GABA-benzodiazepine Receptor Complex Research Articles

Interaction of benzodiazepine receptor agonists and inverse agonists with the GABA benzodiazepine receptor complex

The effects of the benzodiazepine receptor agonists, antagonists and inverse agonists on the in vitro binding of several ligands which label different recognition sites of the GABA benzodiazepine receptor complex are summarized. Also, results with a novel biochemical in vitro functional model of the GABA benzodiazepine receptor complex are presented. They are compatible with the concept that drugs which act on benzodiazepine receptors can lead to a bidirectional modulation of the gain of GABAergic neurotransmission.

The effects of putative anxiolytic compounds (PK 8165, PK 9084 and tracazolate) on the rat corticosterone response

The effects were investigated of three putative anxiolytics, thought to act at the GABA-benzodiazepine receptor complex, on basal corticosterone and novelty-induced rise in plasma corticosterone concentrations. The two phenylquinolines, PK 8165 and PK 9084, increased basal concentrations, but reduced the novelty-induced rise in corticosterone; these actions resemble those found with high doses of benzodiazepines. Tracazolate, a pyrazolopyridazine that enhances the binding of benzodiazepines to their receptors, was without significant effect in either situation.

Benzodiazepines and behavior

This article sets the background for the following series of articles on the behavioral actions of drugs acting at the GABA-benzodiazepine receptor complex. The articles provide detailed analyses of particular behaviors and emphasise the danger of interpreting all behavioral effects in terms of anxiety. What also emerges clearly from several of the articles is that any agonist/inverse agonist classification is test-dependent and that global categorisation is not possible for many of the novel compounds.

What can be learned from the effects of benzodiazepines on exploratory behavior?

The purpose of this review is to assess the value of using tests of exploratory behavior to study the actions of benzodiazepines. The methods of measuring exploration and the factors influencing it are briefly described. The effects of benzodiazepines on exploratory behavior of rats and mice are reviewed; and the dangers of interpreting the results of such tests in terms of any of the clinical effects of the benzodiazepines is stressed. Finally, the interactions between benzodiazepines and other drugs acting at the GABA-benzodiazepine receptor complex are described. The results of these experiments caution against global classification of compounds as benzodiazepine “antagonists”.

Isonicotinic hydrazide causes seizures in scrapie-infected hamstesr with shorter latency than in control animals: A possible GABAergic defect

Isonicotinic hydrazide, a drug that decreases the level of GABA, when injected subcutaneously in control and scrapie-infected hamsters induced tonic-clonic seizures in scrapie hamsters significantly earlier ( P<0.0001) than in control animals. This suggests depression of the GABAergic system in scrapie-infected hamsters. To determine whether this lesion is pre or postsynaptic we measured the level of GABA, glutamate, cGMP and cAMP and the GABA-benzodiazepine receptor complex.

The anxiogenic action of RO 5-4864 in the social interaction test: effect of chlordiazepoxide, RO 15-1788 and CGS 8216

RO 5-4864 (20 mg/kg), a benzodiazepine with high affinity for peripheral-type benzodiazepine binding sites in rat kidney and brain, but not for the "classical" CNS sites, reduced the time spent by pairs of rats in active social interaction, without reducing locomotor activity, possibly reflecting an anxiogenic action. This anxiogenic effect was not reversed by chlordiazepoxide (5 or 10 mg/kg) given acutely, but was reversed by chlordiazepoxide (5 mg/kg) given for 5 days prior to testing. RO 15-1788 (10 mg/kg), a drug that antagonises several effects of benzodiazepines but has little affinity for peripheral-type sites, had no action on the reduction in social interaction induced by RO 5-4864. However, CGS 8216 (10 mg/kg) which also antagonises the effects of benzodiazepines and has little affinity for RO 5-4864 recognition sites, significantly enhanced the reduction in social interaction caused by RO 5-4864, and the combination produced a significant decrease in locomotor activity. These results are discussed in terms of possible sites of action of RO 5-4864 on the GABA-benzodiazepine receptor complex.

Behavioural effects of PK 8165 that are not mediated by benzodiazepine binding sites

The phenylquinoline, PK 8165 (5–25 mg/kg), produced dose-related reductions in locomotor activity, rearing and exploratory head-dipping in a holeboard. Neither Ro 15-1788 (1, 10 or 20 mg/kg) nor CGS 8216 (1 or 10 mg/kg) was able to reverse the reductions in locomotor activity or rearing. This suggests that at least some of the behavioural effects of PK 8165 are not mediated by a site on the GABA-benzodiazepine receptor complex. This conclusion is supported by the recent report [9] that, whereas PK 8165 potently displaces benzodiazepines from their binding site in vitro, it is without effect in vivo.

Characteristics of an atypical benzodiazepine, Ro 5-4864

Ro 5-4864 is a 1,4 benzodiazepine which, atypically, does not bind to the classical CNS benzodiazepine receptors, but has high affinity for the peripheral type of binding site found both in the periphery and in the brain. Biochemical evidence for alternative sites of action for this compound is discussed. We review the behavioral profile of Ro 5-4864 (sedative, convulsant and anxiogenic in rodents) and also describe the behavioral effects of combining Ro 5-4864 treatment with benzodiazepines (e.g., diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). In the light of these interactions and electrophysiological evidence we conclude that the actions of Ro 5-4864 are most likely to be mediated at the GABA-benzodiazepine receptor complex in the CNS.

Evidence for association of a high affinity avermectin binding site with the benzodiazepine receptor

Avermectin B1a concentrations corresponding to the KD value of a specific high affinity binding site for [3H]avermectin B1a are sufficient to stimulate the specific high affinity binding of [3H]flunitrazepam to its receptors. This stimulation of [3H]flunitrazepam binding by low concentrations of avermectin B1a was enhanced by chloride ions and picrotoxinin and reduced by the GABA agonists THIP and PSA. The similar modulation by chloride ions, GABA agonists and picrotoxinin and the resistance against washing of membranes of avermectin B1a bound to its specific high affinity binding site or to the site modulating [3H]flunitrazepam binding indicate a close association of the specific high affinity binding site for [3H]avermectin B1a with the GABa-benzodiazepine receptor complex.

Absence of intrinsic antagonist actions of benzodiazepine antagonists on an exploratory model of anxiety in the mouse

Recent reports suggest that antagonists of benzodiazepine receptors have intrinsic anxiogenic and convulsant properties. An exploratory model in the mouse, specific for anxiolytics, was employed to test further, the hypothesis that antagonists have intrinsic actions pharmacologically opposite to those of the benzodiazepines. The drugs Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidiazo-[1,5-a][1,4]benzodiazepine-3-carboxylate), CGS-8216 (2-phenylpyrazolo[4,3-c]quinolin-3[5H]one),β-CCM (3-carbomethoxy-β-carboline) and FG-7142 ( N 1-methyl- β-carbo]ine-3-carboxamide) all effectively blocked the anxiolytic actions of diazepam. None of these antagonists demonstrated intrinsic or anxiogenic or anxiolytic activity in this model system. These findings suggest that both the species and paradigm employed will determine the pharmacological profile of drugs which act at the GABA-benzodiazepine receptor complex.

Taurine modulation of the benzodiazepine-gamma-aminobutyric acid receptor complex in brain membranes.

In unwashed brain membranes taurine produced an inhibition of [3H]flunitrazepam [( 3H]FNZ) binding with IC50 ranging between 31.5 and 11.9 microM; the IC20 varied between 18 and 26 nM. This inhibitory effect was of a mixed type, with a reduction in Bmax and an increase in KD. Various precursors and metabolites of taurine have a less inhibitory effect. Taurine also has little inhibitory effect (IC50 above 500 microM) on the binding of [3H]ethyl-beta-carboline-3-carboxylate. In extensively washed membranes, 10(-5) M taurine produces a 16-21% increase in the binding of [3H]FNZ while 10(-5) M gamma-aminobutyric acid (GABA) increases it between 31 and 42%. However, if 10(-5) M GABA plus 10(-5) M taurine is included in the assay there is a dramatic inhibitory effect. Taurine causes an inhibition of the GABAergic enhancement of [3H]FNZ binding with an IC50 between 7.3 and 7.8 microM. Binding experiments with [3H]taurine done under different conditions failed to detect a Na+-independent and specific [3H]taurine receptor. These results suggest that endogenous taurine, the second most abundant free amino acid in brain, may play an important modulatory role in the GABA-benzodiazepine receptor complex.

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

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

Ethanol and the gamma-aminobutyric acid-benzodiazepine receptor complex.

Ethanol appears to enhance gamma-aminobutyric acid (GABA)-mediated synaptic transmission. Using radioligand binding techniques, we investigated the possibility that the GABA-benzodiazepine receptor complex is the site responsible for this effect. Ethanol at concentrations up to 100 mM failed to alter binding of [3H]flunitrazepam (FNZ), [3H]Ro 15-1788, or [3H]methyl-beta-carboline-3-carboxylate (MBCC) to benzodiazepine receptors, or of [3H]muscimol to GABA receptors in rat brain membranes. Scatchard analyses of the binding of these radioligands at 4 degrees C and 37 degrees C revealed no significant effects of 100 mM ethanol on receptor affinity or number. A variety of drugs as well as chloride ion increased binding of [3H]FNZ and/or [3H]muscimol, but these influences were not modified by ethanol. These findings indicate that ethanol probably potentiates GABAergic neurotransmission at a signal transduction site beyond the GABA-benzodiazepine receptor complex.

Effects of valproic acid on spinal reflexes and [3H]muscimol and [3H]diazepam binding in brain membranes in rats.

It was examined whether the anticonvulsant activity of valproic acid (di-n-propylacetic acid, DPA) was exerted via the inhibitory GABA system in the central nervous systems (CNS). Dorsal root reflexes (DR-DRR) were not augmented but intensely suppressed following the administration of DPA (50 and 200 mg/kg, i.v.). DPA did not depolarize the resting dorsal root potentials. These electrophysiological findings may indicate that DPA does not potentiate GABA system in the spinal cord of rats. DPA (1 and 10 mM) neither affected the binding of 2 or 40 nM of [3H]muscimol nor the binding of [3H]diazepam in the rat brain membranes; the same concentrations of DPA did not affect the enhancement of binding of [3H]diazepam induced by the addition of GABA (100 microM). The result obtained from binding assays indicates that DPA does not interact with the GABA-benzodiazepine receptor complex. The findings in the present study do not support the proposal that the anticonvulsant action of DPA is exerted by potentiation of the GABA system in the CNS.

Modulation of GABA binding to rat brain membranes by alkyl β-carboline-3-carboxylate esters

The effects of the methyl, ethyl and propyl esters of β-carboline-3-carboxylic acid were assessed on low affinity binding of GABA to rat brain membranes, and the enhancement of such binding by diazepam. The propyl ester acted as a benzodiazepine agonist in enhancing low affinity GABA binding, while the methyl and ethyl esters acted as benzodiazepine antagonists in reversing the stimulation of GABA bindingby diazepam. These effects on low affinity GABA binding in vitro are consistent with pharmacological and behavioural actions of these esters in vivo and support the hypothesis that such actions are mediated via a GABA-benzodiazepine receptor complex.

Central toxic effects of chronic ethanol treatment: Actions on GABA and benzodiazepine recognition sites

Prolonged ethanol treatment modifies various neurotransmitter systems. GABAergic neuronal function was particularly affected. On the other hand, clinical reports have indicated an interaction between ethyl alcohol and benzodiazepine receptors. These observations suggest a possible site of action of ethanol at the level of the GABA-benzodiazepine receptor complex. Our results showed that ethanol treatment differentially affected GABA receptor function and benzodiazepine binding sites. When [ 3H]GABA binding in the cerebellum, striatum and hippocampus was increased, [ 3H]diazepam binding remained unchanged in the same areas. The possibility of modulation of ethanol effects on GABAergic neurons through benzodiazepine receptors is discussed.

Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro

When membranes of rat cerebellum are exposed to UV light in the presence of flunitrazepam this ligand can be incorporated into one of the assumed 4 benzodiazepine binding sites of the GABA-benzodiazepine receptor complex. This irreversible incorporation of flunitrazepam, in contrast to reversible binding of this substance, leads to conformational changes of the remaining 3 benzodiazepine binding sites which result in a decreased affinity of benzodiazepine agonists, but not of benzodiazepine antagonists. The investigation of the affinity of drugs for [ 3H]benzodiazepine antagonist binding before and after photoaffinity labelling of benzodiazepine receptors with flunitrazepam can therefore be used as a sensitive and simple test to distinguish between agonists and antagonists in vitro.

Interaction of anticonvulsants with the barbiturate-benzodiazpine-gaba receptor complex

Unlike the anesthetic barbiturate pentobarbital and the anxiolytic pyrazolopyridine etazolate, which enhance [ 3H]diazepam binding to rat brain membranes, the anticonvulsant barbiturates phenobarbital and metharbital, and also chlormethiazole, at therapeutic concentrations (10–1000 μM), do not stimulate [ 3H]diazepam binding, but instead block the enhancement by both pentobarbital and etazolate. The same anticonvulsants at similar concentrations inhibit [ 3H]α-dihydropicrotoxinin (DHP) binding, suggesting that these anticonvulsants compete for the same receptor sites as pentobarbital and etazolate, designated the barbiturate-picrotoxinin receptor component of the benzodiazepine-GABA receptor complex.