Benzodiazepine Receptor Sites Research Articles

Effects of diazepam on125I-iomazenil-benzodiazepine receptor binding and epileptic seizures in the El mouse

To investigate changes in free benzodiazepine receptor density in response to repeated, long-term administration of diazepam in epilepsy, we assessed 125I-iomazenil (125I-IMZ) binding in a mouse model. El mice were divided into two groups of 12 mice each which received either no diazepam (E1(D[-]) group) or 2 mg/kg of diazepam per week (El(D[+]) group). Nine ddY mice were used as a control. Once each week from the age of 5 to 19 weeks, the El mice received stimulation to produce epileptic seizures 20 minutes after receiving intraperitoneal injections. At 20 weeks of age, a total dose of 0.37 MBq of 125I-IMZ was injected in all mice and their brains were rapidly removed 3 hours later. The incidence of epileptic seizures at the age of 19 weeks and the autoradiograms of the brain were compared. The incidence of epileptic seizures in response to weekly stimulation was significantly lower in the E1(D[+]) group than in the E1(D[-]) group (p < 0.001). The percent injected doses of 125I-IMZ per gram of tissue in the cortex, hippocampus and amygdala were significantly lower in the E1(D[+]) group than in the E1(D[-]) group (p < 0.05). The results suggest that diazepam binds competitively to 125I-IMZ as an agonist to free benzodiazepine receptor sites in the cortex, hippocampus and amygdala and shows anticonvulsant effect in E1 mice.

Anxiolytic properties of green tea polyphenol (−)-epigallocatechin gallate (EGCG)

Naturally occurring polyphenols are potent antioxidants. Some of these compounds are also ligands for the GABA A receptor benzodiazepine site. This feature endows them with sedative properties. Here, the anxiolytic activity of the green tea polyphenol (−)-epigallocatechin gallate (EGCG) was investigated after acute administration in mice, using behavioral tests (elevated plus-maze and passive avoidance tests) and by electrophysiology on cultured hippocampal neurons. Patch-clamp experiments revealed that EGCG (1–10 μM) had no effect on GABA currents. However, EGCG reversed GABA A receptor negative modulator methyl β-carboline-3-carboxylate (β-CCM) inhibition on GABA currents in a concentration dependent manner. This was also observed at the level of synaptic GABA A receptors by recording spontaneous inhibitory synaptic transmission. In addition, EGCG consistently inhibited spontaneous excitatory synaptic transmission. Behavioral tests indicated that EGCG exerted both anxiolytic and amnesic effects just like the benzodiazepine drug, chlordiazepoxide. Indeed, EGCG in a dose-dependent manner both increased the time spent in open arms of the plus-maze and decreased the step-down latency in the passive avoidance test. GABA A negative modulator β-CCM antagonized EGCG-induced amnesia. Finally, state-dependent learning was observable after chlordiazepoxide and EGCG administration using a modified passive avoidance procedure. Optimal retention was observed only when animals were trained and tested in the same state (veh–veh or drug–drug) and significant retrieval alteration was observed in different states (veh–drug or drug–veh). Moreover, EGCG and chlordiazepoxide fully generalized in substitution studies, indicating that they induced indistinguishable chemical states for the brain. Therefore, our data support that EGCG can induce anxiolytic activity which could result from an interaction with GABA A receptors.

Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function

The peripheral-type benzodiazepine receptor or recognition site (PBR) is a widely distributed transmembrane protein that is located mainly in the outer mitochondrial membrane. The PBR binds to high-affinity drug ligands and cholesterol. Many functions are associated directly or indirectly with the PBR, including the regulation of cholesterol transport and the synthesis of steroid hormones, porphyrin transport and heme synthesis, apoptosis, cell proliferation, anion transport, regulation of mitochondrial functions and immunomodulation. Based on these functions, there are many potential clinical applications of PBR modulation, such as in oncologic, endocrine, neuropsychiatric and neurodegenerative diseases. Although "PBR" is a widely used and accepted name in the scientific community, recent data regarding the structure and molecular function of this protein increasingly support renaming it to represent more accurately its subcellular role (or roles) and putative tissue-specific function (or functions). Translocator protein (18kDa) is proposed as a new name, regardless of the subcellular localization of the protein.

Evaluation of the Abuse Potential of Pagoclone, a Partial GABAA Agonist

This study assessed the abuse potential of pagoclone, a partial agonist at the gamma-aminobutyric acid type A (GABAA) benzodiazepine receptor site, in healthy recreational drug users. Twenty-three young adults, who reported past recreational use of sedative drugs or alcohol, participated in 4 sessions during which capsules containing pagoclone (doses: 1.2 mg, the higher end of the proposed therapeutic dose range, and 4.8 mg, a 4-fold higher dose), diazepam (dose, 30 mg), or placebo were randomly administered under double-blind conditions. Subjective ratings of mood, drug effects, and psychomotor tests were completed at regular intervals after ingesting the capsules. On most of the standardized measures of abuse potential, pagoclone (dose, 4.8 mg) was rated as being similar to diazepam. Both drugs increased the ratings of good effects and drug liking. However, pagoclone also produced some adverse mood effects that might limit its potential to be used recreationally, and it produced fewer sedativelike effects on some measures. In general, the results with these doses indicate that the abuse potential of pagoclone is similar to that of diazepam, although its profile as a partial agonist suggests that differences between the drugs may emerge at higher doses.

Refinement of the Benzodiazepine Receptor Site Topology by Structure−Activity Relationships of NewN-(Heteroarylmethyl)indol-3-ylglyoxylamides

N-(heteroarylmethyl)indol-3-ylglyoxylamides (1-26) were synthesized and evaluated as ligands of the benzodiazepine receptor (BzR) to probe the hydrogen bonding properties of the so-called S(1) site of the BzR by means of suitable heterocyclic side chains. SARs were developed in light of our hypothesis of binding modes A and B. Pyrrole and furan derivatives adopting mode A (2, 8, 10, 20, 22) turned out to be more potent (K(i) values < 35 nM) than their analogues lacking hydrogen bonding heterocyclic side chains. These data suggest that the most potent indoles interact with a hydrogen bond acceptor/donor (HBA/D) group located within the S(1) site of the BzR. Compounds 1, 2, 8, 19, 20, and 22, tested at recombinant rat alpha(1)beta(2)gamma(2), alpha(2)beta(2)gamma(2), and alpha(5)beta(3)gamma(2) BzRs, elicited selectivity for the alpha(1)beta(2)gamma(2) isoform. On the basis of published mutagenesis studies and the present SARs, we speculate that the S(1) HBA/D group might be identified as the hydroxyl of alpha(1)-Tyr209 or of other neighboring amino acids.

Biflavones from Rhus species with affinity for the GABA A/benzodiazepine receptor

In South Africa Rhus pyroides is traditionally used in the treatment of epilepsy. In the present study two biflavonoids with activity in the 3H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Rhus pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of 3H-Ro 15-1788 with a K i of 28 and 37 nM, respectively. Extracts of Rhus dentata and Rhus pentheri were not as active as the extract from Rhus pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABA A receptor benzodiazepine site. This reflected the affinities of the compounds in the [ 3H]-flumazenil binding assay.

Facilitation of cholinergic transmission by combined treatment of ondansetron with flumazenil after cortical cholinergic deafferentation

We have studied the effects of concomitant blockade of 5-HT 3 and GABA A receptors on acetylcholine (ACh) release in the frontal cortex of rats with a selective cholinergic lesion. Lesions were performed by microinjection of the cholinergic toxin 192 IgG-saporin into the nucleus basalis magnocellularis. Single treatment with either the 5-HT 3 receptor antagonist ondansetron, 0.1 μg/kg, or the GABA A receptor benzodiazepine site antagonist flumazenil, 10 mg/kg, did not affect ACh release. However, the combined ondansetron+flumazenil administration significantly increased ACh release to a similar extent as a depolarising stimulus with K +, 100 mM, at both 7 and 30 days post-lesion. Cortical perfusion with the combined ondansetron+flumazenil treatment also increased [ 3H]ACh efflux “in vitro” 30 days after lesion, suggesting that local events within the frontal cortex may participate in the interaction of ondansetron with GABAergic neurons, modulating ACh release in situations of cholinergic hypoactivity. No differences in the expression of 5-HT 3 and GABA A receptors in the frontal cortex were found after the cholinergic lesion. These results suggest that a combined ondansetron+flumazenil treatment would contribute to restoring a diminished cholinergic function and may provide a basis for using this treatment in the therapy of cognitive disorders associated with degeneration of the cholinergic system.

Facilitation of cholinergic transmission by combined treatment of ondansetron with flumazenil after cortical cholinergic deafferentation

We have studied the effects of concomitant blockade of 5-HT 3 and GABA A receptors on acetylcholine (ACh) release in the frontal cortex of rats with a selective cholinergic lesion. Lesions were performed by microinjection of the cholinergic toxin 192 IgG-saporin into the nucleus basalis magnocellularis. Single treatment with either the 5-HT 3 receptor antagonist ondansetron, 0.1 μg/kg, or the GABA A receptor benzodiazepine site antagonist flumazenil, 10 mg/kg, did not affect ACh release. However, the combined ondansetron+flumazenil administration significantly increased ACh release to a similar extent as a depolarising stimulus with K + , 100 mM, at both 7 and 30 days post-lesion. Cortical perfusion with the combined ondansetron+flumazenil treatment also increased [ 3 H]ACh efflux “in vitro” 30 days after lesion, suggesting that local events within the frontal cortex may participate in the interaction of ondansetron with GABAergic neurons, modulating ACh release in situations of cholinergic hypoactivity. No differences in the expression of 5-HT 3 and GABA A receptors in the frontal cortex were found after the cholinergic lesion. These results suggest that a combined ondansetron+flumazenil treatment would contribute to restoring a diminished cholinergic function and may provide a basis for using this treatment in the therapy of cognitive disorders associated with degeneration of the cholinergic system.

Effects of midazolam on equine innate immune response: a flow cytometric study

Benzodiazepines (BDZ) are among the most frequently used class of psychotropic drugs employed in veterinary medicine in Brazil and worldwide due to their anxiolytic, muscle relaxant and anticonvulsant effects [J. Clin. Pharmacol. 33 (1993) 124]. Peripheral benzodiazepine receptor (PBR) sites were described in peripheral organs, endocrine steroidogenic tissues and immune organs and cells. Midazolam is a mixed-type agonist of PBRs. The present study is focused on the effects of midazolam on equine peripheral blood neutrophils, peritoneal macrophages and cortisol levels in plasma. Adult horses were treated with a single dose of midazolam (0.06 or 0.1 mg/kg) or with 0.9% NaCl. Immune cells were collected 24 h after treatment for flow cytometry analysis of Staphylococcus aureus-induced phagocytosis and oxidative burst. Plasma cortisol concentration was measured 30, 90, 180 and 360 min after midazolam treatment. Midazolam induced a dose-dependent reduction on: (1) peripheral blood neutrophil and peritoneal macrophage oxidative burst; (2) the capacity of both peripheral blood neutrophils and peritoneal macrophages to phagocyte S. aureus. Increments on plasma cortisol concentration were not found after 0.06 and 0.1 mg/kg of midazolam. The effects on oxidative burst of neutrophils and macrophages from horses treated with midazolam were interpreted as a consequence of an impairment of S. aureus-induced phagocytosis. The present data suggest that midazolam, most probably acting on PBRs present on equine macrophage and neutrophil membranes, might have changed some mechanisms related to both phagocytosis and oxidative burst. These results support the use of flow cytometry to identify functional properties and dysfunction of equine immune cells. They also confirm the notion that changes in the functional capacity of the immune system may represent an important hazard of exposure to drugs or chemicals.

Anxiolytic-like action of orally administered dl-tetrahydropalmatine in elevated plus-maze

dl-Tetrahydropalmatine ( dl-THP), a naturally occurring alkaloid, has been intensively studied for its sedative and hypnotic effects. Putative explanation for its mechanism and target of action involves the dopaminergic neurotransmission system. In view of the close interactions between the dopaminergic and the GABAergic neurons in the amygdala, pharmacological effects of dl-THP were tested for activity at the GABA A receptor benzodiazepine site (BDS). Effects of dl-THP were examined in mice employing the elevated plus-maze, the holeboard and the horizontal-wire tests. In the elevated plus-maze, mice treated with low doses of dl-THP (0.5–10 mg/kg) exhibited significant increase in the percentage of entries and time spent in open arms without altering the number of closed-arm entries when compared to the control group, indicative of its selective anxiolytic effect. In the holeboard and horizontal wire tests, where exploratory behavior and potential muscle relaxant effect were assessed, respectively, only mice treated with as much as 50 mg/kg dl-THP manifested sedation and myorelaxation, as observed in the significant decrease in the number of head dips and the decrease in the percentage of mice grasping wire in comparison to control. Notably, coadministration of the BDS antagonist flumazenil abolished the dl-THP-induced anxiolysis as seen in the reversal of the increase of both the number of entries and time spent in open arms back to basal levels in the elevated plus-maze test. The results suggest that dl-THP at defined low dosages acts as anxiolytics in mice, and the BDS mediates, at least in part, such anxiolytic effect of dl-THP.

3D‐Pharmacophores of Flavonoid Binding at the Benzodiazepine GABAA Receptor Site Using 4D‐QSAR Analysis.

4D-QSAR analysis was applied to a training set of 38 flavonoids where affinity constants, Ki, to the GABAA benzodiazepine receptor site, BzR, were determined. It was found that the −logKi values of...

Reduction of inflammation in rats by diazepam: tolerance development

High doses of diazepam (10–20 mg/kg) were shown to reduce the volume of acute carrageenan-induced inflammatory paw edema in rats. This effect was not observed after adrenalectomy or long-term use of similar doses of diazepam. The present experiment was undertaken to analyze the effects of long-term (21 daily injections) treatment with diazepam (10 mg/kg) on both carrageenan-induced paw edema (CIPE) and corticosterone serum levels. For comparison, the effects of a single and acute 10 mg/kg dose of diazepam were also analyzed. Results showed that: 1- long-term diazepam treatment induced no changes in CIPE values and corticosterone serum levels; 2- acute diazepam treatment reduced CIPE values and increased corticosterone serum levels; 3- the plasmatic levels of diazepam measured 1 hour after the single treatment or 1 hour after the last dose of long-term diazepam administration were not different. These results indicate the development of tolerance to diazepam effects on both CIPE and corticosterone serum levels and suggest a relevant role for corticosterone on diazepam-induced inhibition of acute inflammation. Data were discussed in the light of peripheral benzodiazepine receptor site (PBR) activation within adrenal gland cells by diazepam, thereby increasing the serum levels of corticosterone and thus reducing CIPE. Possible actions of diazepam on HPA axis activity and/or on cytokine network were also discussed.

3D-pharmacophores of flavonoid binding at the benzodiazepine GABA(A) receptor site using 4D-QSAR analysis.

4D-QSAR analysis was applied to a training set of 38 flavonoids where affinity constants, Ki, to the GABA(A) benzodiazepine receptor site, BzR, were determined. It was found that the -logKi values of the compounds are highly dependent on the size and electrostatics character of the substituents at the R(3') and R(6) positions of the flavonoid scaffold. Polar negative groups correctly embedded in the R(3') and/or R(6) substituents are predicted to increase -logKi values. A planar conformation of the flavonoid scaffold was found not to be a requirement for the flavonoids to be active. A test set of four compounds was used to evaluate the predictivity of the 4D-QSAR models.

In vivo imaging of anaesthetic action in humans: approaches with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI)

In vivo imaging of anaesthetic action in humans: approaches with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI)

Reduction of motor seizures in rats induced by the ethyl bicyclophosphate trimethylolpropane phosphate (TMPP)

1. Trimethylolpropane phosphate (TMPP) is a potent cage convulsant, reported to act through binding to the picrotoxinin and/or benzodiazepine receptor sites of the gamma-aminobutyric A (GABA A) ionophore complex. 2. Adult male Fischer-344 rats were pretreated by intraperitoneal (ip) injection with either diazepam (DZP) [0.5–5.0 mg/kg], Phenobarbital (PB) [5–20 mg/kg], dizocilpine maleate (MK-801) [0.5–3.0 mg/kg], Tiagabine (TGB) [0.5–5.0 mg/kg], 6,7-dinitro-quinoxaline-2,3-dione (DNQX), [5–20 mg/kg], or scopolamine [SCP] (0.25–1.0 mg/kg) 30 min prior to ip injection with a convulsive dose of TMPP (0.6 mg/kg). 3. Rats were rated for occurrence of convulsive activity for 120 min post-injection. Time from TMPP injection to observation of subclinical seizures, generalized (tonic-clonic) seizures, and lethality was rated for each pretreatment group. 4. In general, DZP = PB > TGB in reduction of TMPP subclinical and/or clinical seizures. MK-801, at dose levels inducing near sedation, was also effective in modulation of TMPP-induced seizures. SCP or DNQX were generally ineffective in reducing or eliminating TMPP-induced seizures.

Mechanism of action of the hypnotic zolpidem in vivo.

Zolpidem is a widely used hypnotic agent acting at the GABA(A) receptor benzodiazepine site. On recombinant receptors, zolpidem displays a high affinity to alpha 1-GABA(A) receptors, an intermediate affinity to alpha(2)- and alpha(3)-GABA(A) receptors and fails to bind to alpha(5)-GABA(A) receptors. However, it is not known which receptor subtype is essential for mediating the sedative-hypnotic action in vivo. Studying alpha1(H101R) mice, which possess zolpidem-insensitive alpha(1)-GABA(A) receptors, we show that the sedative action of zolpidem is exclusively mediated by alpha(1)-GABA(A) receptors. Similarly, the activity of zolpidem against pentylenetetrazole-induced tonic convulsions is also completely mediated by alpha(1)-GABA(A) receptors. These results establish that the sedative-hypnotic and anticonvulsant activities of zolpidem are due to its action on alpha(1)-GABA(A) receptors and not on alpha(2)- or alpha(3)-GABA(A) receptors.

Diazepam-potentiated [ 3H]phenytoin binding is associated with peripheral-type benzodiazepine receptors and not with voltage-dependent sodium channels

In the presence of diazepam, [ 3H]phenytoin binds with high affinity to brain membranes. The present experiments examined whether this high affinity [ 3H]phenytoin-binding site co-localized with the standard [ 3H]phenytoin-binding site on the voltage-dependent sodium channel (VDSC). Veratridine, a pharmacological activator of the voltage-dependent sodium channel, that inhibits standard [ 3H]phenytoin binding, failed to affect the high affinity diazepam-potentiated [ 3H]phenytoin binding in brain membranes, suggesting that the potentiated binding interaction resides at a site distinct from the voltage-dependent sodium channel. This possibility was confirmed by anion exchange chromatography of digitonin-solubilized rat brain membranes, as diazepam-potentiated high affinity [ 3H]phenytoin binding eluted in column fractions that were distinct from [ 3H]saxitoxin-defined voltage-dependent sodium channels. To examine whether diazepam-potentiated [ 3H]phenytoin binding might be associated with other ‘classic’ benzodiazepine receptor sites, we tested whether specific ligands for benzodiazepine receptors would either produce or block potentiated [ 3H]phenytoin binding. Neither agonists, nor antagonists, of the high affinity central-type benzodiazepine receptor affected potentiated [ 3H]phenytoin binding, suggesting that the high affinity potentiated binding site is not likely associated with central benzodiazepine receptors. Peripheral-type benzodiazepine receptor agonists, however, did potentiate [ 3H]phenytoin binding, and a specific receptor antagonist (PK11195) attenuated the potentiation seen with diazepam. Overall, these data illustrate that [ 3H]phenytoin interacts with a novel site in brain membranes that is distinct from the voltage-dependent sodium channel and is allosterically revealed by peripheral-type, but not central-type, benzodiazepine receptor agonists.

Two mouse lines selected for differential sensitivities to beta-carboline-induced seizures are also differentially sensitive to various pharmacological effects of other GABA(A) receptor ligands.

Two mouse lines were selectively bred according to their sensitivity (BS line) or resistance (BR line) to seizures induced by a single i.p. injection of methyl beta-carboline-3-carboxylate (beta-CCM), an inverse agonist of the GABA(A) receptor benzodiazepine site. Our aim was to characterize both lines' sensitivities to various physiological effects of other ligands of the GABA(A) receptor. We measured diazepam-induced anxiolysis with the elevated plus-maze test, diazepam-induced sedation by recording the vigilance states, and picrotoxin- and pentylenetetrazol-induced seizures after i.p. injections. Results presented here show that the differential sensitivities of BS and BR lines to beta-CCM can be extended to diazepam, picrotoxin, and pentylenetetrazol, suggesting a genetic selection of a general sensitivity and resistance to several ligands of the GABA(A) receptor.

Effects of Treatment with Amphetamine and Diazepam on Mycobacterium Bovis-Induced Infection in Hamsters

Tuberculosis is an example of an infection with an intracellular bacterium in which sensitivity is determined mainly by the host response. Macrophages are the architectural and functional units of the granulomas described in tuberculosis. Treatment with amphetamine (AMPH) and diazepam has been reported to decrease macrophage activity The present experiment was undertaken to investigate the effects of AMPH and/or diazepam given alone or in combination on hamster resistance to Mycobacterium bovis. The effects of these treatments on serum Cortisol levels were also studied. Adult hamsters were treated i.p. with AMPH (group E1 = 1.0 or 2.0 mg/kg/day), with AMPH (group E2 = 1.0 or 2.0 mg/kg/day) plus diazepam (2.0 mg/kg/day), with diazepam (group E3 = 2.0 mg/kg/day), or with control vehicles (1.0 ml/kg/day) for 40 days. Six days after the beginning of the treatments, the animals received identical inoculum concentrations of M. bovis. Hamsters treated with AMPH plus diazepam exhibited: 1) increased weight loss; 2) increased mortality; 3) increased scores of M. bovis colony forming units (CFU) isolated from liver, lung and spleen; 4) increased granuloma areas measured in the liver, lung and spleen. These effects were not induced by AMPH (1.0 or 2.0 mg/kg/day) given alone and were produced by diazepam (2.0 mg/kg/day) treatment per se. Furthermore, AMPH (2.0 mg/kg/day) and diazepam (2.0 mg/kg/day) given alone or in combination for 20 days increased the serum levels of Cortisol in relation to control hamsters, with the effect being higher in the animals treated with both drugs. The present data, which demonstrate an impaired defense against M. bovis in hamsters treated with AMPH plus diazepam or with diazepam alone, were tentatively explained on the basis of a direct and/or indirect action of the drugs on macrophage/lymphocyte activity. In the former case, the effects may be related to stimulation of peripheral benzodiazepine receptor sites (PBR) present on macrophages/lymphocytes and/or to a direct effect of ACTH on immune cells, while in the latter they may be mediated by Cortisol via PBR and ACTH stimulation of the adrenals.

Increased “peripheral-type” benzodiazepine receptor sites and mRNA in thalamus of thiamine-deficient rats

“Peripheral-type” benzodiazepine receptors (PTBRs) are highly expressed on the outer mitochondrial membrane of several types of glial cells. In order to further elucidate the nature of the early glial cell changes in thiamine deficiency, PTBR sites and PTBR mRNA were measured in thalamus, a brain structure which is particularly vulnerable to thiamine deficiency, of thiamine-deficient rats at presymptomatic and symptomatic stages of deficiency. PTBR sites were measured using an in vitro binding technique and the selective radio ligand [ 3H]-PK11195. PTBR gene expression was measured by RT-PCR using oligonucleotide primers based upon the published sequence of the cloned rat PTBR. Microglial and astrocytic changes in thalamus due to thiamine deficiency were assessed using immunohistochemistry and antibodies to specific microglial (ED-1) and astrocytic (GFAP) proteins respectively. Significant increases of [ 3H]-PK11195 binding sites and concomitantly increased PTBR mRNA were observed in thalamus at the symptomatic stage of thiamine deficiency, coincident with severe neuronal cell loss and increased GFAP-immunolabelling (indicative of reactive gliosis). Positron Emission Tomography using 11C-PK11195 could provide a novel approach to the diagnosis and assessment of the extent of thalamic damage due to thiamine deficiency in humans with Wernicke’s Encephalopathy.