Isolation and identification in bovine cerebral cortex of n-butyl beta-carboline-3-carboxylate, a potent benzodiazepine binding inhibitor.

Abstract

A substance having benzodiazepine-binding inhibitory activity has been extracted from 18 kg of gray matter of bovine cerebral cortex and purified to homogeneity. This substance Inhibits competitively PHlflunitrazepam and ethyl The demonstration that benzodiazepines (BZDs) bind specifically and with high affinity to receptors in the central nervous system (1, 2) led to the search for a possible endogenous ligand that could recognize such receptor sites and modulate y-aminobutyric acid (GABA) neurotransmission. A number of substances extracted from brain were found to displace BZD from the receptor; however, in most cases the binding had low affinity-for example, purines (3, 4) and nicotinamide (5) had a Ki in the millimolar range. A number ofpeptides and proteins ofMr 1500-70,000 and other nonidentified substances were also isolated from brain (6-9). The best-characterized polypeptide is the diazepam-binding inhibitor, which is anxiogenic, has a Mr of 11,000, and has a relatively weak affinity (10). This substance has been submitted to controlled hydrolysis and an 18-amino acid fragment was isolated, which keeps the anxiogenic properties while partially losing affinity for the BZD receptor (11). So far, the most active compound isolated from urine and brain is ethyl ,-carboline-3-carboxylate (P-CCE), which has a Ki in the nanomolar range, similar to some BZDs (12). However, 3-CCE was found to be formed during the extraction in a hot ethanol HCO step. While recognizing that it was an artifact, Braestrup et al. (12) suggested that the true endogenous ligand could be a derivative of f-carboline-3-carboxylic acid. The complexities encountered in trying to demonstrate the existence of an endogenous ligand for the BZD receptor (see ref. 13) have led some investigators to disregard the presence of an endogenous ligand and to attribute the effect of BZDs only to their modulatory action on the GABA receptor (14).In the preliminary experiments, we used an aqueous extraction of rat or bovine cerebral cortices and found a heat-stable substance of Mr <1000, which specifically andto central BZD receptors (15). This substance was later extracted from larger quantities of bovine cerebral cortex by using a mild acetic acid and methanol extraction, followed by Sephadex G-25 filtration, countercurrent distributions, and several rounds of preparative and analytical high-performance liquid chromatography (HPLC) (16). We could detect several fractions having BZD binding inhibitory activity, one of which was prominent. This particular inhibitor is specific for the central BZD receptor and displaces [3H]FNZ binding with a Ki in the nanomolar range. Furthermore, it has no effect on other receptors such as the a1-, a2-, and 3-adrenoceptors; the cholinergic muscarinic and the GABAA receptors; as well as on the "peripheral" BZD binding site.In the present work, we describe the experiments done to identify the chemical nature of this major and most-purified peak of BZD inhibitory activity that we have isolated from bovine brain (16...

Axon-to-Glia Interaction Regulates GABAAReceptor Expression in Oligodendrocytes

“…Nonetheless, the presence of the benzodiazepine site in GABA A receptors in OLGs is further supported by the effects of b-carbolines, which are postulated to be endogenous benzodiazepine site modulators (Peña et al, 1986). Thus, DMCM reduced the GABA response as a typical inverse agonist, in agreement with its known properties in other benzodiazepine sites, whereas b-CCB acted to increase the OLG GABA response, an effect that deserves further study (Peña et al, 1986;Rigo et al, 1994).…”

“…In one case using # EC 30 (Von Blankenfeld et al, 1991), potentiation was reported for FZP, and in other cases applying GABA $ EC 50 , no effect was observed for FZP (Williamson et al, 1998) or DZP (Bronstein et al, 1998) (see also Vélez-Fort et al, 2012). Nonetheless, the presence of the benzodiazepine site in GABA A receptors in OLGs is further supported by the effects of b-carbolines, which are postulated to be endogenous benzodiazepine site modulators (Peña et al, 1986). Thus, DMCM reduced the GABA response as a typical inverse agonist, in agreement with its known properties in other benzodiazepine sites, whereas b-CCB acted to increase the OLG GABA response, an effect that deserves further study (Peña et al, 1986;Rigo et al, 1994).…”

Limitations of the Benzodiazepine Receptor Nomenclature: A Proposal for a Pharmacological Classification as Omega Receptor Subtypes

“…The recognition sites for benzodiazepines (Mohler and Okada, 1977 ;Squires and Braestrup, 1977) however, are historically associated with this chemical class and, in spite of several proposals (Armando et al, 1986;Braestrup et al, 1980;Crawley et al, 1986;Marquardt et a!., 1986;Pena et al, 1986;Sangameswaran and De Blas, 1985 ;Verma et al, 1987;Wildmann et al, 1986) there are at present no established endogenous ligands acting on either the central or peripheral types of receptors. The recognition sites for benzodiazepines (Mohler and Okada, 1977 ;Squires and Braestrup, 1977) however, are historically associated with this chemical class and, in spite of several proposals (Armando et al, 1986;Braestrup et al, 1980;Crawley et al, 1986;Marquardt et a!., 1986;Pena et al, 1986;Sangameswaran and De Blas, 1985 ;Verma et al, 1987;Wildmann et al, 1986) there are at present no established endogenous ligands acting on either the central or peripheral types of receptors.…”

Identification of Inosine as an Endogenous Modulator for the Benzodiazepine Binding Site of the GABA_A Receptors

“…The search for naturally occurring ligands for BZ binding site is, therefore, conceptually analogous to the work involving opiate receptors in which the discovery of a functional receptor for the non-physiologic opiates have led to the isolation and identification of endorphins and enkephalins as being endogenous substrates for these receptors [8,26]. Indeed, several substances have been isolated and proposed as endogenous ligands for the BZ binding site, referred to as EBZs [1,4,6,19,25,28,30]. Similarly, several endogenous substances which inhibit the binding of [ 3 H]muscimol to the GABA binding site have been isolated and proposed to be involved in the modulation of the GABA receptor [4,12,13,15,24,29,32,[35][36][37][38].…”