Effect of the beta-carboline derivative FG 7142 on inhibition in hippocampal sections

The action of anxiogenetic derivative of beta-carboline FG7142 on evoked activity of neurons in hippocampal sections was investigated using extra- and intracellular recordings. The activity in CAI area was registered upon stimulation of Schaffer's collaterals (SC). Excitatory effect of FG7142 (5 microM) on population spike (PS) was blocked by simultaneous diazepam (5 microM) or RO15-1788 (5 microM) application. This suggests that FG7142 action is mediated by benzodiazepine receptors. To evaluate the action of FG7142 on hippocampal inhibitory processes paired-pulse stimulation of SC was used. It was shown that FG7142 (5 microM) increased 4-5 times the amplitude of the second PS, the amplitude of the first one being much less augmented (10-20%). Such an effect may probably be associated with specific action of GABA inhibition. The following application of diazepam (5 microM) partially reversed disinhibitory effect of FG7142. The main intracellular change observed was the appearance of the local depolarization following the single action potential (AP). With the application continued, this depolarization gave rise to the second AP. The data suggest that suppression of hippocampal inhibitory circuits may contribute to the development of the anxiety feeling.     

Benzodiazepine inhibition of the stimulation-evoked catecholamine release from bovine adrenomedullary cells in culture

Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 μ M) inhibited CA release evoked by acetylcholine (ACh), excess K⁺ and veratridine but not by A23187 or caffeine in Ca²⁺ -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-β-carboline-3-carboxylate (βCCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K⁺ was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked ⁴⁵Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca²⁺ influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed.     

Electrophysiological and pharmacological characterization of peripheral benzodiazepine receptors in a guinea pig heart preparation

R05-4864 decreased in a dose-dependent manner, from 3 × 10^?9 M to 3 × 10^?6 M, the duration of intracellular action potential and the contractility in a guinea pig preparation. Diazepam was less effective and clonazepam inactive. The effects of R05-4864 were GABA-independent and antagonized by PK 11195 but not by the selective antagonist of the brain type benzodiazepine receptors R015-1788. These results show the pharmacological relevance of peripheral type benzodiazepine binding sites at the cardiac level.     

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7-96 microM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.     

Subcellular Localization of "Peripheral-Type" Binding Sites for Benzodiazepines in Rat Brain

The binding of [3H]Ro 5-4864, a specific ligand for "peripheral-type" benzodiazepine binding sites and [3H]Ro 15-1788, a specific ligand for the central benzodiazepine receptors, was determined in subcellular fractions of rat brain. As previously reported, the highest levels of "peripheral-type" benzodiazepine binding sites and benzodiazepine receptors were found in the crude P1 and P2 fractions, respectively. Purification of these crude fractions revealed that high levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding were present in the mitochondrial and synaptosomal fractions. In contrast, the purified nuclei and myelin contained low levels of both [3H]Ro 5-4864 and [3H]Ro 15-1788 binding.     

Effect of amino acid derivatives of beta-carboline on the evoked activity of hippocampal neurons

The action of methylamide-beta-carboline-3-carboxylate (1), glycinamide-beta-carboline-3-carboxylate methyl ester (2), leucinamide-beta-carboline-3-carboxylate methyl ester (3) on evoked activity of hippocampal neurons was investigated using brain slice technique. Both 1 and 2 application (5 mkM, 15 min) reduced paired-pulse inhibition (PPI). Single-pulse and frequency stimulation revealed population spike (PS) increase or generation of additional PS after 1 or 2 application, the former drug being more active at all stimulation patterns. 3 had no definitive activity in the test-system used. The data obtained suggest similarity of pharmacological profiles of 1 and 2, and anxiogenic activity of 2 in vivo.     

Opposite effects of two ligands for peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, in a conflict situation in the rat

The effects of two drugs acting at the peripheral type benzodiazepine binding sites, PK 11195 and RO5-4864, were examined in shock-induced suppression of drinking in rats. These two compounds have opposite effects : RO5-4864 (3.1-1205 mg/kg i.p.) enhanced whereas PK 11195 (25-50 mg/kg i.p.) decreased the punished responding, and PK 11195 (6.25 mg/kg, a dose which did not alter the punished responding) blocked the proconflict action of RO5-4864 (6.25 and 12.5 mg/kg). The effects of RO5-4864 and PK 11195 were not antagonized by RO15-1788, a selective antagonist of the central benzodiazepine site. In addition, PK 11195 (6.25 mg/kg) did not reverse the proconflict effect of two beta-carbolines : beta-CEE and FG 7142. AS picrotoxin did not change the punished responding, these data imply that the effects of RO5-4864 and PK 11195 on the one hand and those of chlordiazepoxide and beta-carbolines on the other hand are differentially mediated and suggest that the peripheral type benzodiazepine binding sites are involved in this conflict model.     

Histidine modification with diethylpyrocarbonate induces a decrease in the binding of an antagonist, PK 11195, but not of an agonist, RO5-4864, of the peripheral benzodiazepine receptors

[3H]PK 11195 binding to peripheral type benzodiazepine binding sites in kidney membranes is inhibited by the histidine blocking agent diethylpyrocarbonate. This reagent irreversibly decreases the Bmax for [3H]PK 11195 without affecting the affinity. By contrast binding of [3H]RO5-4864 is not affected by diethylpyrocarbonate treatment. However RO5-4864 can protect in a concentration dependent manner the [3H]PK 11195 binding site from diethylpyrocarbonate whereas clonazepam and RO15-1788 are not active. These results suggest that PK 11195 and RO5-4864 interact with different conformational states of the receptors that RO5-4864. This is in agreement with our previous hypothesis that PK 11195 is an antagonist and RO5-4864 an agonist at the "peripheral type" benzodiazepine receptors.     

Peripheral benzodiazepine binding sites: Effect of PK 11195, 1-(2-chlorophenyl)-n-methyl-n-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies

[³H] R05-4864 binding sites have been characterized in kidney, heart, brain, adrenals and platelets in the rat. In all these organs the following order of potency in the R05-4864 displacement was found : R05-4864 > diazepam > clonazepam indicating that they correspond to the “peripheral type” of benzodiazepine binding sites. PK 11195, an isoquinoline carboxamide derivative, displaces [³H] R05-4864 from its binding sites in all the organs. PK 11195 was as potent as R05-4864 in the platelets, heart, adrenals, kidney and several brain regions (midbrain, hypothalamus, medulla + pons and hippocampus. However it was 5 to 10 times more effective in cortex and striatum. In conclusion PK 11195 might represent a new tool to elucidate the physiological relevance of “peripheral type” benzodiazepine binding sites and might help to discriminate the hypothetical subclasses of these binding sites.     

Specific binding of benzodiazepines to human breast cancer cell lines

Binding of [3H]Ro5-4864, a peripheral benzodiazepine receptor (PBR) agonist, to BT-20 human, estrogen- (ER) and progesterone- (PR) receptor negative breast cancer cells was characterized. It was found to be specific, dose-dependent and saturable with a single population of binding sites. Dissociation constant (K(D)) was 8.5 nM, maximal binding capacity (Bmax) 339 fM/10(6) cells. Ro5-4864 (IC50 17.3 nM) and PK 11195 (IC50 12.3 nM) were able to compete with [3H]Ro5-4864 for binding, indicating specificity of interaction with PBR. Diazepam was able to displace [3H]Ro5-4864 from binding only at high concentrations (>1 microM), while ODN did not compete for PBR binding. Thymidine-uptake assay showed a biphasic response of cell proliferation. While low concentrations (100 nM) of Ro5-4864, PK 11195 and diazepam increased cell growth by 10 to 20%, higher concentrations (10-100 microM) significantly inhibited cell proliferation. PK 11195, a potent PBR ligand, was able to attenuate growth of BT-20 cells stimulated by 100 nM Ro5-4864 and to reverse growth reduction caused by 1 and 10 microM Ro5-4864, but not by 50 microM and 100 microM. This indicates that the antimitotic activity of higher concentrations of Ro5-4864 is independent of PBR binding. It is suggested, that PBR are involved in growth regulation of certain human breast cancer cell lines, possibly by supplying proliferating cells with energy, as their endogenous ligand is a polypeptide transporting Acyl-CoA.     

Benzodiazepine receptors on human blood platelets

Binding studies conducted on membrane preparation from human platelets using (3H) Ro5-4864 and (3H) diazepam showed specific and saturable binding. Scatchard analysis revealed a single class of binding sites with KD = 10.8 +/- 0.9 nM and Bmax = 775 +/- 105 fmol/mg protein for (3H) Ro5-4864 and KD = 10.5 +/- 1.1 nM and Bmax = 133 +/- 19 fmol/mg for (3H) diazepam. We were unable to detect any GABA binding site on crude membrane preparation, nor did GABA enhance the binding of (3H) Ro5-4864 or (3H) diazepam. This suggests that benzodiazepine receptors are uncoupled to GABA system on human platelets. Ro15-1788, a specific antagonist for "central type" benzodiazepine (BDZ) binding sites was inactive in displacing (3H) Ro5-4864 from membrane receptors, while PK 11195 (a specific ligand for the "peripheral type" receptor) was the most potent of the drugs tested in inhibiting (3H) Ro5-4864 binding. These results indicate that human blood platelets bear "peripheral-type" BDZ receptor. Moreover, we could not detect any (3H) propyl beta carboline specific binding on platelet membranes. Results on benzodiazepine receptors on human circulating lymphocytes are also reported and similarity in pharmacological properties with platelet benzodiazepine receptors is suggested.     

Behavioural actions of Ro 5-4864: A peripheral-type benzodiazepine?

Ro 5-4864 is a 1,4 benzodiazepine lacking typical benzodiazepine behavioural actions, and which has very low affinity for the “classical” CNS benzodiazepine binding sites. However, Ro 5-4864 has very high affinity for the peripheral type of binding site in the periphery and in the brain. Evidence is reviewed that Ro 5-4864 is sedative, convulsant and anxiogenic in rodents. We also describe the effects of combining Ro 5-4864 treatment with benzodiazepines (e.g. diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). The binding sites that might be mediating these behavioural actions of Ro 5-4864 are discussed.     

RO5-4864 inhibits the binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes

RO5-4864, a 1,4-benzodiazepine, has recently been shown to possess anticonvulsant, convulsant and anxiogenic properties and to inhibit Ca++-calmodulin-stimulated membrane phosphorylation. RO5-4864 inhibited the binding of [35S]t-butylbicyclophosphorothionate (TBPT) to cerebral cortex, cerebellar and hippocampus membranes, with an IC50 value of approximately 20 microM. TBPT binds apparently to the picrotoxinin site of the benzodiazepine-GABA receptor-ionophore complex and appears to be a site of action for several classes of convulsant, depressant and anxiolytic drugs that modulate GABAergic transmission. RO5-4864 inhibited [35S] TBPT binding in cerebral cortex, apparently competitively. Antagonists of GABA and central benzodiazepine sites did not interfere with the ability of RO5-4864 to inhibit [35S] TBPT binding. The properties of RO5-4864 to inhibit TBPT binding are similar to other convulsants and GABA antagonists (except bicuculline) which inhibit TBPT binding. These results suggest that RO5-4864 interacts with the TBPT binding sites of the oligomeric GABA receptor complex.     

Differential effects of benzodiazepines on phospholipid methylation in hippocampus and cerebellum of rats

To elucidate the relationship between the occupancy of BDZ binding sites and phospholipid methylation in brain, we examined phosphatidylethanolamine-N-methyltransferase (PEMT) activity in synaptosomes of rat hippocampi and cerebella in the presence of BDZ ligands with different modes of action. We found that Ro 5-4864, a specific ligand for "peripheral type" receptors, increased PL methylation in hippocampal and cerebellar synaptosomes. This effect was directly related to receptor occupancy, since the specific antagonist PK 11195 inhibited the rise in PEMT activity induced by Ro 5-4864. Clonazepam, on the other hand, tended to reduce PL production in cerebellum and hippocampus except for hippocampal (3H)-phosphatidyl-N-monomethylethanolamine which was elevated by 40 to 70% at doses ranging from 10(-9) to 10(-6) M. When equimolar concentrations of the antagonist Ro 15-1788 were given in association the clonazepam-induced phosphatidyl-N-monomethylethanolamine increase was reduced by 70%. These data support the involvement of structural and functional membrane alterations in the action of BDZ.     

Coexistence of central and peripheral benzodiazepine binding sites in the human pineal gland

The pineal gland and particularly its major hormone, melatonin, may participate in several physiological functions, including sleep promotion, anticonvulsant activity and the modulation of biological rhythms and affective disorders. These effects may be related to an interaction with benzodiazepine receptors, which have been demonstrated to be present in the pineal gland of several species including man. The present study examined the characteristics of benzodiazepine binding site subtypes in the human pineal gland, using [³H] flunitrazepam and [³H] PK 11195 as specific ligands for central and peripheral type benzodiazepine binding sites respectively. Scatchard analysis of [³H] flunitrazepam binding to pineal membrane preparations was linear, indicating the presence of a single population of sites. Clonazepam and RO 15-1788, which have a high affinity for central benzodiazepine binding sites, were potent competitors for [³H] flunitrazepam binding in the human pineal, whereas RO 5-4864 had a low affinity for these sites. Analyses of [³H] PK 11195 binding to pineal membranes also revealed the presence of a single population of sites. RO 5-4864, a specific ligand for peripheral benzodiazepine binding sites was the most potent of the drugs tested in displacing [³H] PK 11195, whereas clonazepam and RO 15-1788 were weak inhibitors of [³H] PK 11195 binding to pineal membranes. Overall, these results demonstrate, for the first time, the coexistence of peripheral and central benzodiazepine binding sites in the human pineal gland.     

Peripheral-type benzodiazepine receptors are involved in the regulation of cholesterol side chain cleavage in adrenocortical mitochondria

In an attempt to elucidate the physiological relevance of the peripheral type of benzodiazepine receptor in adrenocortical mitochondria, we examined the effect of three different benzodiazepines (diazepam, Ro5-4864, and chlordiazepoxide) on the conversion of cholesterol to pregnenolone, the rate-limiting step in steroidogenesis, by using cholesterol-loaded mitochondria from bovine adrenal zona fasciculata. These benzodiazepines, except chlordiazepoxide, caused a dose-dependent stimulation of the cholesterol side chain cleavage in the mitochondria. The stimulatory effect of Ro5-4864 was approximately 10 times more potent than that of diazepam. No inhibitory effect of YM-684 (Ro15-1788), a potent antagonist to central-type benzodiazepine receptors, was observed in the stimulation induced by diazepam and Ro5-4864. Both external calcium ion and voltage-dependent calcium channel blocker, (+)-PN200-110, were without effect on the diazepam-induced steroidogenesis. By contrast, pretreatment of mitochondria with digitonin abolished the stimulatory effect of diazepam on the mitochondrial steroidogenesis. The present results indicate that the peripheral-type benzodiazepine receptor of adrenocortical mitochondria plays an essential role in regulating cholesterol side chain cleavage without any change of calcium channels.     

Antagonism of RO 15-1788 with benzodiazepines in the effect on motivated aggression and the action of analgesics

The influence of Ro 15-1788 and bicuculline on the action of GABA-positive drugs (muscimol), GABA cethyl ester, piracetam and depakine and benzodiazepine tranquilizers (diazepam, phenazepam) on motivated aggression has been studied. It has been shown that Ro 15-1788 which has a weak antiaggressive effect selectively antagonizes the anti-aggressive effect of tranquilizers but not that of GABA-positive drugs. Bicuculline antagonizes antiaggressive activity of the drugs of both types. The action of these antagonists on the effect of the drugs under study as regards the analgetic activity of morphine was also studied. It has been shown that Ro 15-1788 antagonizes the potentiation of morphine analgesia caused by diazepam. At the same time Ro 15-1788 does not influence morphine analgesia potentiated by muscimol. Bicuculline removes the potentiation of morphine analgesia caused both by diazepam and muscimol it is concluded that bicuculline-sensitive GABA receptors modulate the antiaggressive effect of benzodiazepines and their influence on the analgetic action of opiates.     

Benzodiazepine Enhancement of γ-Aminobutyric Acid-Mediated Chloride Ion Flux in Rat Brain Synaptoneurosomes

Benzodiazepine agonists such as Ro 11-6896 [B10(+)], diazepam, clonazepam, and flurazepam were found to enhance muscimol-stimulated 36Cl- uptake into rat cerebral cortical synaptoneurosomes. The rank order of potentiation was B10(+) greater than diazepam greater than clonazepam greater than flurazepam. These benzodiazepines had no effect on 36Cl-uptake in the absence of muscimol. Further, the inactive enantiomer, Ro 11-6893 [B10(-)], and the peripheral benzodiazepine receptor ligand Ro 5-4864 did not potentiate muscimol-stimulated 36Cl- uptake at concentrations up to 10 microM. In contrast, the benzodiazepine receptor inverse agonists ethyl-beta-carboline-3-carboxylate and 6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylic acid methyl ester inhibited muscimol stimulated 36Cl- uptake. Benzodiazepines and beta-carbolines altered the apparent K0.5 of muscimol-stimulated 36Cl- uptake, without affecting the Vmax. The effects of both benzodiazepine receptor agonists and inverse agonists were reversed by the benzodiazepine antagonists Ro 15-1788 and CGS-8216. These data further confirm that central benzodiazepine receptors modulate the capacity of gamma-aminobutyric acid receptor agonists to enhance chloride transport and provide a biochemical technique for studying benzodiazepine receptor function in vitro.     

Benzodiazepine Binding Characteristics of Embryonic Rat Brain Neurons Grown in Culture

The binding of [3H]diazepam to cell homogenates of embryonic rat brain neurons grown in culture was examined. Under the conditions used to prepare and maintain these neurons, only a single, saturable, high-affinity binding site was observed. The binding of [3H]diazepam was potently inhibited by the CNS-specific benzodiazepine clonazepam (Ki = 0.56 +/- 0.08 nM) but was not affected by the peripheral-type receptor ligand Ro5-4864. The KD for [3H]diazepam bound specifically to cell homogenates was 2.64 +/- 0.24 nM, and the Bmax was 952 +/- 43 fmol/mg of protein. [3H]Diazepam binding to cell membranes washed three times was stimulated dose-dependently by gamma-aminobutyric acid (GABA), reaching 112 +/- 7.5% above control values at 10(-4) M. The rank order for potency of drug binding to the benzodiazepine receptor site in cultured neurons was clonazepam greater than diazepam greater than beta-carboline-3-carboxylate ethyl ester greater than Ro15-1788 greater than CL218,872 much greater than Ro5-4864. The binding characteristics of this site are very similar to those of the Type II benzodiazepine receptors present in rat brain. These data demonstrate that part, if not all, of the benzodiazepine-GABA-chloride ionophore receptor complex is being expressed by cultured embryonic rat brain neurons in the absence of accompanying glial cells and suggest that these cultures may serve as a model system for the study of Type II benzodiazepine receptor function.     

Presence of a peripheral type benzodiazepine binding site on the macrophage; its possible role in immunomodulation

Saturable binding site for 3H-flunitrazepam (KD = 43 +/- 7 nM, Bmax = 391 +/- 58 fmoles/cell, i.e. 250,000 sites/cell) is characterized on Mouse peritoneal inflammatory macrophages. The affinity for different ligands (PK 11195 greater than Ro 5-4864 greater than diazepam greater than flunitrazepam greater than clonazepam greater than Ro 15-1788) shows that this site is of peripheral type. In vivo the humoral response in Mice to Sheep red blood cells was stimulated by administration of 1 mg/kg of PK 11195 (+85%), Ro 5-4864 (+80%) and diazepam (+58%). Clonazepam and Ro 15-1788 are devoid of activity. This suggests that molecules which show affinity for the "peripheral type" benzodiazepine binding site might modulate the immune response.