withdrawal of the pentobarbital pellets without altering intrinsic coupling activity of barbiturate recognition sites and convulsant binding sites and that these increases in [35S]TBPS binding are related to the increased susceptibility to seizures induced by PTZ in rats made dependent on pentobarbital.     

Foot-Shock Stress and Anxiogenic β-Carbolines Increase t-[35S]Butylbicyclophosphorothionate Binding in the Rat Cerebral Cortex, an Effect Opposite to Anxiolytics and γ-Aminobutyric Acid Mimetics

The effect of foot-shock stress on t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) binding to fresh unwashed membrane preparations from rat cerebral cortex was studied and was compared to those of GABAA receptor agonists and antagonists and to positive and negative modulators of the GABAergic transmission. [35S]TBPS binding was increased in the cerebral cortex of rats exposed to foot shock compared to that of nonstressed rats. Scatchard analysis revealed that the effect of foot shock was due to an increase in the total number of [35S]TBPS binding sites. In contrast, the in vitro addition of muscimol or GABA induced a dose-dependent inhibition of [35S]TBPS binding, an effect abolished by the concomitant addition of the GABA receptor antagonist, bicuculline, which, per se, enhanced [35S]TBPS binding by 73%. Thus, bicuculline, similar to stress, increased [35S]TBPS binding in the same membrane preparation. In contrast to stress, the anxiolytic and positive modulators of the GABAergic transmission (ZK 93423, ZK 91296, and diazepam) inhibited the specific binding of [35S]TBPS in a concentration-dependent manner. The greatest inhibitory effect was produced by ZK 93423 at 30 microM (31% of control), followed by diazepam (54% of control) and by the partial agonist ZK 91296 (61% of control). Scatchard plot analysis indicated that the inhibition induced by ZK 93423 and diazepam was due to a decrease in the density of [35S]TBPS recognition sites. On the other hand, the anxiogenic beta-carbolines DMCM and FG 7142 mimicked the effect of stress. Thus, at a 10 microM concentration, DMCM and FG 7142 increased [35S]TBPS binding by 22% and 26%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

t-[3H]Butylbicycloorthobenzoate: New Radioligand Probe for the γ-Aminobutyric Acid–Regulated Chloride Ionophore

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.     

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.     

In Vivo Administration of Ethanol Enhances the Function of the γ-Aminobutyric Acid-Dependent Chloride Channel in the Rat Cerebral Cortex

The effect of in vivo administration of ethanol on the gamma-aminobutyric acidA (GABAA) receptor-coupled chloride channel was studied by measuring ex vivo t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in the rat cerebral cortex. Intragastric administration of ethanol (0.5-1 g/kg) elicited in 40 min a significant decrease of [35S]TBPS binding to unwashed cortical membrane preparations, an effect mimicked by diazepam (0.5-1 mg/kg, i.p.). However, Scatchard plot analysis indicated that, unlike the case with diazepam, the decrease was entirely due to a reduction in the apparent affinity of [35S]TBPS receptors with no change in the total number of binding sites. Moreover, ethanol, like diazepam, reduced the increase of [35S]TBPS binding elicited by isoniazid (350 mg/kg, s.c.), an inhibitor of the GABAergic transmission. Finally, ethanol markedly potentiated the inhibitory action of diazepam on [35S]TBPS binding. The results suggest that ethanol, like benzodiazepines, enhances the function of the GABAA-coupled chloride channel.     

Enhancement of t-[35S]Butylbicyclophosphorothionate and [3H]Strychnine Binding by Monovalent Anions Reveals Similarities Between γ-Aminobutyric Acid- and Glycine-Gated Chloride Channels

The characteristics of [3H]strychnine and t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding to sites associated with glycine- and gamma-aminobutyric acid (GABA)-gated chloride channels were compared in the presence of a series of anions with known permeabilities through these channels. Good correlations were found between (a) the potencies (EC50) of these anions to stimulate radioligand binding and their permeabilities relative to chloride; (b) the affinities (KD) of these radioligands in the presence of fixed concentrations of these anions and their relative permeabilities; (c) the potencies (EC50) of these anions to stimulate [35S]TBPS and [3H]strychnine binding; and (d) the affinities (KD) of [3H]strychnine and [35S]TBPS measured at a fixed concentration of these anions. These studies support electrophysiological and biochemical observations demonstrating similarities between glycine- and GABA-gated chloride channels, and suggest that anions enhance [3H]strychnine and [35S]TBPS binding through specific anion binding sites located at the channels.