The GABA/benzodiazepine receptor chloride channel complex during repeated episodes of physical ethanol dependence in the rat

The GABA/benzodiazepine (BZ) receptor chloride channel complex was investigated during repeated episodes of ethanol intoxication and withdrawal in the rat; the intragastric intoxication technique was applied and the severity of intoxication, withdrawal and number of seizures were recorded. The following groups were studied after decapitation during withdrawal 10–16 h after the last ethanol feeding: A) isocalorically fed controls not receiving ethanol; B) isocalorical controls subjected to a single ethanol intoxication period; C) animals subjected to 15 intoxication-withdrawal episodes (spontaneous seizures); D) same as C, but without developing seizures. A radio receptor technique was applied in the characterization of the receptor complex comprising specific binding to the BZ-receptor, the chloride channel and the GABA receptor by ³H-diazepam, ³⁵S-TBPS and ³H-muscimol, respectively. The allosteric couplings among the components of the receptor complex were studied by ³H-diazepam and ³⁵S-TBPS binding enhancement tests involving muscimol, ZK 93423 and DMCM. Cortex, hippocampus and cerebellum were the brain regions studied. Except for a reduced specific binding of ³H-diazepam in cerebellum, there were no indications of changes in specific binding to any part of the receptor complex. The allosteric coupling of BZ and GABA receptors as well as chloride channel-BZ receptors were unchanged in all groups. It is notable that no changes at all could be related to number of intoxication-withdrawal episodes or to the development of seizures. Thus, the present study gave no indication that the GABA/benzodiazepine receptor chloride channel complex is directly involved in the augmentation of cerebral nervous system excitability (seizures) during repeated episodes of physical ethanol dependence.     

GABA and benzodiazepine receptors in the offspring of dams receiving diazepam: ontogenetic studies

Ontogenesis of the regulation of ³H-GABA and ³H-diazepam binding to rat brain plasma membranes treated with 0.05% Triton X-100 has been studied. The density of ³H-diazepam and ³H-GABA binding in cortex, cerebellum and corpus striatum at birth was approximately one third of the adult values. They increased at the same rate and reached the adult values between 14–21 days after birth. Study of the binding characteristics showed that the K_D for high and low affinity for ³H-GABA, and for ³H-diazepam did not change during ontogenesis and the increase reflects only an increase of Bmax. The number of Triton X-100 treatments of crude synaptic membrane (CSM) required to maximize ³H-GABA for the high affinity component were different at various postnatal days: only one treatment was required in 1-day old rats, two in 7- and 14-day old rats and three in adult animals. In addition, the capability of muscimol to stimulate ³H-diazepam binding in both frozen-thawed and Triton X-100 treated membrane preparations decreased with increasing age. Binding of ³H-GABA and ³H-diazepam to brain of newborn rats whose dams received diazepam throughout pregnancy (100 mg/kg, × os, bid) was also studied. No significant differences were observed in the ontogenetic development of both bindings. However, in the cortex of these newborn rats the capability of muscimol to stimulate ³H-diazepam binding was greatly reduced in Triton X-100-treated membranes.     

GABA receptor-mediated modulation of 3H-diazepam binding in rat cortex.

Three membrane preparations of rat cortex were used to examine the effects of GABA, bicuculline and bicucine on specific 3H-diazepam binding. In the crude synaptosomal fraction, GABA had no effect on either the maximal binding capacity (Bmax) or dissociation constant (KD) of 3H-diazepam binding. Bicuculline and bicucine both decreased binding affinity. This was antagonized by adding GABA. In the repeatedly washed membrane preparation, and in the washed, frozen and thawed preparation, GABA increased binding affinity and, at high concentrations, increased Bmax. Increased binding affinity was observed with as little as 10(-8) M GABA in the washed, frozen and thawed preparation. Bicuculline inhibited the effect of GABA on 3H-diazepam binding. It was found that about 3 X 10(-5) M GABA was present in the assay medium containing crude synaptosomal fraction. These results suggest endogenous GABA is present in, and influences the results of 3H-diazepam binding assays. Furthermore, it appears that GABA and bicuculline affect 3H-diazepam binding through their binding to the specific GABA binding site.     

Pharmacological profile of the imidazopyridine zolpidem at benzodiazepine receptors and electrocorticogram in rats

Summary Zolpidem is a novel non-benzodiazepine related hypnotic, which possesses an imidazopyridine structure. This drug has preferential affinity for the³H-diazepam binding site in the rat cerebellum, while it is only weakly active at inhibiting³H-Ro 5-4864 binding to the rat kidney. The potency of zolpidem at displacing³H-Ro 15-1788 binding to rat cerebral cortex membranes is enhanced in the presence of GABA. On the sleep pattern of the electrocorticogram in the curarised rat, zolpidem induces a physiological type of slow wave sleep with rapid onset of action. Zolpidem differs from classical benzodiazepine drugs, in possessing an atypical binding profile to³H-benzodiazepine receptors, and because it does not affect the sleep patterns.     

A steroid derivative,R 5135, antagonizes the GABA/benzodiazepine receptor interaction

A novel steroid derivative, R 5135 (3α-hydroxy-16-imino-5β-17-aza-androstan-11-one) showed high affinity for both the GABA and glycine receptors in vitro. It also displaced [³H]diazepam from the benzodiazepine receptor in a rat cortex membrane preparation, but in this case a plateau occurred in the displacement curve at a concentration of R 5135 between 10^?7 and 10^?6 M, where binding was decreased by about 50 %. The “high affinity” component of R 5135 inhibition no longer appeared when the endogenous GABA concentration was reduced by extensive washing of the membrane preparation and it reappeared when GABA was re-introduced. Thus R 5135 behaves as a powerful antagonist of the GABA stimulation of [³H]diazepam binding, being 500 times more active than the GABA-antagonist bicuculline. The dual interaction between R 5135 and GABA and glycine receptors suggests that these may share some common structural feature or that they have overlapping specificity.     

1,4-Benzodiazepines and gamma-aminobutyric acid: pharmacological and biochemical correlates

The high affinity receptors or GABA present in brain interact with an endogenous thermostable inhibitor (GABA modulin) which allosterically modifies GABA binding sites. This is the type of GABA receptor that we term GABA2 receptor in comparison to GABA1 receptor which has low affinity for GABA and is not regulated by GABA modulin. The 1,4-benzodiazepines interact competitively with GABA modulin and thereby modify GABA2 receptor binding. In contrast the occupancy of GABA receptor increases the affinity of 1,4-benzodiazepine receptors for their specific agonist. The GABA modulin and both GABA receptors are located on the membranes of C6 and NB2a cells. The NB2a cell membranes also contain CL- ionophore, thus the complete receptor complex is present in the membranes of NB2a cell clone. It was proposed that the inability of clonazepam to displace 3H-diazepam from specific binding sites characterizes the nonneuronal 1,4-benzodiazepine receptor. This characterization was shown to relate to the properties of other membrane components rather than to the characteristics of the specific benzodiazepine receptors.     

Occupation of brain receptors by benzodiazepines and β-carbolines: Multiple mechanisms and responses

High affinity for the brain benzodiazepine receptor can no longer be considered predictive of benzodiazepine-like pharmacology in vivo. Studies of ³H-diazepam (³H-DZ) and ³H-propyl-β-carboline-3-carboxylate (³H-PrCC) binding were performed to investigate how occupation of the benzodiazepine receptor by these two high-affinity ligands leads to their significantly different pharmacological effects. In mouse hypothalamus, there are four times as many ³H-DZ binding sites as ³H-PrCC binding sites (B_max values = 1,025 and 265 fmol/mg protein, respectively) and both types of site form a distinct anterior-to-posterior gradient in this tissue. Physiological Ca⁺² concentrations do not regulate ³H-ligand binding in Ca⁺²-depleted whole mouse brain membranes, but in the presence of increasing concentrations of guanosine 5′-triphosphate (GTP), Ca⁺² stimulates ³H-DZ binding up to 25%. In contrast, ³H-PrCC binding is unaffected by Ca⁺² and GTP. Diazepam (DZ) and ethyl β-carboline-3-carboxylate (βCCE) both stimulate ³H-muscimol binding to GABA receptors in whole mouse brain membranes (DZ = +57%, βCCE = +27%), and βCCE partially blocks the effect of the benzodiazepine. Moreover, alkyl β-carboline-3-carboxylates prevent a temperature-induced thermodynamic transition in the benzodiazepine receptor that occurs in the presence of GABA. Studies of 8-anilino-1-naphthalene sulfonic acid fluorescence (ANS) perturbation have also suggested that occupation of the receptor by a benzodiazepine results in a conformational change. Although benzodiazepines and β-carbolines are high-affinity ligands for the benzodiazepine receptor, these results suggest that their different pharmacological profiles may result from significant differences in binding mechanisms and receptor responses.     

Muscimol and related GABA receptor agonists: the potency of GABAergic drugs in vivo determined after intranigral injection

Contralateral turning behaviour following unilateral intranigral injection of a large series of GABA analogues was investigated. The results indicated that the turning behaviour was induced stereospecifically and was selectively antagonized by the GABA antagonist bicuculline methochloride. The comparative potencies of a series of GABA agonists related to muscimol in general corresponded well to the affinity for ³H-GABA receptor sites and to the depressant action on single neurons using microelectrophoretic administration. However, the GABA agonists trans-aminocrotonic acid and 3-aminopropanesulphonic acid were much weaker than expected from in vitro studies. The GABA-uptake inhibitors nipecotic acid and guvacine showed only weak and short-lasting effects. The GABA-transaminase inhibitor γ-acetylenic GABA showed delayed effects compared with the agonists which acted immediately. It is proposed that this behavioural effect may be a sensitive and quantitative method for evaluation of GABA agonists in vivo.     

Neuroactive steroid modulation of [3H]muscimol binding to the GABAA receptor complex in rat cortex

Epalons are a subclass of neuroactive steroids which are positive allosteric modulators of the GABA_A receptor acting via a unique site on the receptor complex. Enhancement of [³H]muscimol binding to the GABA recognition site was observed to be either full (>150%) or limited (110–135%) and specific for epalons. Both one and two component modulation was observed. Saturation studies performed in the presence of 3α-hydroxy-5α-pregnan-20-one (3α,5α-P) showed that 3α,5α-P increased the density of high affinity [³H]muscimol sites and doubled the affinity of low affinity sites. 3α,5α-P had no effect on the affinity of the high affinity site or the density of the low affinity site. These data indicate that: (1) epalons are potent, stereoselective enhancers of [³H]muscimol binding; (2) epalons display varying levels of efficacy and some exhibit two-component enhancement; and (3) 3α,5α-P enhancement of [³H]muscimol binding results from both an increased density of high affinity sites and an increased affinity for low affinity sites.     

Pharmacology of Morphine and Morphine-3-glucuronide at Opioid,Excitatory Amino Acid,GABA and Glycine Binding Sites

Abstract: Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of ³H-MK801 (non-competitive antagonist) and ¹²⁵I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of ³H-muscimol (GABA receptor agonist), ³H-diazepam and ³H-flunitrazepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABA_A, receptors. Mor-phine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of ³H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors.     

A study on benzodiazepine receptor binding in audiogenic seizure-susceptible rats

Benzodiazepine receptors were investigated in the cerebral cortex, the hippocampus, the brainstem, and the cerebellum of audiogenic seizure (AGS)-susceptible and seizure-resistant (ER) control rats. In AGS-susceptible rats of Sprague-Dawley descent, muscimol (10-6 M and 3 x 10-5 M) activated the binding of 3H-diazepam (0.4 nM) significantly less than in ER-rats. This finding may be strain selective, since it was not observed in AGS-susceptible rats of Wistar descent. Specific binding of the convulsant benzodiazepine receptor ligand methyl 6,7-dimethoxy-4-ethyl carboline-3-carboxylate (3H-DMCM), the benzodiazepine receptor ligand 3H-diazepam and the chloride channel directed cage convulsant t-butylbicyclophosphorothionate 35S-TBPS were not significantly changed in AGS-susceptible as compared to control rats. Our findings indicate that a disturbance at the level of the benzodiazepine receptor/GABA receptor/chloride channel complex is not a likely general aetiological factor for audigenic seizures in rats.     

Neurosteroids may differentially affect the function of two native GABAA receptor subtypes in the rat brain

Hippocampal noradrenergic and cerebellar glutamatergic axon terminals are known to possess GABA_A receptors mediating, respectively, enhancement of noradrenaline (NA) and glutamate release. It has been recently found that the hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar receptor is insensitive to benzodiazepine agonists. We here tested the effects of neurosteroids on these two native GABA_A receptors using superfused rat hippocampal and cerebellar synaptosomes. Allopregnanolone (3α,5α-P), at nanomolar concentrations, potentiated the GABA-induced [³H]-NA release from superfused hippocampal synaptosomes; in the absence of GABA, the steroid was ineffective up to 10μM. The enhancement by GABA of the K⁺-evoked [³H]-D-aspartate release from cerebellar synaptosomes also was potentiated by nanomolar 3α,5α-P; in addition, at 1–10μM, the steroid increased [³H]-D-aspartate release in the absence of GABA. Both in hippocampus and cerebellum the potentiations of the GABA effects produced by nanomolar 3α,5α-P were abolished by dehydroepiandrosterone sulphate (DHEAS). Added up to 10μM, DHEAS could not inhibit the effects of GABA alone. The enhancement of [³H]-D-aspartate release elicited by 3μM 3α,5α-P in the absence of added GABA was antagonized completely by bicuculline and picrotoxin and halved by DHEAS. To conclude, 3α,5α-P, at nanomolar concentrations, behaves as a positive allosteric GABA modulator at both the GABA_A receptors under study. Low micromolar 3α,5α-P can directly activate the cerebellar receptor, whereas the hippocampal GABA_A receptor is insensitive to the neurosteroid alone. DHEAS appears to be a pure antagonist at the neurosteroid allosteric sites. Along with the previously observed differential sensitivity to benzodiazepines, the present data strengthen the idea that the two receptors investigated represent native subtypes of the GABA_A receptor having distinct pharmacology, neuronal localization and function. Received: 13 October 1997 / Accepted: 30 December 1997     

Receptor-mediated model relating anticonvulsant effect to brain levels of camazepam in the presence of its active metabolites

In a displacement test using³H-diazepam as a radioligand, the in vitro affinities of metabolites of camazepam (CZ) for the benzodiazepine receptors were 1–50 times more potent than that of CZ. In contrast, only three metabolites (temazepam, oxazepam, and hydroxy CZ), as well as CZ itself, exhibited an in vivo affinity parallel to their ability to protect against pentylenetetrazole-induced clonic convulsion in rats. In addition, CZ and these active metabolites displaced the radioligand from their receptor sites in a concentration-dependent saturable manner, indicating the competitive bimolecular interaction of these molecules with their receptors. The percent anticonvulsant effect was a nonlinear, single-valued function of the in vivo percent displacement of specific³H-diazepam binding, independent of these displacers after i.v. dosing; this relationship could be approximated by the Hill equation. On the basis of these findings, a receptor-mediated model, including the Langmuir equation to describe the receptor binding-brain concentration relationship and the Hill equation to accommodate the anticonvulsant effect-receptor binding relationship, was constructed. This model was found to adequately relate the time course values of anticonvulsant effect and of brain levels of CZ and its active metabolites after oral administration. These results demonstrate that CZ and its active metabolites exert anticonvulsant effect by competitive binding to the benzodiazepine receptors.     

Enhancement of 3H-diazepam binding by SQ 65,396: a novel anti-anxiety agent.

SQ 65,396, a clinically active anti-anxiety agent, enhanced the binding of 3H-diazepam at 1.5 nM. This effect was due to an increase in the affinity for the ligand, without a change in the number of 3H-diazepam binding sites. This action of SQ 65,396 may mediate its anti-anxiety effects by affecting the action of an endogenous modulator of the "benzodiazepine receptor." Several other substances and treatments increase the affinity of 3H-diazepam for its receptors by mechanisms which may be related to the effect produced by SQ 65,396.     

Radioligand binding characteristics of the chicken cardiac muscarinic receptor

Summary The muscarinic receptor present in chicken cardiac membranes was characterised using a ligand binding approach and compared to the M1, M2 and M3 receptors that can be identified in ligand binding studies at present. [³H]N-methylscopolamine and [³H]pirenzepine appeared to label the same population of muscarinic receptors in chicken cardiac membranes since the density of sites labeled by the two radioligands was similar. Furthermore, affinity estimates of 8 muscarinic receptor antagonists for chicken cardiac muscarinic receptors were the same irrespective of whether [³H]N-methylscopolamine or [³H]pirenzepine was used as the radioligand. The chicken cardiac muscarinic receptor displayed high affinity for pirenzepine (pK _i = 7.9) and so did not appear to represent an M2 receptor. Despite the high affinity of chicken cardiac muscarinic receptors for pirenzepine, affinity estimates for dicyclomine (pK _i = 8.0), CPPS (pK _i = 8.4) and 4DAMP (pK _i = 8.6) in chicken heart were not consistent with the presence of M1 receptors. The chicken cardiac muscarinic receptor also displayed significant differences to the M3 receptor since it displayed high affinity for AF-DX 116 (pK _i = 7.1) and methoctramine (pK _i = 8.4). Finally, chicken heart muscarinic receptors displayed high affinity for gallamine (pK _i = 7.0) and pirenzepine suggesting that the receptor was different to the M4 muscarinic receptor of the NG108-15 cell line. These findings suggest that chicken heart expresses a novel muscarinic receptor subtype distinct from the M1, M2, M3, and M4 subtypes already described. Send offprint requests to A. Michel.     

Pharmacological characterization of benzodiazepine receptors in the brain

Receptors in rat brain membranes which specifically bind ³H-diazepam were characterized pharmacologically using reference substances representing several pharmacological classes of drugs. Of 28 benzodiazepines tested, several “classical” ones (diazepam, clonazepam, lorazepam, oxazepam, nitrazepam, flurazepam, bromazepam and chlorazepate) with known clinical efficacy, as well as three newer “triazolo” benzodiazepines (estazolam, U 35,005, U 31,957), one new “imidazolo” benzodiazepine, U 31,219, and one new 2-carbamoylmethylene-benzodiazepine, displaced ³H-diazepam binding at low concentrations (K_i = 1–60 nM). For these benzodiazepines there was a statistically significant correlation between K_i values for displacement and ED₅₀ (or MED) values in several pharmacological tests predictive of anxiolytic activity in man. More than 100 nonbenzodiazepines, representing 22 distinct pharmacological classes as well as 14 presumed neurotransmitters in the CNS, including 4 peptides, were much weaker as ³H-diazepam displacers (K_i > 0.1 nM). These results that in vitro ³H-diazepam binding represents the physiologically relevant binding to hitherto unknown receptors in the CNS.     

Characteristics of type 1 and type 2 benzodiazepine receptors in the ovine brain

Analysis of the displacement of 3H-diazepam binding to membranes prepared from the ovine frontal cortex by the triazolopyradiazine CL218,872 yielded a Hill coefficient significantly below unity. By analogy with similar studies of this drug in rat brain this suggested the existence of Types 1 and 2 benzodiazepine receptors. The degree of displacement of 3H-diazepam by CL218,872 (200 nM, Type 1; 800 nM, Type 2) in homogenates of brain regions differed, the rank order being cerebellum greater than parietal cortex greater than frontal cortex congruent to temporal cortex congruent to hippocampus greater than striatum. Displacement of 3H-diazepam by CL218,872 was enhanced by 10(-5) M GABA in the striatum (at 200 nM and 800 nM CL218,872) and cerebellum (at 200 nM CL218,872). Benzodiazepine receptors in the ovine frontal cortex were least sensitive to CL218,872 (200 nM) in young fetuses (54-68 days gestation) and achieved adult levels of sensitivity by late gestation. Finally, the potency of CL218,872 to displace 3H-diazepam was not effected by the 3H-ligand concentration (0.5 nM or 5.0 nM), suggesting that Types 1 and 2 benzodiazepine receptors are not identical to the high and low affinity 3H-diazepam binding sites we have previously identified in the ovine brain.     

Putative endogenous ligands to the benzodiazepine receptor: What can they tell us?

The endogenous ligand for the benzodiazepine receptor has not been convincingly demonstrated, nor is it clear what role this drug receptor actually plays in brain function. A number of putative endogenous ligands for the benzodiazepine receptor have been identified and reported in the literature. These putative ligands, at various stages of purification, have been typically identified by their ability to displace ³H-diazepam in a radioreceptor binding assay. Taken together, these studies provide useful information on the role that the benzodiazepine receptor and its ligand might play in brain function. The purpose of this paper is to review this area of research and attempt to provide a unifying hypothesis. In summary, calling this bioactive site the benzodiazepine receptor may be a misnomer, as it appears to effect both anxiogenic and anxiolytic activities involved in the maintenance of homeostatic levels of vigilance. Moreover, it can be suggested that endogenous ligands of reciprocal action could exist, mediating each function (relaxation and vigilance) and, thereby, maintain a balance of neural activity.     

Environmentally-induced modification of the benzodiazepine/GABA receptor coupled chloride ionophore

The benzodiazepine/GABA receptor coupled chloride ionophore was examined in brain membranes of rats maintained in either a conventional animal facility or a protected (low-stress) environment. Following a 10 min ambient temperature swim, animals maintained in both environments had qualitatively similar increases in the number (B _max) of [³⁵S]t-butylbicyclophosphorothionate (TBPS) binding sites, the apparent affinity of this radioligand, and the efficacy and potency of Cl^– to enhance [³H]flunitrazepam binding. Nonetheless, the B _max of [³⁵S]TBPS and efficacy of Cl^– to enhance [³H]flunitrazepam binding were significantly lower in animals housed in the protected environment compared to those maintained in a conventional facility both before and after swim stress. Furthermore, in rats housed in a protected environment, sequential removal of animals from a common cage (cohort removal), produced a very rapid increase (15 s) in Cl^–-enhanced [³H]flunitrazepam binding in cortical and hippocampal but not cerebellar membranes. Cohort removal also produced a sequential increase in the number of [³⁵S]TBPS binding sites and apparent affinity of this radioligand in cerebral cortical membranes. The effects of cohort removal were not observed in animals subjected to ambient temperature swim or if animals were removed from different cages. Changes in the benzodiazepine/GABA receptor coupled chloride ionophore produced by cohort removal from a common cage preceded any statistically significant changes in circulating levels of -MSH, -endorphin, ACTH or corticosterone. These findings suggest that the benzodiazepine/GABA receptor chloride ionophore complex (supramolecular complex) is under both tonic and acute regulation by the environment, and may subserve a physiologically relevant function in the response to stressful or anxiety producing stimuli.     

An endogenous substance from porcine brain antagonizes the anticonvulsant effect of diazepam

An endogenous substance /ES/ obtained from porcine brain /MW~3000/ partially inhibited the specific ³H-diazepam binding in the P₂-fraction but not in the synaptic membrane fraction of rat brain. ES inhibited specific ³H-GABA binding to synaptic membranes in a dose-related manner.Intraamygdaloid injection of diazepam fully inhibited the convulsions evoked by systemic administration of pentylenetetrazol in the rat. This anticonvulsant action of diazepam was antagonized by a previous intraamygdaloid injection of ES but not of myoglobin.ES does not seem to be an endogenous ligand of benzodiazepine receptors; it probably exerts its effect via GABA receptors.