GABA Receptor Binding Research Articles

Inhibition of benzodiazepine and GABA receptor binding by amino-γ-carbolines and other amino acid pyrolysate mutagens

The effect of several pyrolysate mutagens on the benzodiazepine and GABA receptors was investigated. Of amino-γ-carbolines, Trp-P-1 antagonized the suppressive effect of diazepam on the pentylenetetrazol-induced convulsions and death, whereas Trp-P-2 by itself precipitated seizures and death in male mice. Both Trp-P-1 and Trp-P-2 inhibited the specific binding of [ 3H]diazepam and [ 3H]muscimol in rat brain membranes mainly by increasing K d, indicating that these γ-carbolines bind on benzodiazepine and GABA receptors. IC 50s of Trp-P-1 and Trp-P-2 on specific [ 3H]flunitrazepam binding were not changed by addition of GABA. The Hill coefficient of Trp-P-1 for displacing [ 3H]diazepam binding was about unity whereas that of Trp-P-2 was less than unity. These results suggest that Trp-P-1 and Trp-P-2 act as active antagonists or inverse agonists at benzodiazepine receptors. The convulsant effect of the γ-carbolines may be mediated by an action on the central benzodiazepine receptors; however, the role of the effect on GABA receptors is not clear.

The action of structural analogues of γ‐aminobutyric acid on binding sites in mouse brain

AbstractNineteen structural analogues of γ‐aminobutyric acid (GABA) and the anxiolytics buspirone and diazepam were tested for their ability to modify GABA function in vitro. Each compound was added to the GABA receptor binding assay, the flunitrazepam receptor binding assay, the sodium‐dependent GABA binding assay, and the assay for GABA aminotransferase. It was anticipated that some of these analogues would substantially affect several of these assays. The most interesting compound to emerge was phenylthiohydantoic acid. This not only inhibited GABA receptor binding (Ki = 121 μM) but also stimulated flunitrazepam binding and inhibited the activity of GABA aminotransferase. In light of the evidence that a reduced GABA function can be a factor in anxiety, phenylthiohydantoic acid has the potential to be a GABA‐mimetic and thus be useful in the treatment of anxiety.

An endogenous inhibitor of GABA receptor binding.

An endogenous inhibitor of GABA receptor binding was prepared from synaptic membrane of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA for GABA binding sites. The inhibition of GABA receptor binding by the endogenous inhibitor was blocked by the allosteric effect of diazepam. In the presence of diazepam, specific [3H]GABA binding was greater in a medium containing the endogenous inhibitor than in one containing an equal inhibitory potency of GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself.

Effect of 2-aminoethanol on the synthesis, binding, uptake and metabolism of GABA

2-Aminoethanol (ethanolamine) was studied for effects on neurochemical assays for GABA synthesis, receptor binding, uptake and metabolism in rat brain preparations. The effects of ethanolamine were compared with those of ethanolamine O-sulphate (EOS), an inhibitor of GABA degradation. Furthermore, the effect of both compounds was compared on GABA metabolism in rat brain in vivo. In vitro, ethanolamine and EOS had no significant effect on the GABA synthesizing enzyme glutamic decarboxylase (GAD) and GABA uptake, but both drugs proved virtually equipotent to inhibit the GABA degrading enzyme GABA aminotransferase (GABA-T). EOS was a relatively potent inhibitor of GABA receptor binding, whereas ethanolamine was not effective in this regard. Following systemic administration in rats, 50% inhibition of GABA-T in the brain was achieved by 500 mg/kg ethanolamine or 2000 mg/kg EOS, respectively. As a consequence of GABA-T inhibition, GABA levels increased significantly. GAD activity remained unchanged after both treatments. The present results suggest that the recently reported enhancement of functional effects of GABA by ethanolamine may relate, at least in part, to the inhibitory effect of the compound on GABA catabolism.

Biochemical and functional alterations of central GABA receptors during chronic estradiol treatment

The characteristics of GABA and benzodiazepine receptors were examined in the hippocampus, striatum and cerebral cortex of female rats at various times (up to 9 months) after the subcutaneous implantation of an estradiol pellet (10 mg). A significant decrease in the B max of the high-affinity binding of [ 3H]muscimol to membranes from these 3 regions was detected as soon as one week after the implantation. Although the characteristics of the high-affinity binding of [ 3H]flunitrazepam remained unaffected during the whole treatment, the stimulatory effect of GABA (and muscimol) on this binding was significantly reduced by estrogenization. The changes in GABA receptor binding appeared functionally relevant since the elevation of striatal acetylcholine levels normally induced by the peripheral administration of muscimol (5 mg/kg) was significantly lower in estradiol-treated than in control female rats. In contrast to that observed in intact female rats, the implantation of estradiol in hypophysectomized animals did not affect the characteristics of [ 3H]muscimol binding to hippocampal, striatal and cortical membranes. [ 3H]muscimol binding was also unchanged in female rats implanted with estradiol and treated chronically with bromocriptine for 3 weeks. Since both hypophysectomy and the chronic administration of bromocriptine suppressed the hyperprolactinemia normally induced by estrogenization, the down-regulation of central GABA receptors very likely involved prolactin in intact animals implanted with 17-β-estradiol.

Alterations in benzodiazepine and GABA receptor binding in rat brain following systemic injection of kainic acid

The binding of γ-aminobutyric acid (GABA) and benzodiazepine to receptors was examined in regions of rat brain at various times after subcutaneous injection of kainic acid (KA, 15 mg/kg). The animals exhibited pronounced convulsions 90 min–4 hr after this treatment. During this period (2 hr after the injection of kainic acid) no alterations in the binding of [ 3H]-GABA or [ 3H]flunitrazepam to receptors were detected in the frontal cortex, the hippocampus or the amygdala-pyriform cortex. After recovery from the acute convulsive phase, the rats appeared to be hyperexcitable, hyperactive, and displayed marked aggression and occasional clonic convulsions one to 80 days later. During this period a marked increase (80–200%) in the number of binding sites for GABA in the amygdala-pyriform cortex occurred but this was associated with a slow decrease in the number of binding sites for [ 3H]flunitrazepam to 70% control value at 3 weeks. Binding of the “peripheral” -type of benzodiazepine ligand, [ 3H]-Ro5-4864, was increased to 450% of control 3 weeks after injection. In addition, the ability of GABA to stimulate the binding of [ 3H]flunitrazepam was reduced when measured 3 days after the injection of kainic acid. It is suggested that the long-term behavioural syndrome observed in kainic acid-treated rats, as well as the reduced effectiveness of diazepam in preventing seizures in animals treated with kainic acid, (Czuczwar, Turski, Turski and Kleinrock, 1981) may be explained in part by a reduction in the number of neuronal benzodiazepine receptors and a “desensitization” of the GABA receptors which are coupled to benzodiazepine receptors.

GABA receptor binding and hemodynamic responses to ICV GABA in adult spontaneously hypertensive rats.

Blood pressure and heart rate responses after central administration of GABA (100–1000 μg, ICV) and after ganglionic blockade with hexamethonium (25 mg/kg, i.a.) were compared in conscious 12 week-old spontaneously hypertensive (SH) and age-matched Wistar-Kyoto (WKY) rats. Ganglionic blockade produced an equivalent change in mean arterial pressure between SH and WKY rats. Thus, the total functional sympathetic nervous system contribution to blood pressure is equivalent in these two strains. Intraventricularly-administered GABA produced a greater absolute decrease in mean arterial pressure in the SHR compared to the WKY. However, the percent changes in arterial pressure produced by GABA were not different between these strains. This greater absolute depressor effect of GABA does not appear to be due to differences in GABA receptor binding constants since GABA receptor affinity and number of binding sites were not significantly different in the forebrain, cerebellum or pons-medulla between SH and WKY rats.

The effects of chronic haloperidol administration on GABA receptor binding

The effect of chronic administration of haloperidol for 30 days and its subsequent withdrawal for 0,4 and 8 days on the binding of [ 3H]-GABA and [ 3H]-muscimol to GABA binding sites in rat brain membranes obtained from six discrete regions of the brain and spinal cord was investigated. The chronic administration of haloperidol resulted in a marked increase in the number of GABA binding sites within the substantia nigra, but did not affect GABA binding sites in other regions of rat brain or spinal cord. The increase in GABA binding sites in the substantia nigra was evident for at least 4 days following termination of haloperidol treatment; however, 8 days following withdrawal from haloperidol, when dopaminergic supersensitivity had become maximally expressed, the number of GABA binding sites in the substantia nigra had declined and was the same as that observed in the control rats. These observations suggest that blockade of dopamine receptors in the striatum by haloperidol results in a compensatory decrease in the activity of the GABAergic strionigral system that leads to an increase in the number of GABA binding sites within the substantia nigra. This increase in the number of GABA binding sites is gradually reversed when dopamine is again allowed to interact with its receptors.

Increased GABA receptor binding in dominantly-inherited cerebellar ataxias

We measured γ-aminobutyric acid (GABA) receptor binding in the cerebellar cortex of 14 patients with dominantly-inherited cerebellar ataxias. Most of these patients had moderate to marked Purkinje cell loss as judged from the subnormal dentate nucleus GABA levels. Mean GABA receptor binding (at 25 nM GABA concentration) was elevated significantly by 37% in the total group of 14 patients studied and by 60% in the largest subgroup ( n=6) analyzed. Possible explanations for the enhanced binding include glial prolife-ration, exposure of normally unexposed receptor sites consequent to neuronal degeneration, and/or denervation supersensitivity.

Neurochemical studies on GABA receptor (VIII). Enhancement of 3H-muscimol binding to synaptic membrane by unsaturated free fatty acids.

Pretreatment of cerebral synaptic membrane with phospholipase (PLase)A2 as well as PLase C resulted in a significant facilitation of 3H-muscimol binding. In addition, it was found that a significant facilitation of 3H-muscimol binding was induced by pretreating synaptic membrane with unsaturated free fatty acids such as arachidonic acid(ARA) and linoleic acid. The stimulatory action of ARA on the binding was significantly enhanced by the addition of lipoxygenase, whereas in vitro addition of various prostaglandins(E1, E2, F2 α, D2 and I2), cyclo-oxygenase products, had no profound effects on the binding. No significant correlation was observed between lipid peroxides formation in the membrane and the enhancement of 3H-muscimol binding induced by PLase A2, PLase C and ARA. On the other hand, 3H-muscimol binding was significantly elevated by the pretreatment of membrane with xanthine and xanthine oxidase, a superoxide anion generating system. The enhancement of 3H-muscimol binding by PLase A2 was also suppressed significantly by the addition of various scavengers for superoxide anion such as superoxide dis-mutase, tiron and nitroblue tetrazolium. These results suggest that PLase A2 may stimulate GABA receptor binding through the production of active oxygens including superoxide anion which can be originated from unsaturated free fatty acids released from membrane phospholipids by the action of this enzyme.

Tofizopam selectively increases the affinity of benzodiazepine binding sites for [ 3H]flunitrazepam but not for β [ 3H] carboline-3-carboxylic acid ethyl ester

Summary The psychoactive properties of tofizopam (TP), a 3,4-benzodiazepine (BZ), differ from those of 1,4-BZ's. TP does not cause sedation or muscle relaxation, although it is claimed to be anxiolytic. TP does not act by occupying BZ binding sites, although similarly to 1,4-BZ's, it increases binding of GABA receptors. In this study the mechanism of the neurochemical action of TP was further studied. TP (300 mg/kg) enhanced the binding of [3H]flunitrazepam (3H-FP) to BZ binding sites in the mouse brain by 13.7 – 22.9 % in vivo, but failed to alter the binding of β [3H]carboline-3-carboxylic acid ethyl ester (3H-BCCE). 3H-BCCE is a selective ligand for a subclass of BZ binding sites that is most abundantly present in the cerebellum. In vitro binding of 3H-BCCE was not affected by TP; but diazepam (DP) decreased 3H-BCCE binding yielding IC50 of 16 nM. In vivo DP (50 mg/kg) also competed with bound 3H-FP and 3H-BCCE, decreasing binding of 3H-FP by 85.4–86.9 % and that of 3H-BCCE by 45.0–51.0 %. The psychoactive effect of TP seems to be mediated by influencing GABAergic transmission. TP is more selective in its action than DP, a 1,4-BZ; TP enhances binding of the GABA receptors and the binding of one or more subclasses of BZ binding sites, but does not affect the cerebellar type of BZ binding sites.

Changes in high affinity sodium independent gamma-aminobutyric acid binding in cerebral cortex and hippocampus of the rat following electroshock

Electroshock induced seizures in the rat enhanced high affinity specific Na + independent binding of 3H-gamma aminobutyric acid (GABA) to frozen, Triton X-100 treated cerebral synaptic membranes 30 minutes after exposures to electroshock, although no change in 3H GABA binding was observed in similarly treated preparations from hippocampus. Scatchard analysis of the binding isotherms from cortical membranes indicated that the increase in 3H GABA binding at 30 minutes was due to a rapid increase (39%) in the number of available GABA receptor binding sites (B max) rather than an alteration in receptor affinity (K D). The number of binding sites returned to control values within 1 hour and remained so throughout the duration of this study.

Selective changes of receptor binding in brain regions of aged rats

Binding to several receptors was compared in brain regions of 3 and. 21–23 month-old rats. In crude membrane preparations of aged rats the number of dopamine antagonist receptors in striatum was much reduced (−53%). β-Noradrenergic receptors (cortex) and benzodiazepine receptors (hippocampus and cerebellum) were less but significantly reduced and serotonergic receptors, α 1 noradrenergic receptors (both in cortex) and dopamine agonist receptors (striatum) were unchanged. For each receptor binding the K D values were the same in young and old animals. GABA receptor binding (hippocampus and cerebellum) evaluated at only one 3H-GABA concentration (8 nM) was similar in both groups when expressed per protein content but significantly reduced in aged rats when expressed per tissue wet weight because of the partial purification of the synaptic membranes used for 3H-GABA binding. In our experimental conditions age-related changes of specific binding sites in the central nervous system were selective for some receptors studied and did not seem to be due to general non-specific modification of brain tissue composition.

3H]GABA binding in developing rabbit retina

We have studied the developmental sequence of the GABA system in the rabbit retina using an in vitro binding assay to monitor developmental changes in the post-synaptic receptor. A variety of tissue treatments including perchlorate and Triton X-100 were employed to optimize binding and remove endogenous factors which inhibit binding. Pre-treatment of the tissue with 0.05% Triton X-100 revealed high affinity binding for [3H]GABA which increased in a sigmoidal fashion with the post-natal age of the animal. A constant level of binding, at about 16% of adult levels, was noted until day 8, at which time a rapid increase occurred. At 16 days post-natal, the amount of specific binding reached a plateau near adult levels. Kinetic analysis of the GABA receptor showed an increase in the number of receptors (Bmax) with little or no change in the apparent affinity (KD). Our results suggest that the onset of post-synaptic receptor activity is delayed approximately 1 to 2 days, relative to the pre-synaptic components, and the period of rapid increase in GABA receptor binding coincides with the period of maximum increase in retinal synaptic density.

The influence of ACTH and corticosterone on [3H]GABA receptor binding in rat brain

Bilateral adrenalectomy (ADX) induces a significant, regionally selective, increase in GABA, but not cholinergic muscarinic orα1-adrenergic, receptor binding in rat brain. The increase in GABA receptor binding in the midbrain occurs within 72 h of surgery, whereas that found in the corpus striatum becomes evident between 1 and 2 weeks later. These ADX-induced receptor changes are counteracted by the administration of corticosterone, a reversal which can occur within 24 h following a single administration of the steroid. Unlike ADX, hypophysectomy causes a significant reduction in [3H]GABA receptor binding in these two brain areas, an action that is not reversed by corticosterone treatment. Furthermore, systemic administration of either ACTH1–39 or ACTH4–10 in unoperated animals causes an increase in midbrain and striatal GABA receptor binding similar to that observed in ADX animals. The increase in [3H]GABA binding observed after ACTH administration appears to be due to the apearance of low affinity, high capacity binding sites not observed in untreated animals. ADX had no effect on high affinity GABA uptake, glutamic acid decar☐ylase or GABA content in the brain regions where receptor modifications were noted. These findings indicate that GABA receptor binding in rat brain can be modified by changes in the circulating levels of ACTH.

(3H)-muscimol, (3H)-nipecotic acid and (3H)-isoguvacine as autoradiographic markers for GABA neurotransmission.

Retinas from rabbit, goldfish and guinea-pig were exposed to (3H) GABA, (3H)-nipecotic acid and (3H)-isoguvacine either by intravitreal injection in vivo or by incubations in a balanced salt solution and the distribution of radioactivity was then studied with autoradiography. All substances labelled a similar set of presumed amacrine cells. Incubating at 0 degrees C, in 10-(5)M ouabain, or in 10-(3)M GABA inhibited the labelling by (3H)-muscimol whereas bicuculline (10-(4)M), and glycine (10-(3)M) were less efficient blockers. The result is interpreted as a neuronal uptake of (3H)-muscimol rather than as a GABA receptor binding. All the substances except (3H)-isoguvacine also labelled glia to such a degree that neuronal labelling was often disguised in rabbits and goldfish. Glial labelling by muscimol was less pronounced in guinea-pig. (3H)-isoguvacine (tested only in rabbits) gave a strong labelling of cells with the distribution of GABA neurons and only little glial labelling.

Stimulation of GABA receptor binding by barbiturates

Barbiturates increase Na +-independent GABA binding to bovine cerebral cortex membranes. The relative activity of a series of compounds to enhance GABA binding correlates significantly with their anesthetic activity and with their ability to reverse bicuculline antagonism of GABA responses. There are regional differences in the maximal percent stimulation of GABA binding by pentobarbital; a low stimulation in cerebellum.

A modulating role of mercurials-sensitive sulfhydryl groups in synaptic GABA receptor binding

The specific binding of GABA (?-aminobutyric acid) agonist (3)H-muscimol, to synaptic membranes from the rat brain showed a significant increase, when the membranous preparations were treated with a low concentration (10(?4)-10(?5)M) of mercurial sulfhydryl reagents such as p-chloromercuribenzoate and mercuric chloride. This activation in GABA receptor binding was bicuculline-sensitive, and was partially restored by subsequent treatments with 10 mM cysteine, penicillamine, or mercaptoethanol. Scatchard analysis of the binding revealed that this activation was due to the increase in the affinity of both high and low affinity bindings sites but not in the B(max) values. On the other hand, the treatment of synaptic membranes with hydrophilic sulfhydryl reagents such as N-ethylmaleimide and iodoacetate had no effect on the binding. These hydrophilic sulfhydryl reagents, however, induced an increase of the binding following the pretreatment of synaptic membranes with 0.01% Triton X-100 or 0.5 U/mg prot. of phospholipase A(2) (EC 3.1.1.4.). These results suggest that mercurials-sensitive sulfhydryl groups, which are normally masked by membrane lipids, may play a modulating role in GABA receptor binding at central synapses.

A relationship between synaptosomal GABA uptake and blood pressure in five inbred strains of mice

Synaptosomal GABA uptake, sodium-dependent GABA binding to synaptic membranes and GABA receptor binding were measured in the cerebral cortex of five inbred strains of mice. These were the SWR/J, BALB/cJ, DBA/1J, C57BL/10J and C3HeB/FeJ strains. A highly significant negative correlation was found to exist between GABA uptake and blood pressure (r = −0.92). A similarly good negative correlation occurred between sodium-dependent GABA binding and blood pressure. A poor correlation was observed between blood pressure and GABA receptor binding (r = −0.46). Taken together with other evidence that exists in the literature, these results suggest that in mice blood pressure could be under the influence of the GABA system.

Differences between GABA receptor binding to membranes from cerebellum during postnatal development and from cultured cerebellar granule cells.

GABA receptor binding sites were determined in membranes from cerebella of 7-, 15- and 60-day-old rats and cerebellar granule cells derived from 7-day-old rats and cultured for 8 days using [3H]GABA as the ligand and nonradioactive GABA to measure nonspecific binding. Membranes from cerebellum at all postnatal stages exhibited at least two binding sites for GABA with binding constants around 7-9 and 150-750 nM, respectively. The total number of binding sites expressed on the basis of total protein increased 10 times between 7 and 60 days of age and 2 times between 15 and 60 days. The latter increase could be quantitatively accounted for by an increase in the number of low affinity sites. In contrast to this, membranes from cultured granule cells only exhibited the high affinity binding site (KD 7.1 +/- 0.5 nM) and the number of binding sites was comparable to that of cerebellar membranes from rats of the corresponding age (15 days old). GABA analogues such as muscimol, piperidine-4-sulphonic acid, homo-beta-proline, THIP, and isoguvacine were potent displacers of GABA binding to cultured granule cells, whereas nipecotic acid and guvacine had no effect.