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

doi:10.1016/0014-2999(79)90264-4

European Journal of Pharmacology

Volume 56, Issue 4, 1 July 1979, Pages 337-345

https://doi.org/10.1016/0014-2999(79)90264-4 Get rights and content

Abstract

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 (B_max) or dissociation constant (K_D) 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 B_max. 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 × 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.

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In a previous study, it was found that down-regulation of benzodiazepine (BZ) binding in rats treated 4 weeks with flurazepam was relatively greater and more widespread when measured with [³H]zolpidem, a selective ‘BZ₁ receptor’ ligand, than that measured with the non-selective ligand, [³H]flunitrazepam. In the present study, the time course for down-regulation of [³H]zolpidem binding was studied in rats treated with flurazepam. [³H]Zolpidem binding was also studied in rats given a midazolam treatment shown to cause tolerance. Rats were chronically treated with flurazepam for 1 or 2 weeks, or with midazolam for 3 weeks, then killed immediately after the treatment. Another group of rats was acutely treated with desalkyl-flurazepam and killed 30 min later. After 2 weeks of flurazepam treatment, the B_max of [³H]zolpidem binding was decreased by 22% in cerebral cortex, 26% in cerebellum and 33% in hippocampus, with no change in the K_d in any region. After 1 week of flurazepam treatment, the B_max was decreased by 23% in cerebellum and 14% in hippocampus, but not changed in cerebral cortex. The K_d was increased in cerebral cortex, but not in cerebellum or hippocampus. Neither the B_max nor the K_d of [³H]zolpidem binding was affected by acute desalkyl-flurazepam treatment, or by 3 weeks of midazolam treatment. These results, in combination with previous findings, which showed no change in [³H]flunitrazepam binding after 1 or 2 week flurazepam treatment, and no change in cerebellum even after the 4 week treatment, may indicate a shift in BZ receptor subtypes in flurazepam-tolerant rats. Such a shift could be based on possible changes in the subunit composition or conformation of GABA_A/BZ receptors after 1 or 2 week flurazepam treatment.
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Folates: epileptogenic effects and enhancing effects on [3H]TBOB binding to the GABA<inf>A</inf>-receptor complex
1990, Epilepsy Research
The biochemical mechanism responsible for the convulsive effects of folates was investigated. The epileptogenic effects of folates were determined in vivo by quantification of the seizures following intracortical application in rats. The rank order of epileptogenic effects is: folic acid ⩾ 5-HCO-H₄ folate > H₂ folate > 5-CH₃-H₄ folate. This sequence of epileptogenicity in vivo is compared to the rank order of the effects of folates on radioligand binding to the GABA_A-recptor complex in vitro. The inhibitory potencies of folates on [³H]muscimol and [³H]diazepam bindings did not correlate with their epileptogenic effects. However, folates reverse the inhibiting effect of GABA on the binding of the cage convulsant [³H]TBOB $(^{3} H]t- butylbicycloorthobenzoate$ ). The rank order of this in vitro effect (folic acid > 5-HCO-H₄ folate > H₂ folate = 5-CH₃-H₄ folate) resembles the rank order of epileptogenicity determined in vivo. A relationship between the in vivo and in vitro effects is therefore suggested.
Chronic exposure to Ro 15-1788: differential effect on flunitrazepam binding to cortex and hippocampus
1989, European Journal of Pharmacology
The time course of changes in specific [³H]flunitrazepam binding following 2 weeks of treatment with the benzodiazepine antagonist Ro 15-1788 (2.7 and 4 mg/kg per day in drinking water) was studied in the rat neocortical and hippocampal synaptosomal membranes. Such a treatment produced regional increases in the density of benzodiazepine sites, which remained for up to 24 and 48 h after drug withdrawal in the hippocampus and cortex, respectively; the dissociation constant (K_d) was unchanged. In addition, a significant reduction in GABA enhanced [³H]flunitrazepam binding to cortical, but not to hippocampal, membranes from Ro 15-1788-treated animals was found 72 h after drug withdrawal. These data show that there is a regional difference in responses of the GABA/benzodiazepine receptor complex following chronic in vivo exposure to Ro 15-1788 and that, beside the increases in the maximal binding (B_max) of [³H]flunitrazepam the coupling between the GABA and the benzodiazepine recognition sites was also affected.
Regional GABA/benzodiazepine receptor/chloride channel coupling after acute and chronic benzodiazepine treatment
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GABA/benzodiazepine coupling was evaluated in 8 regions of rat brain by the ability of GABA to stimulate 0.5 nM [³H]flunitrazepam binding. Rats were treated acutely with diazepam (p.o) or chronically with flurazepam, offered in the drinking water for 4 weeks, and compared to a pair-handled vehicle-treated control group. Regional variations in GABA/benzodiazepine coupling were found in control membranes. GABA increased benzodiazepine binding maximally (40%) in cerebellum and medulla, and least (25%) in olfactory bulb. A significant decrease in the effect of GABA was found in cortex of chronically treated rats immediately after, but not 2 days following treatment. The E_max for GABA stimulation of [³H]flunitrazepam binding was significantly increased in medulla after acute treatment but was not altered after acute or chronic treatment in other brain areas evaluated. Treatment had no effect on the ability of bicuculline to inhibit [³H]flunitrazepam binding in cortex. Benzodiazepine /Cl⁻ couping in cortex or hippocampus of acutely and chronically treated rats, evaluated by the ability of Cl⁻ to stimulate specific [³H]flunitrazepam binding, was not changed. The results support the hypothesis that a functional uncoupling of the benzodiazepine recognition site from the GABA receptor in cortex, but not from the anion recognition site, may play a role in tolerance development.

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Regular paperGaba receptor-mediated modulation of 3H-diazepam binding in rat cortex

Abstract

FEBS Letters

European J. Pharmacol.

European J. Pharmacol.

European J. Pharmacol.

Life Sci.

Brain Res.

Biochem. Pharmacol.

Anal. Biochem.

Life Sci.

J. Biol. Chem.

Neuropharmacol.

Life Sci.

Life Sci.

Life Sci.

Life Sci.

Life Sci.

Regular paper
Gaba receptor-mediated modulation of ³H-diazepam binding in rat cortex