Receptor-chloride Ionophore Complex Research Articles

Age‐Related Loss of t‐[35S]Butylbicyclophosphorothionate Binding to the γ‐Aminobutyric AcidA Receptor‐Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.

Anticonvulsant profile of the progesterone metabolite 5α-pregnan-3α-ol-20-one

5α-Pregnan-3α-ol-20-one (3α-OH-DHP) is a naturally occuring metabolite of progesterone that can modulate brain excitability through a specific steroid recognition site on the GABA/benzodiazepine receptor-chloride ionophore complex. The anticonvulsant properties of 3α-OH-DHP were determined using standardized anticonvulsant screening tests in mice. This steroid was found to be effective against metrazol-, (+)-bicuculline- and picrotoxin-induced seizures. The steroid has maximum potency against (+)-bicuculline-induced convulsions and no activity against maximal electroshock and strychnine-induced seizures. These findings support the hypothesis that therapeutically useful anticonvulsant steroids active at the putative steroid recognition site associated with the GABA/benzodiazepine receptor-chloride ionophore complex can be identified.

Molecular neurobiology: Implications for insecticide action and resistance

Recent advances in molecular neurobiology have provided an unprecedented insight into the structure and function of the three principal target sites for neurotoxic insecticides: acetylcholinesterase, the 4-aminobutyric acid (GABA) receptor–chloride ionophore complex, and the voltage-sensitive sodium channel. This paper reviews some of these advances and their current or potential application to problems in insecticide resistance. It particularly emphasizes studies of the molecular biology of voltage-dependent sodium channels in the context of resistance to DDT and pyrethroids resulting from reduced neuronal sensitivity.

Steroid modulation of the GABA/benzodiazepine receptor-linked chloride ionophore.

Recent findings suggest that steroids with sedative-hypnotic properties interact specifically with the gamma-aminobutyric acidA/benzodiazepine receptor-chloride ionophore complex (GBRC). They show positive heterotropic cooperativity by allosterically enhancing the binding of GABA agonists and the clinically useful benzodiazepines (BZs) to their respective recognition sites. These steroids have stringent structural requirements for activity at the GBRC, with the essential requirements for high potency being a 3 alpha-hydroxyl group and a 5 alpha-reduced A-ring. Some of these steroids are naturally occurring metabolites of progesterone and deoxycorticosterone and have nanomolar potencies as potentiators of chloride channel conductance. These 3 alpha-hydroxylated, 5 alpha-reduced steroids do not act through any known sites on the GBRC. Thus, the exact site and mechanism of action remain to be determined. Together with the observation that physiological levels of these metabolites are sufficient to influence the function of the GBRC, the evidence clearly suggests a role for these steroids in the normal regulation of brain excitability by potentiating the postsynaptic effects of gamma-aminobutyric acid (GABA). Pharmacological studies of the GBRC-active steroids show that they possess anxiolytic and anticonvulsant activities. The potential therapeutic application of these steroids in the treatment of mood disorders and catamenial exacerbation of seizures associated with the menstrual cycle is discussed. Collectively, the evidence from the studies of these steroids imply that another mechanism by which the endocrine system influences brain function has been identified. Its characterization will provide important insight into how steroids modulate brain excitability under normal and pathophysiological states.

Asymmetrical activation of GABA-gated chloride channels in cerebral cortex

A pronounced left/right asymmetry of GABA-gated chloride channels was observed in rat cerebral cortex. This asymmetry was manifest as a higher apparent affinity and density of binding sites for the “cage” convulsant [ 35S]t-butylbicyclophosphorothionate (TBPS) in the right compared to left cortex. Asymmetries in [ 35S]TBPS binding were not observed in other brain areas, and were restricted to the occipital and entorhinal/pyriform areas of cerebral cortex. Evaluation of other components of the benzodiazepine/GABA receptor chloride ionophore complex in cerebral cortex suggests that this asymmetry is not present in either benzodiazepine receptors or that population of GABA receptors linked to benzodiazepine receptors. Brief restraint-stress significantly increased both the apparent affinity and number of [ 35S]TBPS binding sites in cortex, but the left/right asymmetry was no longer apparent. These findings indicate a time-dependent, asymmetric response of cortical GABA-gated chloride channels to stress rather than an anatomical hemispheric difference, and suggest that GABA-gated chloride channels may be involved in a differential processing of stressful stimuli by the cerebral hemispheres.

Effects of chronic cortical seizures on GABA and benzodiazepine receptors within seizure pathways

The effects of chronic cortical seizures on the autoradiographic distributions of two markers of the γ-aminobutyric acid (GABA)/benzodiazepine receptor-chloride ionophore complex within local and long circuits connected to the focus were examined. Rats were subjected to electrically triggered seizures of the forelimb-sensorimotor overlap zone either daily or once every other day. At the time of sacrifice the rats had received a mean of 39 ± 3 stimulations and their seizure responses had grown in intensity and duration. [ 3H]Muscimol binding and [ 3H]flunitrazepam binding, at near saturating ligand concentrations were unchanged in the focus, mirror focus, dorsolateral caudate, globus pallidus, ventrolateral and ventrobasal thalamic nuclei, and the substantia nigra pars reticularis. These results indicate that the progressive increases in strength and duration of recurrent focal cortical seizures are not accompanied by changes in the density of either GABA receptors or benzodiazepine receptors within the focus or projection pathways.

Is the benzodiazepine/GABA receptor chloride ionophore complex involved in physical and emotional stress?

The response of an organism to physical and emotional stressors requires a complex integration of somatic, endocrine and behavioral systems. While causal relationships between specific receptor/effector systems in the central nervous system (CNS) and stress-induced somatic, endocrine and behavioral responses have not been firmly established, both physical and emotional stressors have been shown to affect a variety of neurotransmitters, neuromodulators, second messenger systems, and their associated recognition sites in the CNS (this volume and [1] for overviews). Thus, it is not surprising that most schemata describing stress-induced alterations in the CNS focus on the hypothalamus, although both the control and generation of stress must be initiated in higher centers.KeywordsChloride ChannelGaba ReceptorSupramolecular ComplexApparent AffinityHabituation PeriodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The effect of agonists at the GABA-benzodiazepine receptor complex on the duration of immobility of mice in the forced swimming test

In the present study, we examined the effect of various agents which affect in a different manner the GABA-benzodiazepine receptor-chloride ionophore complex system in relation to the immobile behavior of mice in the forced swimming test. The benzodiazepines diazepam and flurazepam, the barbiturates pentobarbital and phenobarbital, zopiclone and β-CCP (propyl-β-carboline-3-carboxylate) enhanced the immobile behavior in a dose-dependent manner. In the doses used here, these agents produced almost no muscle relaxant action. Ro15-1788 and β-CCM (methyl-β-carboline-3-carboxylate) themselves had no effect on the duration of immobility. However, Ro15-1788 and β-CCM reversed the enhancing effect produced by all 6 drugs. These results indicate that the enhancement of the duration of immobility of mice may be somehow correlated to the anxiolytic action but not to the muscle relaxant action. The effect may be mainly mediated by the benzodiazepine receptor, which forms a part of the GABA-benzodiazepine receptor-chloride ionophore complex. Furthermore it is suggested that there were behavioral similarities in the effects of β-CCP and benzodiazepines.

Different genes specify hyporesponsiveness to seizures induced by caffeine and the benzodiazepine inverse agonist, DMCM

Two strains of inbred mice differed significantly in their susceptibility to tonic seizures induced by caffeine and the benzodiazepine inverse agonist, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). The hyporesponsive strain, SWR, was not less susceptible to the convulsant action of other chemical convulsants, an observation which indicated that the response differences between the strains were pharmacologically specific. These observations and drug interaction studies suggested that caffeine-induced seizures might be mediated through an “inverse” agonist-like action of caffeine on benzodiazepine receptors associated with GABA receptor-benzodiazepine receptor-chloride ionophore complex. To determine whether the coincident alteration in susceptibility to DMCM and caffeine resulted from a single mutational change or was the result of two different genetic changes occurring coincidentally between these two strains of mice, progeny from conventional Mendelian crosses (F 1, F 2 and reciprocal backcrosses) were analyzed for the co-segregation of susceptibility to DMCM and caffeine. The inheritance of DMCM sensitivity was consistent with a single autosomal gene determinant in which the allele specifying increased responsiveness was dominant to the allele determining hyporesponsiveness. The frequent occurrence of recombinant phenotypes (e.g., caffeine hyporesponsive but DMCM sensitive mice) among F 2 and backcross established that different genetic determinants encode DMCM susceptibility and caffeine susceptibility in these two strains of mice. Thus, while these data establish a simply inherited difference in benzodiazepine responsiveness between the two mouse strains, they also indicate that this pair of strains is inappropriate for a genetic analysis aimed at probing the relationship between caffeine-induced seizures and the benzodiazepine receptor.

Neurophysiologische und neurochemische Grundlagen der Wirkung von Antiepileptika

This article surveys neurophysiological and neurochemical findings on the mechanism of action of clinically useful antiepileptics. Two mechanisms appear to be particularly important: reducing the hyperexcitability of the cell membranes by a direct action at ion channels; changes in synaptic transmission by means of an intervention in neurotransmitter systems; it seems that in this regard an important neurotransmitter is the inhibitory gamma-aminobutyric acid GABA. With these mechanisms as basis, antiepileptics can be classified into several groups. In anticonvulsively effective concentrations, phenytoin and carbamazepine will stabilise the cell membranes and thus inhibit their hyperexcitability. This effect seems to be due to a blocking effect on Na+ permeability. If administered in anticonvulsively effective concentrations, phenobarbital and benzodiazepines enhance the GABAerg inhibition by direct attachment to the GABA receptor chloride ionophore complex of the postsynaptic neuronal membrane. In addition, phenobarbital produces a reduction of the postsynaptic effect of glutamic acid which is an excitatory neurotransmitter. In higher concentrations, barbiturates and benzodiazepines produce direct changes in ion conductivity, and this seems to be an important factor determining the sedative/hypnotic effects of these substances. Primidone seems to act mainly (in long-term treatment) via its active metabolite phenobarbital. Valproic acid will lead both to a stabilisation of the membranes and to an amplification of the GABAerg transmission; in this connection, both presynaptic and postsynaptic sites of action are discussed. On the other hand, ethosuximide, given in anticonvulsively effective concentrations, does not affect membrane excitability and synaptic transmission. It can be expected that clarification of the mechanisms of action of clinically effective antiepileptics results in a stricter on-target search for new active substances.

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 (Bmax) 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 Bmax 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 (less than or equal to 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 alpha-MSH, beta-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.

The gaba/benzodiazepine receptor — chloride ionophore complex: Nature and modulation

Bruun-Meyer, Sturla E.: The GABA/Benzodiazepine Receptor-Chloride Ionophore Complex: Nature and Modulation. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1987, 11 : 365–387 1. 1. A high affinity, saturable, stereospecific binding site for Benzodiazepines has been found to be functionally and possibly structurally related to a GABA receptor-chloride ionophore complex. 2. 2. There are both central(CNS) as well as “peripheral” binding sites, involving multiple organs. 3. 3. Evidence strongly suggests that mutually exclusive Benezodiazepine agonists and antagonists bind to the same receptor, possibly in an agonist-antagonist-inverse agonist continuum. 4. 4. The search for an endogenous ligand has been inconclusive and the question of such a substance remains open. 5. 5. Although the relationship between this receptor and the Limbic System remains unclear, it seems certain that the Benzodiazepine receptor plays an important role in the modulation of Limbic System excitability.

A physiological role of the benzodiazepine/GABA receptor-chloride ionophore complex in stress.

Both direct and correlative evidence suggests that the principal pharmacologic actions of the benzodiazepines are mediated by high affinity, stereospecific recognition sites (receptors) found exclusively in tissue derived from the neural crest. The rapid advances in our understanding of the relationship of benzodiazepine receptors to (a subpopulation of) γ-aminobutyric acid (GABA, the principal inhibitory neurotransmitter in mammalian brain) receptors and an associated chloride channel have resulted in new insights about the molecular pharmacology of the benzodiazepines and other psychoactive drugs which share pharmacologic actions with the benzodiazepines (e.g. barbiturates, ethanol). In contrast, the physiological roles of this “benzodiazepine receptor complex” are not well understood, and the physiological relevance of benzodiazepine receptors has been questioned (cf. Guidotti, et al., 1983). This chapter will briefly summarize the evidence implicating the benzodiazepine receptor complex in the response to stress and anxiety, and present some recent findings from our laboratory which supports this hypothesis.KeywordsGaba ReceptorVisual Evoke PotentialInverse AgonistApparent AffinityColony RoomThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Modulation of the benzodiazepine-GABA receptor chloride ionophore complex by multiple allosteric sites: evidence for a barbiturate "receptor".

The discovery of specific recognition sites (receptors) for benzodiazepines in the central nervous system has resulted in a better understanding of the molecular pharmacology of benzodiazepines, barbiturates, and related compounds. Within the past 5 years it has become apparent that the benzodiazepine receptor is one component of a “supramolecular complex” consisting of multiple allosteric recognition sites which play a critical role in the regulation of transmembrane potential. Both neurochemical and electrophysiological studies support the notion that occupation of either the benzodiazepine-GABA receptor complex or distinct, allosteric sites for compounds such as barbiturates and cage convulsants (which are probably located on or near the chloride ionophore) can regulate the properties of this chloride channel. This chapter will review neurochemical evidence that demonstrates a functional coupling of the recognition components of the benzo-diazepine-GABA receptor chloride ionophore complex (supramolecular complex) and summarize recent findings from our laboratory demonstrating that the “effector” component of this complex, the chloride ionophore, is rapidly altered by stress.KeywordsChloride ChannelSupramolecular ComplexApparent AffinityNaive AnimalGaba BindingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A quantitative relationship between Cl − enhanced [ 3H]flunitrazepam and [formula omitted] binding to the benzodiazepine/GABA receptor chloride ionophore complex

A quantitative relationship between the efficacy (i.e. maximum enhancement) of Cl − to increase [ 3H]flunitrazepam binding and the density of [ 35S] -t- butylbicyclophosphorothionate binding sites was observed in well-washed membrane fragments of rat cerebral cortex previously exposed to phospholipase A 2. This relationship (described by the equation y = AB x ) was maintained when [ 3H]flunitrazepam was assayed at Cl − concentrations between 100 and 600 mM, and was not qualitatively altered by the presence of 100 μM pentobarbital. However, under experimental conditions that reduced the ratio of [ 35S]TBPS binding sites/benzodiazepine receptors, the effects of pentobarbital suggest that the conductance state of benzodiazepine receptor-coupled chloride channels may be the primary determinant of Cl − enhanced [ 3H]flunitrazepam binding.

II. Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex

The autoradiographic localization of subcomponents of the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex has provided insight into the distribution of this macromolecular system. GABA inhibits neurons by preferentially increasing the permeability of the affected membrane to chloride ions. This inhibition can be modified by the presence of other substances which bind to the GABA receptor complex. Autoradiographic localization of specific receptor subtypes associated with this complex has been accomplished in the central nervous system. This type of analysis has been performed on high and low affinity GABA A, benzodiazepine (BZ; both BZ 1 and BZ 2) and convulsant sites. These receptor sites are situated in distinct brain regions and co-exist in several areas. Other receptor subtypes, which may be influenced by the presence of GABA, can be analyzed for comparison in order to define regions of the brain where GABA may be exerting independent effects (i.e., those not associated with chloride channels). Microscopic localization of receptor sites indicates specific areas to investigate in further studies concerning the characterization of subcomponents of the macromolecular GABA complex associated with chloride ion channels.

Convulsant effect of Ro 5-4864, a peripheral type benzodiazepine, on the baboon ( Papio papio)

The effects of Ro 5-4864, a 1,4-benzodiazepine with a high affinity for the peripheral-type benzodiazepine (Bz) binding site, were investigated in the baboon ( Papio papio), which is genetically predisposed to epilepsy. A proconvulsant effect of low doses (1–3 mg/kg, i.v.) of Ro 5-4864 was observed by studying its effect on the photic responses induced by intermittent light stimulation in non-photosensitive baboons. Higher doses of Ro 5-4864 (10 mg/kg, i.v.) were overtly convulsant. The Bzs clonazepam and diazepam blocked these convulsant actions of Ro 5-4864 whereas neither Ro 15-1788, an antagonist of central Bz binding sites, nor PK 11 195, an antagonist of peripheral Bz binding sites, had any effect. It thus appeared that the convulsant effect of Ro 5-4864 was not mediated by Bz binding sites of either the central or the peripheral type. It is possible that Ro 5-4864 exerts its convulsant action at the picrotoxin site of the central Bz receptor - γ-aminobutyric acid receptor-chloride ionophore complex.

Are the toxicities of pentobarbital and ethanol mediated by the GABA-benzodiazepine receptor-chloride ionophore complex?

Both barbiturates and ethanol have been reported to interact with the GABA-benzodiazepine receptor-chloride ionophere ‘supramolecular complex’. These observations raise the possibility that some of the pharmacologic actions of barbiturates and ethanol may be mediated through this complex. In this study we have administered a series of drugs which bind to various components of the complex in an attempt to antagonize the lethality of sodium pentobarbital, and ethanol-induced loss of righting reflex in mice. It was found that isopropylbicyclophosphate (IPPO), a cage convulsant which binds at or near the chloride ionophore, greatly reduces the overall mortality (and increases latency to death) of animals pretreated with a lethal dose of pentobarbital. Picrotoxin also decreases pentobarbital lethality, but only at doses which were usually lethal when given alone. Picrotoxin shortened, rather than increased, latency to death. Strychnine did not prevent pentobarbital lethality, suggesting that the IPPO effect is not shared by convulsants in general. IPPO did not prevent ketamine-induced deaths, which supports the notion that the protective actions of IPPO are specific for depressant drugs which act at the chloride ionophore. IPPO also significantly reduced the duration of loss of righting reflex induced by ethanol. These observations suggest that the use of compounds which have a high affinity for the chloride ionophore in vitro might be fruitful in developing a clinical treatment for barbiturate or ethanol toxicity.

Preclinical pharmacology of buspirone hydrochloride.

Buspirone is an azaspirodecanedione that has anxiolytic actions in man and anticonflict actions in experimental animals. In contrast to many commonly used anxiolytic agents, buspirone does not appear to have significant sedative, muscle relaxant or anticonvulsant actions. Consequently, it has been termed an "anxioselective" agent. The effects of buspirone and related compounds have been examined in a number of neurotransmitter systems that have been linked to the actions of other anxiolytic agents. Buspirone has been shown to affect dopaminergic, serotonergic and noradrenergic pathways, as well as the GABA-benzodiazepine receptor chloride ionophore complex. Nonetheless, the doses (in vivo) and/or concentrations (in vitro) of buspirone (and related compounds) needed to affect these systems are not consistent with a causal relationship between the anxiolytic actions of buspirone and any of these systems. The neurochemical mechanism by which buspirone and related compounds exert both anticonflict and anxiolytic actions remains unknown.

Electrophysiological and pharmacological actions of the convulsant benzodiazepine Ro 5-4864

Ro 5-4864 (4′-chlorodiazepam) elicited convulsions in mice with a CD 50 of 23.5 mg/kg (i.p.) and increased the firing rate of substantia nigra zona reticula neurons in a dose dependent fashion (0.5–4 mg/kg i.v.). Diazepam and clonazepam, but not Ro 15-1788, were potent inhibitors of Ro 5-4864 induced convulsions. Ro 15-1788 was also ineffective in reversing Ro 5-4864 induced increases in cell firing of zone reticula neurons. Muscimol potently inhibited the seizures and reversed increases in cell firing elicited by Ro 5-4864. Phenobarbital and pentobarbital inhibited Ro 5-4864 induced convulsions with moderate potencies, while phenytoin and carbamazepine were ineffective at doses of up to 100 mg/kg. These observations suggest that Ro 5-4864 does not elicit its pharmacologic actions through a direct action at a ‘brain-type’ benzodiazepine receptor. However, both the profile and potency of compounds effective in inhibiting the electrophysiological and pharmacological effects of Ro 5-4864 suggest that this compound may act by perturbation of a component of the GABA-benzodiazepine receptor chloride ionophore complex. These findings do not, however, rule out a direct involvement of the high affinity ‘peripheral-type’ benzodiazepine receptors found in brain.