Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain

doi:10.1016/0024-3205(78)90476-9

Life Sciences

Volume 22, Issue 21, 5 June 1978, Pages 1893-1900

https://doi.org/10.1016/0024-3205(78)90476-9 Get rights and content

Abstract

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.

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Cited by (55)

Endozepines and their receptors: Structure, functions and pathophysiological significance
2020, Pharmacology and Therapeutics
Citation Excerpt :
This assumption has prompted several groups to start looking for possible “benzodiazepineoid” peptides as naturally occurring ligands for BZ-binding sites. Thus, as soon as 1978, two groups reported the existence of an endogenous protein that reduces the affinity of [3H]diazepam for crude synaptic membranes from rat cerebral cortex (Guidotti, Toffano, & Costa, 1978; Marangos, Paul, Greenlaw, Goodwin, & Skolnick, 1978; Toffano, Guidotti, & Costa, 1978). This protein, termed DBI, was purified to homogeneity and partially sequenced (Guidotti et al., 1983).
The existence of specific binding sites for benzodiazepines (BZs) in the brain has prompted the search for endogenous BZ receptor ligands designated by the generic term « endozepines ». This has led to the identification of an 86-amino acid polypeptide capable of displacing [³H]diazepam binding to brain membranes, thus called diazepam-binding inhibitor (DBI). It was subsequently found that the sequence of DBI is identical to that of a lipid carrier protein termed acyl-CoA-binding protein (ACBP). The primary structure of DBI/ACBP has been well preserved, suggesting that endozepines exert vital functions. The DBI/ACBP gene is expressed by astroglial cells in the central nervous system, and by various cell types in peripheral organs. Endoproteolytic cleavage of DBI/ACBP generates several bioactive peptides including a triakontatetraneuropeptide that acts as a selective ligand of peripheral BZ receptors/translocator protein, and an octadecaneuropeptide that activates a G protein-coupled receptor and behaves as an allosteric modulator of the GABA_AR. Although DBI/ACBP is devoid of a signal peptide, endozepines are released by astrocytes in a regulated manner. Consistent with the diversity and wide distribution of BZ-binding sites, endozepines appear to exert a large array of biological functions and pharmacological effects. Thus, intracerebroventricular administration of DBI or derived peptides induces proconflict and anxiety-like behaviors, and reduces food intake. Reciprocally, the expression of DBI/ACBP mRNA is regulated by stress and metabolic signals. In vitro, endozepines stimulate astrocyte proliferation and protect neurons and astrocytes from apoptotic cell death. Endozepines also regulate neurosteroid biosynthesis and neuropeptide expression, and promote neurogenesis. In peripheral organs, endozepines activate steroid hormone production, stimulate acyl chain ceramide synthesis and trigger pro-inflammatory cytokine secretion. The expression of the DBI/ACBP gene is enhanced in addiction/withdrawal animal models, in patients with neurodegenerative disorders and in various types of tumors. We review herein the current knowledge concerning the various actions of endozepines and discuss the physiopathological implications of these regulatory gliopeptides.
Isolation of endogenous modulators for the GABA<inf>A</inf> and taurine receptors
1993, Neurochemistry International
Several endogenous brain substances which inhibit [³H]muscimol binding were isolated, and one of them has been purified to apparent homogeneity. The purification involved the extraction of brain tissue with water, followed by several steps of gel filtration column chromatography and high performance liquid chromatography (HPLC). The muscimol binding inhibitor (MBI) thus obtained appeared to be homogeneous as judged from the elution profile of an HPLC column, in which a symmetrical peak was obtained when the eluate was monitored at either 220 or 280 nm. Furthermore, the MBI activity coincided with the absorption peak. The purified MBI is not γ-amino butyric acid (GABA), β-alanine or taurine since these amino acids are clearly separated from the MBI in the purification procedures. The MBI has no effect on benzodiazepine (BZ) binding or glutamate binding to their respective receptors. However, the MBI is a more potent inhibitor for [³H]taurine binding than that of [³H]muscimol binding. The MBI appears to be a small molecule (<2000 Da) that is heat and acid/base stable. The chemical nature of the MBI is currently under investigation.
Behavioral effects induced by beta CCE in free or restrained rhesus monkeys (Macaca mulatta)
1990, Pharmacology, Biochemistry and Behavior
Behavioral effects of the IV injection of beta carboline acid carboxylic acid ethyl ester (beta CCE) were studied in chair-restrained rhesus monkeys and in rhesus monkeys freely moving in their cages. Observation made during the administration of increasing doses of beta CCE (0.5, 1 and 2 mg/kg) evidence behaviors reflecting a state of anxiety. The similarity of results obtained under the two experimental conditions excludes a potential effect of restraint on the behavioral expression of effect induced by the administered molecule. Convulsions observed in three subjects out of twelve should call for great caution when using this molecule.
Molecular aspects of the action of benzodiazepine and nonbenzodiazepine anxiolytics: A hypothetical allosteric model of the benzodiazepine receptor complex
1984, Progress in Neuropsychopharmacology and Biological Psychiatry
- 1.
  1. The availability of radiolabeled benzodiazepines has resulted in the identification of high affinity receptors in the central nervous system for this class of psychotherapeutic agent which are linked to recognition sites for the inhibitory neurotransmitter, GABA.
- 2.
  2. Evaluation of new, synthetic compounds in the benzodiazepine radioligand binding assay has resulted in the identification of nine classes of non-benzodiazepine putative nxiolytic agents, some of which may be more anxioseiective than the benzodiazepines.
- 3.
  3. At least three and possibly five subclasses of benzodiazepine receptor have been identified in mammalian tissues using radioligand binding assays. The possibility exists that one of these receptor subclasses may mediate the anxiolytic effects of the benzodiazepines while the remainder may be involved in the mediation of the sedative, ataxic and anticonvulsant properties associated with benzodiazepine-like agents.
- 4.
  4. Several endogenous ligands for the benzodiazepine receptor(s) have been postulated. These include various proteins and peptides, purines and the beta-carbolines. This latter group, which competitively antagonizes the pharmacological and biochemical effects of the benzodiazepines, has the highest affinity for the benzodiazepine receptor of an compounds thus far examined; however, none of these compounds has been conclusively identified as the endogenous ligand akin to the enkephalins and endorphins at the opiate receptor.
- 5.
  5. The majority of available evidence would indicate that the endogenous ligand for the benzodiazepine receptor(s) is an antagonist of the benzodiazepines and other putative anxiolytic agents.
Chronic clonazepam administration induced benzodiazepine receptor subsensitivity
1982, Neuropharmacology
Clonazepam and chlordiazepoxide were administered chronically in increasing doses for three weeks in two different strains of mice. Forebrain [³H]diazepam binding was assayed in groups of mice sacrified at 2, 26, 50 hr and 10 days following the last dose. Scatchard and single point analyses revealed a significant decrease in the number of [³H]diazepam binding sites [B_max] which persisted for at least two days following chronic clonazepam treatment. The B_max changes observed following chlordiazepoxide treatment were less pronounced than those elicited by clonazepam. No significant changes in receptor binding affinity (K_d) were detected with either drug. In the clonazepam-treated animals, B_max values returned to normal by day 10 after drug treatment. Chronic benzodiazepine administration therefore induced a decrease in the apparent number of benzodiazepine binding sites in the mouse forebrain. The magnitude and duration of the observed subsensitivity appears to depend on the potency of the administered benzodiazepine.
Inhibition of [<sup>3</sup>H]adenosine binding by stereoisomers of oxazepam hemisuccinate in guinea-pig brain synaptosomes
1982, General Pharmacology
- 1.
  1. The stereoisomers of oxazepam sodium hemisuccinate have been tested for their ability to inhibit the [³H]adenosine binding in guinea-pig brain synaptosomes.
- 2.
  2. All three stereoisomers were able to inhibit the [³H]adenosine binding in a dose-dependent fashion.
- 3.
  3. The IC₂₀ values of the d-, dl- and l-stereoisomers were approximately 5 × 10⁻⁵ M, 7.5 × 10⁻⁵ M and 10⁻⁴ M respectively.
- 4.
  4. The results are consistent with hypothesis that adenosine may be the endogenous substrate mediating some actions of the benzodiazepines.

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Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain

Abstract

Life Sci.

FEBS Letters

Life Sci.

J. Biol. Chem.

Life Sci.

Demonstration of an endogenous, competitive inhibitor(s) of [³H] diazepam binding in bovine brain