4-Chlorodiazepam

Mechanism of action

Mitochondria isolated from rat brain were found to cleave cholesterol to produce pregnenolone, the precursor for hormonal steroids, at a mean rate of 21.0 pmol pregnenolone.mg protein-1.min-1. This rate-limiting step in steroidogenesis was significantly stimulated by PK 11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide) and Ro5 4864 (4'-chlorodiazepam), ligands which bind to peripheral benzodiazepine receptors with high affinity. Low-affinity ligands for the peripheral benzodiazepine receptor such as Ro15 1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5 alpha][1,4] benzo-3-carboxylate) and clonazepam had no significant effect on the rate of pregnenolone synthesis. Furthermore, the rank order of potency of these compounds as inhibitors of [3H]Ro5 4864 binding was identical to the rank order for steroid production. Since the 86-amino acid peptide diazepam binding inhibitor is also thought to bind to the peripheral benzodiazepine receptor, four fragments of this peptide, a random sequence and steroidogenesis activator peptide were also evaluated for their ability to interact with peripheral benzodiazepine receptors and to stimulate steroidogenesis in rat brain mitochondria. Steroidogenesis activator peptide and two fragments of diazepam binding inhibitor significantly stimulated pregnenolone biosynthesis. In contrast to the peripheral benzodiazepine receptor ligands, no correlation between peptide potency in displacing [3H]Ro5 4864 binding and steroidogenesis was observed.
Peripheral benzodiazepine receptors mediate cholesterol translocation between the outer and inner mitochondrial membranes in steroidogenic tissues. They are found in many other tissues too, including liver. We studied the effect of the peripheral benzodiazepine receptor ligands PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxa mid e], Ro 5-4864 (4-chlorodiazepam), hemin, protoporphyrin IX and N-methyl protoporphyrin IX on cholesterol mitochondrial intermembrane transport of cholesterol in vitro in rat liver. Endogenous cholesterol translocation from outer to inner mitochondrial membranes was significantly increased by PK11195 and N-methyl protoporphyrin IX (140% and 150% increase, respectively, at 1 microM, P<0.01). 5 microM protoporphyrin IX, 1 microM Ro 5-4864 and 5 microM hemin was ineffective. When mitochondria were labeled with exogenous [4-14C]cholesterol, PK11195 and N-methyl protoporphyrin IX were the most effective in increasing total cholesterol incorporation and cholesterol translocation into inner membranes, and their effect was dose-dependent. These data suggest that in liver the binding to peripheral benzodiazepine receptors is related to cholesterol translocation and the interaction of ligands with these receptors may play a role in the complex mechanism of regulation of cholesterol traffic between liver mitochondrial membranes.
Effects of various benzodiazepines were investigated in ovariectomized rat isolated uterus which had been chronically pre-treated with different female sex hormones: oestrogen, progesterone and oestrogen + progesterone. Uteri obtained from all groups developed a spontaneous, rhythmic activity. The spontaneous activity observed in control uterus was either inhibited in a concentration-dependent manner by diazepam, 4'-chlorodiazepam, clonazepam or 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195), or was abolished in [Ca2+]o-free solution. Diazepam, 4'-chlorodiazepam, clonazepam and PK 11195 all caused a concentration-dependent relaxation of the [K+]o-pre-contracted uterus with the relative order of potency: PK 11195 > 4'-chlorodiazepam > diazepam > clonazepam. Administration of [Ca2+]o (1 microM to 10 mM) caused a concentration-dependent contraction of uterus, bathed in [Ca2+]o-free physiological salt solution obtained from different pre-treatment groups. Incubation with different concentrations (uM) of diazepam, 4'-chlorodiazepam, clonazepam and PK 11195 caused a decrease in response to [Ca2+]o-induced contraction in all groups of rat uteri. These results indicate that micromolar benzodiazepine binding sites exist in rat uterus. Diazepam, 4'-chlorodiazepam, clonazepam and PK 11195 caused relaxation of pre-contracted rat uterus and this effect may involve the inhibition of influx of [Ca2+]o and the relaxing effects of different benzodiazepines observed in this study can be modulated by pre-treatment with different female hormones.
The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABAA receptor cDNAs were determined. Cotransfection of human alpha 2, beta 1, and gamma 2 subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t-[35S]butylbicyclophosphorothionate ([35S]-TBPS) binding to the GABA-activated chloride channel. This site is found on GABAA receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the alpha subunit was further demonstrated as substitution of either alpha 1 or alpha 3 for the alpha 2 subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [35S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of alpha 2 and beta 1 alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [35S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the alpha and beta polypeptides.
For more Mechanism of Action (Complete) data for 4-CHLORODIAZEPAM (6 total), please visit the HSDB record page.

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