Stress-induced decrement in the plasticity of the physical properties of chick brain membranes

The molecular basis underlying the stress-induced increment in the density of central benzodiazepine receptor from chick forebrain, observed previously at 4 degrees C, was studied from a biophysical perspective. The thermal dependence of [3H]flunitrazepam binding to the central benzodiazepine receptor and the supramolecular organization were studied in forebrain membranes from chicks submitted to partial water immersion. The equilibrium dissociation constants increased with temperature in membrane from both control and stressed chicks. The heat capacity values in control samples (deltaC(p, CON)) were significantly less negative than deltaC(p STR). Changes in deltaH and deltaS between 4-37 degrees C were greater in stressed chicks compared to control; however, the binding was exothermic and driven by enthalpy in both conditions. At 4 degrees C, the receptor density (B(max)) was higher in stressed chicks compared to control. Such a difference was lost irreversibly upon temperature elevation, possibly due to the hysteresis between the heating and cooling behaviour of B(max, CON) and the constancy in B(max, STR). The fluorescence anisotropy of diphenylhexatriene was higher in control samples with respect to stressed chicks below 10 degrees C. A temperature-induced increment in protein intrinsic-fluorescence was observed only in control, and was quenched by acrylamide more easily at 4 degrees C than at 25 degrees C. A higher microviscosity at 4 degrees C in control favoured more external localizations of integral proteins; at higher temperatures, tryptophan residues moved to hydrophobic membrane-regions. Changes in the membrane-organization towards more fluid states favoured the accessibility of benzodiazepine to the central benzodiazepine receptor, expressed by the higher values of B(max) found in stressed samples at low temperatures with respect to control samples.     

Biphasic action of midazolam on GABAA receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of the benzodiazepine agonist midazolam on gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated currents was investigated in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. Midazolam displayed a biphasic effect on GABA responses. Low concentrations of midazolam (1nM-10 microM) reversibly potentiated GABA (3 microM)-activated Cl(-) currents (I(GABA)) in a bell-shaped manner, with the maximal facilitary effect at 0.1 microM; whereas at higher concentrations (above 10 microM), midazolam had an antagonistic effect on I(GABA). Our further study indicated that midazolam changed GABA(A) receptor affinity to GABA and the effects of midazolam on I(GABA) were voltage-independent. The benzodiazepine receptor antagonist, flumazenil, abolished the facilitary effect of low concentrations of midazolam rather than the antagonism of I(GABA) induced by high doses of midazolam. In addition, activation of protein kinase C prevented the inhibitory effect of midazolam at higher concentrations, but did not influence the effect of midazolam at low concentrations. These results indicate that midazolam interacts with another distinct site other than the central benzodiazepine receptors on GABA(A) receptors as an antagonist at higher concentrations in SDCN neurons.     

Molecular modeling and QSAR analysis of the interaction of flavone derivatives with the benzodiazepine binding site of the GABA(A) receptor complex

A large number of structurally different classes of ligands, many of them sharing the main characteristics of the benzodiazepine (BDZ) nucleus, are active in the modulation of anxiety, sedation, convulsion, myorelaxation, hypnotic and amnesic states in mammals. These compounds have high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Since 1989 onwards our laboratories established that some natural flavonoids were ligands for the BDZ-bs which exhibit medium to high affinity in vitro and anxiolytic activity in vivo. Further research resulted in the production of synthetic flavonoid derivatives with increased biochemical and pharmacological activities. The currently accepted receptor/pharmacophore model of the BDZ-bs (Zhang, W.; Koeler, K. F.; Zhang, P.; Cook, J. M. Drug Des. Dev. 1995, 12, 193) accounts for the general requirements that should be met by this receptor for ligand recognition. In this paper we present a model pharmacophore which defines the characteristics for a ligand to be able to interact and bind to a flavone site, in the GABA(A) receptor. closely related to the BDZ-bs. A model of a flavone binding site has already been described (Dekermendjian, K.; Kahnberg, P.; Witt, M. R.; Sterner, O.; Nielsen, M.; Liljerfors, T. J. Med. Chem. 1999, 42, 4343). However, this alternative model is based only on graphic superposition techniques using as template a non-BDZ agonist. In this investigation all the natural and synthetic flavonoids found to be ligands for the BDZ-bs have been compared with the classical BDZ diazepam. A QSAR regression analysis of the parameters that describe the interaction demonstrates the relevance of the electronic effects for the ligand binding, and shows that they are associated with the negatively charged oxygen atom of the carbonyl group of the flavonoids and with the nature of the substituent in position 3'.     

Peripheral-type benzodiazepine receptors in human blood cells of patients affected by migraine without aura

The kinetic parameters at equilibrium of peripheral benzodiazepine receptors in platelets, lymphocytes and granulocytes of 15 patients affected by migraine without aura were tested using [3H]PK 11195, a specific radioligand for this receptor and compared with the same number of healthy controls: a statistically significant increase (platelets 212%, lymphocytes 203%, granulocytes 171%, as absolute percentage) in the maximal number of binding sites (B(max)) in all three patient samples, compared with healthy controls was detected; on the contrary, the values of the dissociation constant (K(d)) at equilibrium do not show any statistically significant variations between the two groups. These data further confirm the presence of peripheral biochemical alterations in migraine without aura. As peripheral benzodiazepine receptors appear to be involved in the regulation of the mitochondrial respiratory chain, the observed increase in B(max) might be related to the mitochondrial anomalies found in migraine disorders.     

Proximity-accelerated chemical coupling reaction in the benzodiazepine-binding site of gamma-aminobutyric acid type A receptors: superposition of different allosteric modulators

Benzodiazepines are widely used drugs. They exert sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects and act through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid type A (GABA(A)) receptor. Ligands of the benzodiazepine-binding site are classified into three groups depending on their mode of action: positive and negative allosteric modulators and antagonists. To rationally design ligands of the benzodiazepine site in different isoforms of the GABA(A) receptor, we need to understand the relative positioning and overlap of modulators of different allosteric properties. To solve these questions, we used a proximity-accelerated irreversible chemical coupling reaction. GABA(A) receptor residues thought to reside in the benzodiazepine-binding site were individually mutated to cysteine and combined with a cysteine-reactive benzodiazepine site ligand. Direct apposition of reaction partners is expected to lead to a covalent reaction. We describe here such a reaction of predominantly alpha(1)H101C and also three other mutants (alpha(1)G157C, alpha(1)V202C, and alpha(1)V211C) with an Imid-NCS derivative in which a reactive isothiocyanate group (-NCS) replaces the azide group (-N(3)) in the partial negative allosteric modulator Ro15-4513. Our results show four contact points of imidazobenzodiazepines with the receptor, alpha(1)H101C being shared by classical benzodiazepines. Taken together with previous data, a similar orientation of these ligands within the benzodiazepine-binding pocket may be proposed.     

The peripheral-type benzodiazepine receptor. Localization to the mitochondrial outer membrane

We have investigated the subcellular localization of the peripheral-type benzodiazepine receptor in rat adrenal gland using the high affinity ligand 3H-labeled 1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide ([3H]PK11195). The autoradiographic pattern of [3H]PK11195 binding sites in tissue sections of adrenal gland is similar to the histochemical distribution of the mitochondrial marker enzymes, cytochrome oxidase and monoamine oxidase, which are present in high concentrations only in the cortex. Subcellular fractionation studies of homogenates of adrenal gland indicate that the recovery and enrichment of [3H]PK11195 binding sites in the nuclear, mitochondrial, microsomal, and soluble fractions correlate closely with cytochrome oxidase activity, but not with markers for the nuclei, lysosomes, peroxysomes, endoplasmic reticulum, plasma membrane, or cytoplasm, indicating an association of the peripheral-type benzodiazepine receptor with the mitochondrial compartment. Titration of isolated mitochondria with digitonin results in the simultaneous release of the peripheral-type benzodiazepine receptor and of monoamine oxidase, but not cytochrome oxidase, indicating association of the peripheral-type benzodiazepine receptor with the mitochondrial outer membrane. Scatchard analysis and drug displacement studies of the binding of [3H] PK11195 to intact mitochondria and to the outer membrane-enriched digitonin extract further confirm the localization of the peripheral-type benzodiazepine receptor to the mitochondrial outer membrane.     

Accelerated benzodiazepine receptor recovery after lorazepam discontinuation.

Benzodiazepine discontinuation can lead to a behavioral syndrome in animals and humans. In a mouse model, this syndrome is associated with benzodiazepine receptor up-regulation. The protein-modifying reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), has been used to irreversibly inactivate a number of neurotransmitter receptors including benzodiazepine receptors, and thus allows estimation of receptor recovery in vivo. To assess benzodiazepine receptor recovery after benzodiazepine discontinuation, we treated mice with lorazepam (LRZ), 2 mg.kg-1.day-1 for 1 wk. After 24 h, EEDQ (12.5 mg/kg) was administered, and benzodiazepine binding in the cortex and cerebellum was determined after 4-144 h. EEDQ treatment decreased receptor density in the cortex in both LRZ- and vehicle-treated groups by approximately 50%, with no change in apparent affinity as previously reported. Binding in both groups returned to control values after 96 h. Kinetic analysis indicated a more rapid increase in binding in LRZ-compared with vehicle-treated animals, with t1/2 for LRZ 19.1 h, and for vehicle, 30.8 h (P less than 0.05). Receptor density was decreased in the cerebellum after EEDQ by approximately 40% in both treatment groups, with no change in apparent affinity. Receptor density returned to control values at 96 h, with no difference in kinetics in LRZ- compared with vehicle-treated mice. The decrease in receptor t1/2 associated with lorazepam discontinuation is consistent with the observed increase in benzodiazepine receptors in this setting.     

QSAR studies on acylated histamine derivatives

H(3)-receptor antagonists activity in terms of -log K(i) for a series of acylated histamine derivatives was modeled using topological indices, namely negentropy (N), molecular redundancy (MRI), and valence connectivity index ((m)x(v)) indices. Excellent results were obtained in multiple regression analysis upon the introduction of a dummy parameter (indicator parameter). Consistent increase in R(2)(A) value indicated that inspite of observed collinearity the proposed models are significant.     

Demonstration and purification of an endogenous benzodiazepine from the mammalian brain with a monoclonal antibody to benzodiazepines

Four hybridoma lines secreting monoclonal antibodies to benzodiazepines were produced after BALB/c mice were immunized with a benzodiazepine-bovine serum albumin conjugate. The monoclonal antibodies were purified from ascites fluids, and their binding affinities for benzodiazepines and other benzodiazepine receptor ligands were determined. These antibodies have very high binding affinities for diazepam, flunitrazepam, Ro5-4864, Ro5-3453, Ro11-6896, and Ro5-3438 (the Kd values are in the 10(-9) M range). However, these antibodies have very low affinities for the benzodiazepine receptor inverse agonists (beta-carbolines) and antagonists (Ro15-1788 and CGS-8216). One of the monoclonal antibodies (21-7F9) has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the peripheral-type benzodiazepine receptor. The neurotransmitter gamma-aminobutyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Preliminary evidence indicates that the purified substance is a benzodiazepine with a molecular structure that is identical or very close to N-desmethyldiazepam.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Requisite structural characteristics for benzodiazepine inhibition of triiodothyronine uptake into a human liver cell line.

Previously, we reported potent inhibition of triiodo-L-thyronine (T3) cellular uptake into a human liver cell line (HepG2) by central and peripheral receptor specific benzodiazepine (BZ) compounds and our working hypothesis of BZ's as direct competitors for the iodothyronine transporter, displacing T3 but not acting as a substrate for transport. In this report, we list other reported uptake inhibitors and compare them to 23 benzodiazepine receptor ligands, in their potency to inhibit cellular uptake of T3. The most potent inhibitors are restricted to the benzodiazepine class. From the BZ structure-activity relationship (SAR) for inhibition, we see that the nonfused phenyl ring may be essential for activity and the strongest relationship is seen with substitution at R2' where Cl greater than F greater than H. Substitution at R4' and hydroxyl substitution at R3 enhances potency as will alkyl groups at R1 or on the imidazole group in the 1,2-annelated series. With R7 substitution, Cl is preferred over NO2 but not necessarily H when R4' = Cl; this may reflect a slightly different orientation of the molecule with large aliphatic R1 groups and/or R4' substitution. The carbonyl at R2 in the 1,4 benzodiazepine series, enhances their potency. The resultant structure-activity relationship highlights the importance of the halogen-substituted nonfused phenyl ring and seems unique relative to other described benzodiazepine sites and/or effects.     

Benzodiazepine Receptor Binding in Cerebellar Cortex: Observations in Olivopontocerebellar Atrophy

Benzodiazepine receptor binding was measured in cerebellar cortex of 15 patients with dominantly inherited olivopontocerebellar atrophy (OPCA). The majority of these patients had a moderate to marked Purkinje cell loss, as judged by the lowered levels of dentate nucleus gamma-aminobutyric acid (GABA), a marker of Purkinje cells. Despite the reduction in Purkinje cell number cerebellar cortical benzodiazepine receptor density was either normal or slightly elevated in the OPCA patients. These results are in contrast to the findings in a mutant strain of mice deficient in Purkinje cells in which the concentration of benzodiazepine receptors in cerebellum is greatly reduced. Our data indicate that in the human, cerebellar cortical benzodiazepine receptors are either not significantly associated with Purkinje cells or that in OPCA Purkinje cell loss triggers a de novo synthesis of extra benzodiazepine binding sites. It is concluded that, in contrast with the rodent, in the human benzodiazepine receptor binding may not serve as a marker for cerebellar Purkinje cells.     

Stimulation of benzodiazepine binding to rat brain membranes and solubilized receptor complex by avermectin B1a and gamma-aminobutyric acid

The binding of [3H]flunitrazepam to benzodiazepine receptors in synaptic membranes and a digitonin-solubilized receptor fraction of rat brain is increased by avermectin B1a and gamma-aminobutyric acid (GABA). The effects of avermectin B1a and GABA are both sensitive to inhibition by (+)-bicuculline. Avermectin B1a and GABA both decrease the Kd and increase the Bmax of [3H]flunitrazepam binding to membranes. Kinetic analysis of the binding of [3H]flunitrazepam to rat brain membranes indicates that avermectin B1a and GABA reduce the rate constants of both association and dissociation between the ligand and the receptor. These results suggest a similar mechanism of modulation of benzodiazepine binding by avermectin B1a and GABA. This modulation may involve in interaction among the receptors for benzodiazepine, GABA and avermectin B1a.     

Hypnotic action of benzodiazepines: a possible mechanism

The objective of this investigation was to determine whether the effects of muscimol on benzodiazepine receptor binding relate to the hypnotic activity of nine benzodiazepines (clonazepam, triazolam, diazepam, flurazepam, nitrazepam, oxazepam, temazepam, clobazam, and chlordiazepoxide) and CL 218,872. There was no correlation between the basal receptor binding affinities of the drugs tested and their hypnotic potencies, whereas the benzodiazepine receptor agonists whose receptor bindings are strongly modulated by muscimol possess potent hypnotic activity. These results indicate that benzodiazepine receptors that couple to GABA receptors are involved in the hypnotic activity of the benzodiazepines.     

Effect of multiple daily administration of fenibut and diazepam on GABA and benzodiazepine receptors in the mouse brain

It was shown in experiments on mice that 25 hours after chronic treatment with fenibut (100 mg/kg, twice daily for 10 days) was discontinued the number of benzodiazepine and GABAA (bicucullin-sensitive) receptor sites was increased and 48 hours after treatment discontinuation the number of GABAB (bicucullin nonsensitive) sites was decreased. The enhanced binding to GABAA and GABAB receptor sites and the decreased binding to benzodiazepine receptors was observed 24 hours after discontinuation of chronic treatment with diazepam (5 mg/kg, twice daily). Forty-eight hours after diazepam chronic treatment was discontinued the number of benzodiazepine receptor sites was increased. The involvement of the increased benzodiazepine receptor sensitivity in the mechanism of therapeutic activity of fenibut is suggested.     

Ivermectin Interactions with Benzodiazepine Receptors in Rat Cortex and Cerebellum In Vitro

Abstract: The anthelmintic macrolide, ivermectin, enhances the binding of benzodiazepine agonist ([³H]-diazepam) and antagonist ([³H]β-carboline ethyl ester) ligands to rat cortical and cerebellar membrane preparations. Enhancement of benzodiazepine agonist binding is partially additive with that of γ-aminobutyric acid (GABA) and is inhibited by etazolate, bicuculline, and the steroid GABA antagonist R5135. Ivermectin-stimulated benzodiazepine antagonist binding is enhanced by bicuculline and inhibited by GABA and etazolate. The modulatory effects of bicuculline are chloride-dependent. The stimulatory effects of ivermectin, while quantitatively different in cortex and cerebellum, are qualitatively similar in both brain regions and are reduced in the presence of chloride. Ivermectin effects on benzodiazepine ligand binding to the benzodiazepine receptor complex and the differences in the effects of GABA, bicuculline, and R5135 on ivermectin-stimulated agonist and antagonist binding may provide evidence for distinct differences in the recognition sites for the two classes of benzodiazepine receptor ligand and their interactions with other components of the receptor complex.     

Electrophysiological actions of benzodiazepines

Electrophysiological investigations have revealed that benzodiazepines, applied either locally or systemically, reduce central nervous system excitability. The studies summarized here indicate that this depression of excitability by benzodiazepines is a result of an increase in gamma-aminobutyric acid (GABA) mediated inhibition. This increase in inhibition may result from benzodiazepines increasing the activity of some GABAergic neurons and also from a modulatory action of benzodiazepines on GABA actions at some postsynaptic receptor sites. The modulatory action is observed with doses of benzodiazepines that do not cause any direct effects on neuronal excitability or membrane polarization. Specificity tests indicate that benzodiazepines do not enhance inhibition mediated by glycine or monoamines such as norepinephrine or serotonin. Results of experiments with a convulsant benzodiazepine compound, which causes a specific reduction in GABA-mediated inhibition, are also presented, The data are discussed in terms of a model in which the benzodiazepine receptor, the GABA receptor, and the chloride ionophore are functionally linked. Furthermore, it is proposed that some postsynaptic actions of GABA may be continually regulated by the occupancy of a benzodiazepine receptor, and that occupancy of the benzodiazepine receptor may be permissive for the GABA-elicited increase in chloride ion permeability.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)