Isolation, purification and partial sequence of a neuropeptide (diazepam-binding inhibitor) precursor of an anxiogenic putative ligand for benzodiazepine recognition site

Corda, Maria Giuseppa; Ferrari, Mariano; Guidotti, Alessandro; Konkel, D.; Costa, E.

doi:10.1016/0304-3940(84)90533-0

Cited by 71 publications

(15 citation statements)

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“…In patch-clamp experiments using whole cells, DBI has been shown to reduce the amplitude of outward chloride currents induced by GABA (Bormann 1991), and this e¤ect could be blocked by ßumazenil. In behavioral tests, the ICV application of DBI has a proconßict action (Guidotti et al 1983;Corda et al 1984). Furthermore, the release of endogenous DBI has been linked to the stress of exposure to an acute loud noise (Ferrarese et al 1991a,b).…”

Section: Discussionmentioning

confidence: 99%

Flumazenil blockade of anxiety following ethanol withdrawal in rats

Moy¹,

Knapp²,

Criswell³

et al. 1997

Psychopharmacology

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In previous research, the drug flumazenil has been categorized both as a pure benzodiazepine antagonist and as a benzodiazepine partial agonist. The following studies used an elevated plus maze to test whether flumazenil would exert any antianxiety action in rats. While chlordiazepoxide (3.0 mg/kg), ethanol (0.75 g/kg), and the atypical benzodiazepine zolpidem (1.0 mg/kg) all significantly increased time spent on the open arms and percent open arm entries, flumazenil (1-10 mg/kg) alone did not produce any anxiolytic effects on the maze. Withdrawal from chronic ethanol treatment led to a decrease in open arm time and percent open arm entries. Flumazenil (3.0 mg/kg) blocked these changes, suggesting that the effects of flumaxenil are at least partially dependent upon the levels of stress or anxiety in the subjects. An anxiolytic action of flumazenil was not seen following the central administration of the neuropeptide corticotropin-releasing factor (CRF), which reduced open arm time on the elevated plus maze. These results support the hypothesis that the mechanism of action for flumazenil effects on the anxiety observed during ethanol withdrawal involves antagonism of an endogenous benzodiazepine inverse agonist, rather than activity as a partial agonist or blockade of CRF-mediated effects.

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Section: Discussionmentioning

confidence: 99%

Flumazenil blockade of anxiety following ethanol withdrawal in rats

Moy¹,

Knapp²,

Criswell³

et al. 1997

Psychopharmacology

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“…We also indicated (1) that the purified substance has properties different from those of the putative endogenous benzodiazepine receptor ligands reported in the literature, including among others the diazepam binding inhibitor (DBI) and related peptides (5)(6)(7)(8)(9)(10). Whereas the DBI peptide binds to the f3-carboline (inverse agonist) site of the benzodiazepine receptor and is anxiogenic and Pronase-sensitive, the substance that we have purified by immunoaffinity chromatography binds to the agonist binding site and is Pronaseresistant.…”

mentioning

confidence: 99%

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Sangameswaran¹,

Fales²,

Friedrich³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoafflmity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam The anti-benzodiazepine mAb 21-7F9 was obtained after immunizing BALB/c mice with 3-hemisuccinyloxyclonazepam coupled to bovine serum albumin. The production, purification, and characterization of mAb 21-7F9 have been reported (2).Immunoaffinity Chromatography. Fifty grams of bovine cerebellum and cerebral cortex were homogenized (Waring blender, 6 min) in 60 ml of 5 mM Tris-HCl, pH 8.1/0.3 mM phenylmethylsulfonyl fluoride/1 mM EDTA containing soybean trypsin inhibitor (20 pug/ml) and pepstatin (1 pug/ml). The preparation of the soluble extract and the procedure for immunoaffinity chromatography have been described (1). The sample was loaded on a 10-ml Affi-Gel 10 (Bio-Rad) column containing 50 mg ofimmobilized anti-benzodiazepine mAb 21-7F9, and 100 ml of each washing solution was passed through the column at a flow rate of 10 ml/hr. The retained material was eluted with 100 ml of 0.2 M acetic acid at a flow rate of 50 ml/hr.Gel Filtration on Sephadex G-25. The immunoaffinity eluate was dried under reduced pressure, dissolved in 0.9 ml of 40% formic acid, and layered on a Sephadex G-25 column (51.5 cm x 1.2 cm i.d.) that had been equilibrated with 40% formic acid. Fractions (2 ml) were collected at a flow rate of 5 ml/hr. The active fractions were pooled, concentrated under reduced pressure, and resuspended in 600 Al of 0.1% trifluoroacetic acid. The benzodiazepine receptor binding activity of the fractions was assayed as indicated below.Reversed-Phase HPLC.Step I. A 200-Al sample of the Sephadex G-25-purified substance was subjected to reversed-phase HPLC on a C8 column [length, 25 cm; particle size, 10 /Lm; pore size, 300 A; Brownlee Laboratory (Santa Clara, CA)] and eluted with a linear gradient (5-60%) of acetonitrile in 0.1% trifluoroacetic acid.Step II. tTo whom all reprint requests should be addressed. 9236The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…DBI has a specific distribution in various brain nuclei and is highly concentrated in hypothalamus and cerebellum (10). When injected intracerebroventricularly, it causes proconflict action in rats (6,11). Thus, it has a pharmacological profile of a naturally occurring anxiogenic compound and, accordingly, it preferentially displaces ,B [3H]carboline over [3H]diazepam (7).…”

mentioning

confidence: 99%

Putative diazepam binding inhibitor peptide: cDNA clones from rat.

Mocchetti¹,

Einstein²,

Brosius³

1986

Proc. Natl. Acad. Sci. U.S.A.

113

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cDNA clones corresponding to the polypeptide that has been shown to be an endogenous diazepam binding inhibitor and may act as a physiological ligand for the benzodiazepine/beta-carboline receptor have been isolated from bacteriophage lambda recombinant libraries from rat hypothalamus, total brain, and liver. The clones contain an open reading frame corresponding to 87 amino acids. A signal sequence is not present. In addition to high levels of mRNA in various brain regions, RNA blot analysis reveals an abundance of diazepam binding inhibitor mRNA in many peripheral organs (e.g., testes, kidney, liver, and heart) that are known to be rich in peripheral benzodiazepine recognition sites. The size of the mRNA in all tissue examined is approximately 0.7 kilobase. Southern blot analysis of genomic DNA suggests the presence of about six genes in the rat, some of which may be pseudogenes.

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Isolation, purification and partial sequence of a neuropeptide (diazepam-binding inhibitor) precursor of an anxiogenic putative ligand for benzodiazepine recognition site

Cited by 71 publications

References 1 publication

Flumazenil blockade of anxiety following ethanol withdrawal in rats

Flumazenil blockade of anxiety following ethanol withdrawal in rats

Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Putative diazepam binding inhibitor peptide: cDNA clones from rat.

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