Binding of [<sup>3</sup>H]ethyl‐β‐carboline‐3‐carboxylate to brain benzodiazepine receptors

[3H]Diazepam and [3H]flunitrazepam ([3H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, gamma-aminobutyric acid (GABA) and NaCl both increased [3H]diazepam binding more than [3H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [3H]diazepam in the absence of NaCl and GABA, the Bmax (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [3H]FNP, which dissociates more slowly, had the same Bmax in both assays. [3H]Diazepam association had two components, and was faster than [3H]FNP association. [3H]Diazepam dissociation, which also had two components, was faster than that of [3H]FNP, and also had a greater fraction of rapidly dissociating species. [3H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [3H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.

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“…The underestimation of the B,,, for [3H]diazepam binding may also occur in other reports (cf. Pong et al, 1981;Williams et al, 1981).…”

Section: Discussionmentioning

confidence: 99%

Comparison of the Kinetics of [³H]Diazepam and [³H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Chiu

Rosenberg

1982

Journal of Neurochemistry

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“…The ability of permeant anions to enhance the apparent affinity of BzR agonists was proposed to reflect an increased coupling between their receptors and associated chloride channels (Costa et al, 1979). This effect appeared restricted to agonists, because chloride did not alter the binding of a BzR antagonist (Ro 15-1788) or an inverse agonist (3-carboethoxy-0-carboline) (Mohler and Richards, 198 1 ;Williams et al, 1981). However, Honor6 et al (1983) reported that permeant anions augmented the binding of the convulsant ,f3-carboline [3H]DMCM to BzRs.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, barbiturate and pyrazolopyridine enhancement of radioligand binding to BzRs and GABA receptors is anion dependent (Leeb-Lundberg et al, 1980, 198 1 ;Asano and Ogasawara, 1981;Skolnick et al, 198 l), as is the binding oft-[ 35S]butylbicyclophosphorothionate ([35S]TBPS), a "cage" convulsant that binds to GABAgated chloride channels (Squires et al, 1983). Initially, the ability of anions to regulate radioligand binding to BzRs appeared to be restricted to agonists, because permeant anions (such as chloride) were reported not to affect the binding of either [3H]Ro 15-1788 (a BzR antagonist) (Mohler and Richards,198 1) or [3H]3-carboethoxy-p-carboline (a BzR inverse agonist) (Williams et al, 1981). However, Honor6 et al (1983) demonstrated that anions (chloride, iodide, and bromide) could increase the binding of the convulsant p-carboline [3H]DMCM to BzRs (Braestrup et al, 1982).…”

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confidence: 99%

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Evoniuk

Skolnick

1988

Journal of Neurochemistry

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Binding of the benzodiazepine inverse agonist [3H]methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate [( 3H]DMCM) and the agonist [3H]flunitrazepam [( 3H]FNZ) was compared in rat cortical membranes. Halide ions enhanced [3H]DMCM binding three- to fourfold, increasing both the apparent affinity and the number of binding sites for this radioligand. The effect was present at both 0 and 37 degrees C. In contrast, the magnitude of halide stimulation of [3H]FNZ binding was much smaller, resulting solely from an increase in the apparent affinity for this radioligand, and was not observed at 37 degrees C. The potencies but not the efficacies of a series of anions to stimulate both [3H]DMCM and [3H]FNZ binding to benzodiazepine receptors were highly correlated with their relative permeabilities through gamma-aminobutyric acid (GABA)-gated chloride channels. Two stress paradigms (10 min of immobilization or ambient-temperature swim stress), previously shown to increase significantly the magnitude of halide-stimulated [3H]FNZ binding, did not significantly affect [3H]DMCM binding. Phospholipase A2 treatment of cortical membrane preparations was equipotent in preventing the stimulatory effect of chloride on both [3H]DMCM and [3H]FNZ binding. These data strongly suggest that anions modify the binding of [3H]DMCM and [3H]FNZ by acting at a common anion binding site that is an integral component of the GABA/benzodiazepine receptor chloride channel complex.

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“…For example, whereas GABA greatly stimulates the binding of 3H-benzodiazepines, it has little or no influence o~[~H ] P -C C E binding (Marangos and Patel, 1981). Furthermore, [3H]p-carboline recognition sites are not modulated by halide ions, barbiturates, and pyrazolopyridines, as has been demonstrated for 3H-benzodiazepine binding sites (Williams et al, 1981). Recently, it was demonstrated that nickel ions and heat exposure produced clear-cut differences in the regulation of these binding sites (Hirsch et al, 1982).…”

mentioning

confidence: 83%

“…A range of 0.5-10 nM [3H]P-CCE was used. Since it has been reported that nonspecific binding was identical with use of either unlabeled p-carbolines or unlabelled benzodiazepines (Williams et al, 1981;Hirsch et al, 1982) parallel experiments were carried out in the presence of 3 pM flunitrazepam. The nonspecific binding represented 10-15% of the total.…”

Section: Binding Assaysmentioning

confidence: 99%

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [³H]Flunitrazepam and [³H]Ethyl‐β‐carboline‐3‐carboxylate Binding Sites in Rat Brain Membranes

Medina

Novas

Robertis

1983

Journal of Neurochemistry

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This study was designed to analyze possible differences in the binding of [3H]flunitrazepam ([3H]FNZP) and [3H]ethyl ‐ β ‐ carboline ‐ 3 ‐ carboxylate ([3H]β‐CCE), to rat brain membranes, in various experimental conditions. In cerebral cortex, hippocampus, cerebellum, and orain stem the number of binding sites for [3H]β‐CCE was higher than for [3H]FNZP; both were displaced by clonazepam. Until the 7th day of postnatal brain development the numbers of [3H]FNZP and [3H]β‐CCE sites were equivalent; but later on, the β‐carboline sites increased to a higher level. Noradrenergic denervation by 6‐hydroxydopamine was followed in the hippocampal formation. Already after 2 days, there was a decrease in [3H]FNZP sites, which reached 70% of control after 14 days. Similar results were obtained with DSP‐4 denervation. This change was only in Bmax and not in KD, In contrast, the [3H]β‐CCE sites did not change with denervation. Neonatal injection of l ‐ 2,4,5 ‐ trihydroxyphenylalamine or DSP‐4 produced in the adult a decrease in [3H]FNZP sites in the cerebral cortex, in parallel with the noradrenergic denervation. On the other hand, there was an increase in the cerebellum and brain stem, in correspondence with the hyperinnervation by sprouting. In these rats, the number of sites for [3H]β‐CCE did not change in the different brain regions. With 0.1% Triton X‐100, applied to synaptosomal membranes, [3H]FNZP binding was reduced by 35%, while that of [3H]β‐CCE was not significantly changed. These results suggest that there is heterogeneity of binding sites for benzodiazepine receptors in rat brain. A tentative interpretation of the experiments involving noradrenergic denervation and hyperinnervation, as well as those with Triton X‐100, is that [3H]FNZP binds to pre‐ and postsynaptic receptors, while [3H]β‐CCE binds mainly to postsynaptic benzodiazepine receptors.

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Binding of [³H]ethyl‐β‐carboline‐3‐carboxylate to brain benzodiazepine receptors

Cited by 15 publications

References 19 publications

Comparison of the Kinetics of [³H]Diazepam and [³H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Comparison of the Kinetics of [³H]Diazepam and [³H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [³H]Flunitrazepam and [³H]Ethyl‐β‐carboline‐3‐carboxylate Binding Sites in Rat Brain Membranes

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Binding of [3H]ethyl‐β‐carboline‐3‐carboxylate to brain benzodiazepine receptors

Cited by 15 publications

References 19 publications

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Anion Regulation of Agonist and Inverse Agonist Binding to Benzodiazepine Receptors

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [3H]Flunitrazepam and [3H]Ethyl‐β‐carboline‐3‐carboxylate Binding Sites in Rat Brain Membranes

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Product

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Binding of [³H]ethyl‐β‐carboline‐3‐carboxylate to brain benzodiazepine receptors

Comparison of the Kinetics of [³H]Diazepam and [³H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Comparison of the Kinetics of [³H]Diazepam and [³H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Heterogeneity of Benzodiazepine Receptors: Experimental Differences Between [³H]Flunitrazepam and [³H]Ethyl‐β‐carboline‐3‐carboxylate Binding Sites in Rat Brain Membranes