Receptor-chloride Ionophore Complex Research Articles

Single dose efficacy evaluation of two partial benzodiazepine receptor agonists in photosensitive epilepsy patients: A placebo-controlled pilot study

Benzodiazepines (BZDs) are highly effective to suppress various types of seizures; however, their clinical use is limited due to adverse effects and tolerance and dependence liability. Drugs that act only as partial agonists at the BZD recognition site (initially termed “BZD receptor”) of the GABAA receptor chloride ionophore complex or exhibit a GABAA receptor subtype-selectivity are thought to have advantages vs. full agonists such as diazepam and most other clinically used BZDs in that such compounds have less adverse effects and reduced or absent tolerance and dependence liability. One of such compounds, abecarnil, has been clinically evaluated as a novel anxiolytic drug, but, despite its potent preclinical anti-seizure activity, it has not yet been evaluated in patients with epilepsy. In the present proof-of-concept study, we performed a within-subject placebo-controlled, single oral dose study of abecarnil in patients with photosensitive epilepsy. Flumazenil, which is generally considered a BZD receptor antagonist, but has slight partial agonistic properties, was used for comparison. In total, 12 patients were enrolled in this study. Abecarnil, 5 or 10mg, completely abolished the photo-paroxysmal EEG response, while flumazenil, 30, 60 or 100mg, was less effective. The anti-epileptic effect of abecarnil was significantly different from both placebo and flumazenil. Sedative adverse effects were observed after abecarnil but not flumazenil. The study substantiates previous pre-clinical experiments that abecarnil exerts pronounced anti-seizure activity. Epilepsy is often associated with anxiety, so that the anxiolytic activity of abecarnil would be an added advantage when using this compound in epilepsy patients.

Alterations in GABAergic function following forced swimming stress

Forced swimming induces alterations in the GABA brain concentration and could change the sensitivity of the GABA/benzodiazepine receptor–chloride ionophore complex to benzodiazepines. This change in sensitivity could be explained by the allopregnanolone release that takes place during stress. The current study was carried out to determine whether forced swimming is able to modify the anti-anxiety effect of diazepam and to explore the possible relation of this change to allopregnanolone, the GABA concentration or/and the GABA/benzodiazepine receptor density. Unstressed and stressed mice, injected with the vehicle or diazepam, were evaluated in the exploratory behavior test. Diazepam induced clear anxiolytic actions at all doses in unstressed animals, but such an effect was not observed in stressed animals. The injection of allopregnanolone 24 h before the anxiety test blocked the effect of this benzodiazepine. Forced swimming decreased GABA concentrations in the hippocampus and the thalamus–hypothalamus region, besides decreasing the [ 3H]flunitrazepam labeling in both the hypothalamus and amygdala. These results show that forced swimming abolishes the anti-anxiety effect of diazepam.

Fe 2+ decreases the taurine-induced Cl − current in acutely dissociated rat hippocampal neurons

The effects of ferrous ions (Fe 2+) on taurine-induced Cl − current ( I tau) recorded from single neurons, which was freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Using standard pharmacological approaches, we found that the currents gated by concentrations of taurine (≤10 mM), which existed in about 90% of the hippocampal neurons tested, were predominantly mediated by strychnine-sensitive glycine receptors. When co-applied with taurine, Fe 2+ effectively depressed I tau in a concentration-dependent manner, with an IC 50 of 3.76 mM and Hill coefficient of 1.01, while preincubation with 1 mM Fe 2+ alone did not affect the following membrane currents elicited by taurine. The result suggests that resting taurine-gated channels are insensitive to Fe 2+. Since internal cell dialysis with 3 mM Fe 2+ failed to modify I tau, it was deduced that the site of action of Fe 2+ is extracellular. Furthermore, the Lineweaver–Burke double reciprocal plot of normalized response to taurine against the concentration of taurine illustrated that the depression of I tau was noncompetitive, therefore Fe 2+ may act on the glycine receptor–chloride ionophore complex at a site distinct from where taurine binds. Various concentrations of Fe 2+ ranging from 0.1 to 20 mM depressed I tau and this extracellular depression was independent of membrane voltage. These results indicate that Fe 2+ decreases I tau in acutely dissociated rat hippocampal neurons and the inhibition of glycine receptors by Fe 2+ might be one possible approach through which Fe 2+ induces seizures.

Antidepressant effects on GABA-stimulated 36Cl − influx in rat cerebral cortex are altered after treatment with GABA A receptor antisense oligodeoxynucleotides

Antidepressants act at the GABA A receptor to inhibit GABA-stimulated 36Cl − influx and GABA reduction of [ 35S]TBPS binding. This study examined how selective knock-down (via antisense oligodeoxynucleotides, aODNs) of GABA A receptor subunits modified antidepressant activity. The specific aODNs used were for the α1, β1, β2 or γ2 subunits of the GABA A receptor. The aODN microinjections reduced corresponding GABA A receptor subunit mRNA levels by 30–40% as assessed by RT-PCR. The inhibitory effect of the antidepressants amitriptyline and mianserin on GABA-stimulated 36Cl − influx was decreased after microinjections of α1, β1, or β2 subunit aODNs but potentiated after microinjections of γ2 subunit aODNs. This pattern of aODNs effect on amitriptyline and mianserin modulation of GABA-stimulated 36Cl − influx was the same for both antidepressants and similar to GABA but different than that of diazepam and bicuculline. We conclude that multiple subunits of the GABA A receptor regulate the effect of amitriptyline and mianserin on the GABA A receptor chloride ionophore complex. However, the exact identity of the subunit mediating the direct or allosteric modulation of the antidepressant effect on GABA-stimulated 36Cl − influx remains unclear.

Cocaine decreases the glycine-induced Cl − current of acutely dissociated rat hippocampal neurons

The effects of cocaine on glycine-induced Cl − current ( I GLY) of single neurons, freshly isolated from the rat hippocampal CA1 area, were studied with conventional whole-cell recording under voltage-clamp conditions. Cocaine depressed I GLY in a concentration-dependent manner, with an IC 50 of 0.78 mM. Preincubation with 1 mM cocaine alone had no effect on I GLY, suggesting that resting glycine channels are insensitive to cocaine. The depression of I GLY by cocaine was independent of membrane voltage. Internal cell dialysis with 1 mM cocaine failed to modify I GLY. Because the depression of I GLY was noncompetitive, cocaine may act on the glycine receptor–chloride ionophore complex at a site distinct from that to which glycine binds. The cocaine suppression of I GLY was unaffected by 1 μM tetrodotoxin and 1 μM strychnine. Blockers of protein kinase C (Chelerythrine), kinase A ( N-[2-(( p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl, (H-89)) and Ca-calmodulin-dependent kinase (1-[ N, O-bis(5-isoquinolinesulfonyl)- N-methyl- l-tyrosyl]-4-phenylpiperazine (KN-62)) were also ineffective, which suggests that these phosphorylating mechanisms do not modulate cocaine-induced suppressant action on I GLY. This extracellular, strychnine-independent depression of I GLY may contribute to cocaine-induced seizures.

The anxiolytic-like effects of the neurosteroid allopregnanolone: interactions with GABA A receptors

The neurosteroid 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) was administered systemically to rats which were tested in the Geller-Seifter conflict paradigm, an established animal model of anxiety. Allopregnanolone was found to produce significant anxiolytic-like effects at a dose of 8 mg/kg. When three ligands that function at different sites on the γ-aminobutyric acid/benzodiazepine receptor-chloride ionophore complex (GABA A receptors) were examined in conjunction with allopregnanolone, the anti-conflict effects of allopregnanolone were effectively reversed only by the benzodiazepine receptor inverse agonist RO15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo [1,5-α]-[1,4]benzodiazepine-3-carboxylate). Since this inverse agonist has been reported to inhibit the GABA A-activated chloride flux in neuronal membranes, it is likely that the stimulation of the chloride channel in GABA A receptors is an important component of the effects of allopregnanolone. In contrast, the benzodiazepine receptor antagonist flumazenil (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo [1,5-α]-[1,4]benzodiazepine-3-carboxylate) did not block the anxiolytic-like actions of allopregnanolone, indicating that allopregnanolone does not bind at the benzodiazepine site directly. Isopropylbicyclophosphate, which binds at the picrotoxinin site on the GABA A receptors and blocks the behavioral actions of ethanol, also dose-dependently reversed the anti-conflict effect of this neurosteroid. The results suggest that allopregnanolone may be working either at a site specific for the benzodiazepine receptor inverse agonist RO15-4513 or at the picrotoxinin site to produce its potent anxiolytic-like behavioral effects. © 1997 Elsevier Science B.V.

GABA and epileptogenesis.

GABA and epileptogenesis.

Ro 15-4513 potentiates, instead of antagonizes, ethanol-induced sleep in mice exposed to small platform stress

The effects of ethanol and the benzodiazepine receptor ligand ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo-[1,5 a] [1,4] benzodiazepine-3-carboxylate (Ro 15-4513), were examined in NMRI mice exposed to small platform stress. This model contains several factors of stress, like rapid eye movement (REM) sleep deprivation, isolation, immobilization, falling into water and soaking. In control mice, ethanol exerted an anxiolytic effect in the plus-maze, but did not further enhance the anxiolytic-like effects induced by small platform stress. Ro 15-4513 antagonized ethanol-induced sleep in control animals, but enhanced the hypnotic and lethal actions of ethanol in small platform stressed mice. Small platform stress did not alter the characteristics ( K D and B max) of [ 3H]Ro 15-4513 binding to cerebellar membranes. Muscimol-stimulated 36Cl − uptake into brain microsacs was significantly reduced in cortex from small platform stressed animals. Ethanol had no effect on 36Cl − uptake into brain microsacs from cortex or cerebellum. It is proposed that small platform stress alters the activity of the γ-aminobutyric acid (GABA) A receptor-chloride ionophore complex, causing changes in the interaction between ethanol and Ro 15-4513.

Is upregulation of benzodiazepine receptors a compensatory reaction to reduced GABAergic tone in the brain of stressed mice?

Effects of various forms of stress on the GABAA receptor-chloride ionophore complex in the brain of NMRI mice were investigated. Male albino mice were subjected to stress by placing them on small platforms (SP; 3.5 cm diameter) surrounded by water for 24 h. This experimental model contains several stress factors like rapid eye movement (REM) sleep deprivation, isolation, immobilization, falling into water and soaking. As additional stress control groups we used animals subjected to isolation, large platform (9.0 cm diameter) and repeated swimming stress. SP stress induced an increase in the number of cortical benzodiazepine (BDZ) receptors and a reduction in the GABA-stimulated 36Cl-uptake by brain microsacs, whereas none of these changes could be observed in animals exposed to isolation, swimming or large platform stresses. Furthermore, the amount of GABA-induced stimulation of [3H]flunitrazepam binding was reduced in cortical brain membranes of SP-stressed animals, an effect due to fact that these animals displayed an increase in the basal [3H]flunitrazepam binding, whereas the absolute level of maximally enhanced BDZ binding in the presence of GABA did not differ from those found in controls. Neither basal [3H]muscimol binding or thiopentone sodium-induced stimulation of [3H]flunitrazepam binding were changed in any group of stressed mice. It is proposed that the observed upregulation in the number (Bmax) of cortical BDZ receptors in SP-stressed mice may represent a compensatory response to a stress-induced attenuation of GABAergic neurotransmission.

Stable high expression of human gamma-aminobutyric acid a receptors composed of alpha and beta subünitS

Multiple classes of pharmacological agents including benzodiazepines, cage convulsants like t-butylbicyclophosphorothionate (TBPS), barbiturates and neuroactive steroids allosterically modulate the y-aminobutyric acidA receptor-chloride ionophore complex (GRC). The function of benzodiazepines requires a GRC comprised of α, β and γ subunits, while TBPS, barbiturates and neuroactive steroids will allosterically modulate GRCs comprised of only a and β subunits. Binary αβ complexes are still hypothesized to be expressed in the mammalian brain particularly during development and could contribute to the pharmacological action of neuroactive steroids and barbiturates. In order to examine binary αβ complexes we report here the establishment of stable cell lines that express high levels of human GABA a receptors comprised of α1β1, α2β1 and a3β1 subunit combinations. The apparent potencies for allosteric modulation of [ 35S]TBPS for most naturally occurring neuroactive steroids for the binary subunit combinations was similar to that of the γ-containing subunit combinations. Also discussed is the usefulness of these cell lines for the biophysical analysis of the GABa a receptor stoichiometry.

Anti-anxiety effects of progesterone and some of its reduced metabolites: an evaluation using the burying behavior test

In this study, pregnenolone sulfate, progesterone and some of its main reduced metabolites were tested for anxiolytic properties in rats using the burying behavior paradigm. All steroids were subcutaneously injected. Progesterone (0.5, 1.0, 2.0 and 4.0 mg/rat) and its 3α-5α-reduced metabolite (0.125, 0.250, 0.5 and 1.0 mg/rat) produced a clear dose-dependent anxiolytic response, without affecting the spontaneous ambulatory behavior. 5α-Pregnanedione decreased burying behavior only at the highest dose (4.0 mg/rat). Pregnenolone sulfate (1.0, 2.0 and 4.0 mg/rat), 5β-pregnanedione and 3β or 5β-pregnanolones were devoid of effects. The 3β-5β-reduced metabolite of progesterone (2.0 and 4.0 mg/rat) decreased motor activity, without altering the burying behavior. These results demonstrate that the 3α-5α metabolite of progesterone shows the highest anxiolytic potency in the burying behavior test, when compared with all steroids evaluated. The data are discussed in terms of the close structure-activity relationship requirements of steroids to stimulate the GABA/benzodiazepine receptor-chloride ionophore complex.

Lack of effect of flumazenil on the reversal of propofol anaesthesia

Propofol, like the benzodiazepines, activates the GABAA receptor-chloride ionophore complex; they potentiate one another. Since neither pharmacodynamic nor pharmacokinetic data concerning drug interaction between flumazenil and propofol is available, and especially considering the relationship of binding sites, flumazenil, the antagonist of benzodiazepines, was investigated to determine its effect upon recovery from propofol anaesthesia. Forty women receiving dilatation and curettage procedures were included in this double-blind test. After 50 micrograms fentanyl, propofol 2 mg.kg-1 was injected for induction and followed by infusion at the rate of 15 mg.kg-1.hr-1. After the operation, patients were given normal saline (Group A) or flumazenil 10 micrograms.kg-1 (Group B) randomly. Recovery time in Group A was 15.2 +/- 5.1 min and Group B 15.8 +/- 4.8 min. Propofol concentrations at the end of infusion were 4.17 +/- 1.33 micrograms.ml-1 (Group A) and 4.03 +/- 1.45 micrograms.ml-1 (Group B); these then declined to 1.22 +/- 0.17 micrograms.ml-1 (Group A) and 1.18 +/- 0.15 micrograms.ml-1 (Group B) when patients were able to open their eyes on command. No significant differences were found between the groups based on propofol concentrations and recovery time, nor did haemodynamic changes differ between them after administration of reversal agents. It was concluded that flumazenil 10 micrograms.kg-1 does not influence recovery from propofol anaesthesia.

Alterations in the brain GABAA/benzodiazepine receptor-chloride ionophore complex in a genetic model of paroxysmal dystonia: A quantitative autoradiographic analysis

Dystonia is a relatively common syndrome of sustained muscle contractions, frequently causing twisting and repetitive movements or abnormal postures. The most frequent type of dystonia is idiopathic generalized dystonia, whose pathophysiology is largely unknown. In this respect, mutant animal strains with inborn dystonia may be helpful to elucidate the pathophysiological defects involved in idiopathic dystonia. The genetically dystonic (dtsz) hamster is an animal model of paroxysmal dystonia that displays attacks of generalized dystonia either spontaneously or in response to mild environmental stimuli. In the present study, a quantitative autoradiographic analysis of ligand binding to different sites of the GABAA/benzodiazepine receptor-chloride ionophore complex was carried out in 123 brain areas from genetically dystonic mutant hamsters and age-matched control hamsters. Animals were killed 2 weeks after their last dystonic attack. Analysis of the GABA-binding site of the receptor complex, using the ligand [3H]muscimol, and the benzodiazepine site labelled with [3H]flunitrazepam revealed no significant alterations in the binding of either ligand in any of the brain regions examined. In contrast, widespread changes were observed in binding densities of [35S]t-butylbicyclophosphorothionate ([35S]t-butylbicyclophosphorothionate), which labels the picrotoxinin site of the GABAA receptor-chloride ionophore complex. Significantly increased [35S]t-butylbicyclophosphorothionate binding was found in several parts of the thalamus, cortex, and hippocampus as well as in the red nucleus, the subthalamic nucleus, and the granular layer of the cerebellum. Since high-affinity [35S]TBPS binding is thought to represent the closed conformation of the GABA-gated chloride ionophore, increased TBPS binding would indicate an impaired GABAergic function. The study is consistent with the concept that dystonia is caused by impaired connections between the basal ganglia, the thalamus, and frontal association areas. The data on increased [35S]TBPS binding are the first evidence implicating alterations in the GABA-gated chloride ion channel function in a movement disorder, i.e. idiopathic generalized dystonia.

Propofol Activates GABAA Receptor-Chloride Ionophore Complex in Dissociated Hippocampal Pyramidal Neurons of the Rat

The molecular mechanism of propofol anesthesia has been related to facilitation of the inhibitory neurotransmission mediated by gamma-aminobutyric acid (GABA). In the current study, the authors examined the direct actions of propofol on the acutely dissociated mammalian central neurons. Hippocampal pyramidal neurons were dissociated after enzymatic treatment of the brain slices of the rat. Single neurons were voltage-clamped using the whole cell configuration of the patch clamp technique, and drugs were applied with a rapid drug-application system. In the pyramidal neurons voltage-clamped at -60 mV, propofol evoked a transmembrane inward current, which desensitized at high concentrations of the anesthetic. The peak amplitude of the current increased sigmoidally with increasing doses of propofol applied. A least-squares fitting gave a dissociation constant of 1.2 x 10(-5) M and a Hill coefficient of 1.8, thereby indicating that clinical concentrations of propofol evoke the current, and that the anesthetic cooperatively activates the channel. The threshold concentration was less than 10(-6) M. The reversal potential for the current shifted according to the chloride equilibrium potential predicted by the Nernst equation, indicating that the current was carried by chloride ions. Bicuculline and strychnine suppressed the current in a concentration-dependent manner, in which the former was almost 40-fold more potent than the latter. The propofol-induced current cross-desensitized with the GABA-induced current, but no such interaction was observed with the glycine-induced current. Ro15-1788 (10(-6) M), an allosteric benzodiazepine antagonist, had no effect on the response. Diazepam (10(-6) M) enhanced the propofol-induced current, but pentobarbital (10(-6) M and 3 x 10(-5) M) did not affect the current. Propofol at clinically relevant concentrations directly activates the GABAA receptor-chloride ionophore complex in the mammalian central neurons and, hence, increases the chloride conductance, which may contribute to anesthesia produced by the agent. The desensitization of the GABAA receptor in the presence of high concentrations of propofol may result in a suppression of the GABAA inhibitory system.

Changes in intrinsic inhibition in isolated hippocampal slices during ethanol withdrawal; lack of correlation with withdrawal hyperexcitability.

1. Intracellular recordings were made from pyramidal cells in area CA1 in mouse isolated hippocampal slices, after chronic ethanol treatment in vivo. 2. Fast i.p.s.ps were isolated by injection of the impaled neurones with QX314 (to block fast sodium currents and the slow i.p.s.p.) and stimulating the interneurones in the presence of the glutamatergic blockers, CNQX and APV. 3. The isolated fast-inhibitory postsynaptic potential (f.-i.p.s.p.) was measured at intervals during the 7 h withdrawal period. The reversal potential and sensitivity to bicuculline suggested that the isolated f.-i.p.s.p. was mediated by activation of the GABAA receptor-chloride ionophore complex. 4. Measurement of stimulus-response relationships for the f.-i.p.s.ps revealed an initial increase in the maximum size of the i.p.s.p., evoked from a membrane potential of -50 mV, seen at 2 h into ethanol withdrawal. This was attributed to a negative shift in the reversal potential, Ei.p.s.p., with no observed change in conductance, Gi.p.s.p. 5. No differences in f.-i.p.s.ps evoked during ethanol withdrawal or in control slices were seen at 4 h or 6 h. At these times, epileptiform activity was seen in previous field potential recordings. 6. Paired pulse depression of the f.-i.p.s.p. was significantly increased at 2 h into withdrawal, when a 150 ms pulse interval was used. No differences were seen at later times in the ethanol withdrawal period. 7. The results suggest that ethanol withdrawal hyperexcitability in isolated hippocampal slices is not caused by primary decreases in inhibition mediated by the GABAA receptor-chloride ionophore complex.4. Measurement of stimulus-response relationships for the f.-i.p.s.ps revealed an initial increase in the maximum size of the i.p.s.p., evoked from a membrane potential of - 50 mV, seen at 2 h into ethanol withdrawal. This was attributed to a negative shift in the reversal potential, Ejp.sp with no observed change in conductance, Gj ps p.5. No differences in f.-i.p.s.ps evoked during ethanol withdrawal or in control slices were seen at 4 h or 6 h. At these times, epileptiform activity was seen in previous field potential recordings.6. Paired pulse depression of the f.-i.p.s.p. was significantly increased at 2 h into withdrawal, when a 150 ms pulse interval was used. No differences were seen at later times in the ethanol withdrawal period.7. The results suggest that ethanol withdrawal hyperexcitability in isolated hippocampal slices is not caused by primary decreases in inhibition mediated by the GABAA receptor-chloride ionophore complex.The increase in the f.-i.p.s.p. during the initial stages of the withdrawal might prevent the overt expression of epileptiform activity at this time.

Bidirectional effect of β-carboline agonists at the benzodiazepine-GABA A receptor chloride ionophore complex on GABA-stimulated 36Cl − uptake

β-Carboline agonists produced a left shift of the GABA concentration-chloride uptake curve or a reduction in the maximal increase in GABA-stimulated 36Cl − uptake depending on their concentration. The enhancement of the GABA effect occurs only at lowerβ-carboline and GABA concentrations and is smaller for the partial agonist ZK 91296 compared to the full agonist ZK 93423. The opposite effect, inhibition of GABA-stimulated chloride conductance, is observed only at higher concentrations of β-carboline agonists and GABA. The reduction of the GABA maximal response by the partial agonist ZK 91296 is greater than by the full agonist ZK 93423. The transformation of GABA-stimulated 36Cl − uptake data to specific chloride influx ( 36Cl − uptake per nM of GABA) reveals that the GABA concentration-response curve consists of three parts characterized by differences in the molar effectiveness of GABA relative to the GABA concentration. The molar effectiveness of GABA is a measure of the sensitivity of the GABA A receptor chloride ionophore complex and shows adaptive changes by this complex to increasing concentrations of GABA and/or β-carboline. We conclude from our data that the change from GABA-sensitive to GABA-insensitive conformation of the GABA A receptor occurs with increasing concentrations of GABA andJor β-carboline. Both conformations maintain positive heterotropic cooperativity with β-carboline binding sites, one responsible for positive and the other responsible for negative effects of β-carboline agonists on chloride uptake.

Gamma-aminobutyric acid (GABAA) receptor-function in a rat model of hepatic encephalopathy

The functional activity of the gamma-aminobutyric acid (GABAA) receptor-chloride ionophore complex was studied in rats with hepatic encephalopathy (HE) secondary to thioacetamide-induced fulminant hepatic failure (FHF). Muscimol stimulation and benzodiazepine potentiation of GABA receptor-mediated 36Cl- uptake into cerebral cortical synaptoneurosomes was compared in HE and control rats. [3H]Flumazenil binding assays were conducted to determine whether the levels of endogenous benzodiazepine-like ligands in extracts of cortex were increased with stages of encephalopathy in this animal model of HE. In both control and HE rats maximal uptake of 36Cl- via the GABAA receptor complex occurred at muscimol concentrations of 30 microM. Potentiation of muscimol-stimulated 36Cl- uptake into synaptoneurosomes by diazepam (5 microM) was equivalent in both groups. Aqueous extracts of proteolytically digested homogenates of cerebral cortices prepared from control and HE rats were effective in stimulating 36Cl- uptake into synaptoneurosomes. Alkaline organic extracts of proteolytically digested homogenates of cerebral cortices from HE rats were more effective than corresponding extracts from controls at inhibiting the binding of [3H]flumazenil. Inhibition of [3H] fumazenil binding by organic extracts derived from the cerebral cortices of HE rats did not increase with progression of encephalopathy. The results show that muscimol-stimulated 36Cl- uptake into synaptoneurosomes and, consequently, GABAA receptor-mediated chloride channel function are not significantly altered in the model of HE studied and are consistent with the hypothesis that HE results in an increased availability of one or more endogenous ligands which can augment GABA receptor-gated chloride conductance.

γ-Aminobutyric acid antagonism produced by an organophosphate-containing combustion product

Results from [ 35S] t-butylbicyclophosphorothionate (TBPS) binding and γ-aminobutyric acid (GABA)-dependent 36Cl − uptake indicated that the pyrolysis product of liquid trimethylol propane (TMP) containing organic phosphates interacts with the GABA/benzodiazepine receptor chloride ionophore complex (GBRC) producing a receptor-mediated reduction in 36Cl − flux. The pyrolysate-ligand interaction was competitive and directly proportional to the trimethylol propane phosphate (TMPP) content of the combustion product. TBPS displacement demonstrated that TMPP had less affinity than did TBPS and picrotoxin, but greater affinity than GABA, racemic dimethylbutyl barbituric acid (DMBB), clonazepam, and phenobarbital for their respective recognition sites. Addition of exogenous GABA was a necessary condition for demonstrating clonazepam-induced ligand displacement, thus being indicative of an allosteric interaction. The same rank order held for altering GABA-dependent 36Cl − uptake with the caveat of inhibitors vs enhancers, and noting that pentobarbital was less potent than DMBB. Concentration-dependent ligand displacement by GBRC agonists and antagonists quantitatively correlated with alterations in GABA-dependent 36Cl − uptake. The median convulsant dose (CD50) of pyrolysate TMPP was higher than that of synthetic TMPP. The presence of combustion by-products altering the dispositional kinetics of the former is felt to be the reason for this difference. Under our experimental conditions, phenobarbital was the most effective antidote for pyrolysate (TMPP) toxicity. The inhalant convulsant, flurothyl, was found not to displace [ 35S]TBPS binding, not to affect GABA-dependent 36Cl − uptake, and not to respond to the anticonvulsants in a manner consistent with a strict GABA A receptor phenomenon.

Differential immune responsiveness in mouse lines selectively bred for high and low sensitivity to ethanol

Natural killer cell activity was compared in the Long-Sleep and Short-Sleep mouse lines. These mice, initially selected for their sensitivities to a hypnotic dose of ethanol, are also differentially sensitive to other agents which act through the benzodiazepine/GABA receptor chloride ionophore complex. Natural killer cell activity was 40–59% lower in Short-Sleep when compared to Long-Sleep mice. Flow cytofluorometric analysis demonstrated that the number of Nk-1 + cells was also lower in the spleens of Short-Sleep than Long-Sleep mice. In addition, the incidence of 3-methylcholanthrene-induced tumors was significantly greater in Short-Sleep (85.7%) than in Long-Sleep (14.3%) mice. These results suggest that the Long-Sleep and Short-Sleep mouse lines may represent a unique model to assess the physiological role of the benzodiazepine/GABA receptor chloride ionophore complex in the neural modulation of immune function.

Antidepressant-anxiolytic interactions: Involvement of the benzodiazepine-GABA and serotonin systems

1. 1. Recent studies have demonstrated that antidepressant drugs are actually more effective than BZ's in the treatment of anxiety states. The role of two major neurochemical substrates that may be implicated in the anxiolytic activity of antidepressants, the benzodiazepine (BZ)-GABA receptor chloride ionophore complex and central serotonergic pathways, are focused on in this review. 2. 2. A wide range of antidepressants elicit a reduction in BZ receptors and display anxiolytic effects within a conflict paradigm. 3. 3. The anxiolytic activity of antidepressants, however, does not appear to be mediated via the BZ receptor, but possibly via another component of the complex such as the chloride channel-associated with the GABA A receptor. 4. 4. Additionally, as possible candidates for the mechanism of anxiolytic activity of these compounds, results of pharmacological, behavioral and clinical studies point to the importance of serotonin (5-HT) 1A receptors and 5-HT transporter sites as targets for the action of antidepressants, triazolobenzodiazepines and anxioselective piperazine derivatives.