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We studied the effects of phenazepam (0.075 mg/kg) after pretreatment (5 minutes before) with naloxone (10 mg/kg) on open-field behavior of C57Bl/6 and BALB/c mice. In ex vivo experiments, we studied the effects of naloxone (1 and 10 mg/kg) on receptor binding of [H]-fl unitrazepam by membranes of brain fraction (P1+P2) of C57Bl/6 and BALB/c mice. It was shown that naloxone increased motor activity in the open field in BALB/c mice and decreased this parameter in C57Bl/6 mice. During combined treatment, naloxone potentiated the activating effects of phenazepam on the open-field behavior of BALB/c mice and slightly increased the sedative effect of this drug in C57Bl/6 mice. Naloxone stimulated reception of [H]-fl unitrazepam in BALB/c mice and slightly increased radioligand binding in C57Bl/6 mice. These data attest to enhanced reception in benzodiazepine site of GABA-receptor under conditions of opioid receptor blockade, the presence of anxiolytic or sedative (depending on the phenotype of the response to emotional stress) effect of naloxone, and co-directed effects of naloxone and benzodiazepine tranquilizer on open-field behavior of C57Bl/6 and BALB/c mice. [ABSTRACT FROM AUTHOR]

Benzodiazepines act at the major isoforms of GABA type A receptors where they potentiate the current evoked by the agonist GABA. The underlying mechanism of this potentiation is poorly understood, but hypothesized to be related to the mechanism that links agonist binding to channel opening in these ligand activated ion channels. The loop F of the α1 and the β2 subunit have been implicated in channel gating, and loop F of the γ2 subunit in the modulation by benzodiazepines. We have identified the conservative point mutation Y168F located N-terminally of loop F in the α1 subunit that fails to affect agonist properties. Interestingly, it disrupts modulation by benzodiazepines, but leaves high affinity binding to the benzodiazepine binding site intact. Modulation by barbiturates and neurosteroids is also unaffected. Residue α1Y168 is not located either near the binding pockets for GABA, or for benzodiazepines, or close to the loop F of the γ2 subunit. Our results support the fact, that broader regions of ligand gated receptors are conformationally affected by the binding of benzodiazepines. We infer that also broader regions could contribute to signaling from GABA agonist binding to channel opening. [ABSTRACT FROM AUTHOR]

Purpose: The astrocytic enzyme glutamine synthetase (GS) is a key regulator of glutamate and γ-aminobutyric acid (GABA) metabolism in the glutamate/glutamine cycle (GGC). Inhibition of GS results in changes of neurotransmitter release and recycling. However, little is known about the influence of GGC on neurotransmitter receptor expression. In the pentylenetetrazole model of epilepsy, GS becomes nitrated and partially inhibited, and we demonstrated alterations of neurotransmitter receptor expression in the same model. Therefore, we hypothesized similar changes of neurotransmitter receptor expression when GS is inhibited in vivo. Methods: Rats were treated with a single dose (100 mg/kg bodyweight) ofl-methionine sulfoximine (MSO), an irreversible inhibitor of GS. We used 3H-receptor autoradiography to measure glutamatergic [α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA)], GABAergic (GABAA, GABAB and GABAA-associated benzodiazepine (BZ) binding sites], dopamine D1, and adenosine A1 receptor subtypes. In addition, we performed saturation analysis of BZ binding sites on cerebral membrane homogenates and investigated the expression of GABAAα1 and γ2 subunits (which primarily mediate BZ binding) by western blot analysis. Results: We demonstrated a significant reduction of BZ binding in the somatosensory, piriform, and entorhinal cortices and in the amygdala, 24 and 72 h after MSO treatment. Saturation analysis revealed decreased BZ binding (Bmax) on cerebral membrane homogenates 72 h after MSO treatment, without changes in binding site affinity (KD). Furthermore, we found differential changes of α1, γ2, and phosphorylated γ2 subunits following MSO treatment. Conclusion: On the basis of our findings, we conclude that the glutamate/glutamine cycle directly influences GABAergic neurotransmission by regulating GABAA subunit composition, thereby affecting its modulation by endogenous benzodiazepines. [ABSTRACT FROM AUTHOR]

Abstract: Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for γ-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABAA receptors are composed of a variable array of subunits forming heteropentameric chloride channels with different pharmacological properties. The γ2 subunit is essential for the formation of the binding site for benzodiazepines, allosteric modulators of GABAA receptors that are clinically often used as sedatives/hypnotics and anxiolytics. There are contradictory reports in regard to the γ2 subunit''s expression and participation in the functional GABAA receptors in the mammalian locus coeruleus. We report here that the γ2 subunit is transcribed and participates in the assembly of functional GABAA receptors in the tyrosine hydroxylase-positive neuromelanin-containing neurons within postmortem human locus coeruleus as demonstrated by in situ hybridization with specific γ2 subunit oligonucleotides and autoradiographic assay for flumazenil-sensitive [3H]Ro 15-4513 binding to benzodiazepine sites. These sites were also sensitive to the α1 subunit-preferring agonist zolpidem. Our data suggest a species difference in the expression profiles of the α1 and γ2 subunits in the locus coeruleus, with the sedation-related benzodiazepine sites being more important in man than rodents. This may explain the repeated failures in the transition of novel drugs with a promising neuropharmacological profile in rodents to human clinical usage, due to intolerable sedative effects. [Copyright &y& Elsevier]

The three most widely expressed subunits of the GABAA receptor are α1, β2, and γ2 subunits, and the major isoform in the human brain is a pentameric receptor composed of 2α12β21γ2. Previously, we overexpressed the extracellular domain Q28-R248 of GABAA receptor α1 subunit. In the present study, the homologous extracellular domains Q25-G243 of GABAA receptor β2 subunit and Q40-G273 of γ2 subunit were also obtained through overexpression in Escherichia coli. Successful production of recombinant β2 and γ2 subunit receptor protein domains facilitates the comparison of structural and functional properties of the three subunits. To this end, the secondary structures of the three fragments were measured using CD spectroscopy and the β-strand contents calculated to be >30%, indicating a β-rich structure for all three fragments. In addition, the benzodiazepine (BZ)-binding affinity of the recombinant fragments were measured using fluorescence polarization to be 2.16 μM, 3.63 μM, and 1.34 μM for the α1, β2, and γ2 subunit fragments, respectively, indicating that all three homomeric assemblies, including that of the β2 subunit, generally not associated with BZ binding, can bind BZ in the micromolar range. The finding that the BZ binding affinity of these recombinant domains was highest for the γ2 subunit and lowest for the β2 subunit is consistent with results from previous binding studies using hetero-oligomeric receptors. The present results exemplify the effective approach to characterize and compare the three major subunits of the GABAA receptor, for two of which the overexpression in E. coli is reported for the first time. [ABSTRACT FROM AUTHOR]

GABAA receptors are chloride ion channels that mediate fast synaptic transmission and belong to a superfamily of pentameric ligand-gated ion channels. The recently published crystal structure of the acetylcholine binding protein can be used as a template for comparative modeling of the extracellular domain of GABAA receptors. In this commentary, difficulties with comparative modeling at low sequence identity are discussed, the degree of structural conservation to be expected within the superfamily is analyzed and numerical estimates of model uncertainties in functional regions are provided. Topography of the binding sites at subunit-interfaces is examined and possible targets for rational mutagenesis studies are suggested. Allosteric motions are considered and a mechanism for mediation of positive cooperativity at the benzodiazepine site is proposed. [Copyright &y& Elsevier]

The molecular basis underlying the stress-induced increment in the density of central benzodiazepine receptor from chick forebrain, observed previously at 4°C, was studied from a biophysical perspective. The thermal dependence of [[sup 3]H] 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 (ΔC[sub p, CON]) were significantly less negative than ΔC[sub p STR]. Changes in ΔH and ΔS between 4–37°C were greater in stressed chicks compared to control; however, the binding was exothermic and driven by enthalpy in both conditions. At 4°C, the receptor density (B[sub 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[sub max, CON] and the constancy in B[sub max, STR]. The fluorescence anisotropy of diphenylhexatriene was higher in control samples with respect to stressed chicks below 10°C. A temperature-induced increment in protein intrinsic-fluorescence was observed only in control, and was quenched by acrylamide more easily at 4°C than at 25°C. A higher microviscosity at 4°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[sub max] found in stressed samples at low temperatures with respect to control samples. [ABSTRACT FROM AUTHOR]

The GABA and benzodiazepine binding sites on GABAA receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug-naive rats was potentiated ∼1.6-fold by 100 μM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA potentiation were observed. Diazepam-induced changes in GABAA receptor γ2-subunit gene transcription and α1-, β2-, and γ2-subunit steady-state mRNA levels did not appear to be temporally related to allosteric uncoupling. [ABSTRACT FROM AUTHOR]

The antipunishment properties of diazepam (DZP) were investigated in mice treated acutely, or following nine daily treatments with either DZP (5 mg/kg, PO) or its vehicle. Acutely, or following chronic vehicle treatment, DZP produced a dose-related increase in activity punished by footshock. Following chronic DZP, test doses of DZP given 24 or 48 h following the last chronic treatment were no longer, or less effective in enhancing punished activity. Effects on unpunished activity were unaffected. In a study of the time course of tolerance development, tolerance was not seen after one or three daily treatments but was present after 6 days. Following establishment of tolerance by 9 days' treatment, the antipunishment activity of DZP reappeared after 8 days' withdrawal and was restored to acute levels after 16 days. Tolerance was not associated with changes in benzodiazepine (BZ) receptor affinity or numbers, but the ability of GABA to enhance BZ binding was increased. There was no change in the ability of DZP or the convulsant β-carboline DMCM to modulate S-TBPS binding. The mechanism of tolerance to the antipunshment properties of DZP therefore remains unknown. [ABSTRACT FROM AUTHOR]

Cultured glial cells were exposed to ferric nitrilotriacetate (Fe-NTA) at varying concentrations. Studies of the exposed glial cells were performed at days 29 and 36 post-conceptional age (culture days 8 and 15). In addition to morphologic studies, biochemical assays including [H]-flunitrazepam (FLU) specific binding, Ro5-4864-displaceableH-FLU binding, and protein determinations were performed. At day 29 post-conceptional age, significant decreases inH-FLU specific binding, Ro5-4864-displaceableH-FLU binding, and protein determinations were discernible only in the presence of 100 μM Fe-NTA. At day 36 post-conceptional ageH-FLU specific binding was significantly decreased at 20, 60, and 100 μM Fe-NTA concentrations, while Ro5-4864-displaceableH-FLU binding and protein determinations were significantly reduced at 60 and 100 μM Fe-NTA concentrations. The effects of Fe-NTA exposure appear to be both concentration and duration-of-exposure related. When compared to previously reported neuronal cell culture, studies utilizingH-FLU specific binding, Ro5-4864-displaceableH-FLU binding, and protein determinations, glial cells appear to be significantly more resistant to chelated iron exposure. [ABSTRACT FROM AUTHOR]

The synthesis, in vitro ligand binding study and in vivo Elevated Plus Maze test (EPM) of a series of pyrazolo[4,3-c]quinolin-3-ones (PQs) are reported. Multistep synthesis of PQs started from anilines and diethyl 2-(ethoxymethylene)malonate to give the quinolin-4-one nucleus, via the Gould-Jacobs reaction. These quinolinones were transformed to 4-chloroquinolines, which react with aryl-hydrazines affording the final compounds. PQs exhibited different potency in displacing specific [ 3 H]Flunitrazepam binding from the benzodiazepine binding site at the γ-aminobutyric acid receptor (GABA A -R) depending on the substitution of the pyrazoloquinolone nucleus. PCA helped determine how different substituents contributed to the differential behavior of the PQs studied. Compounds with high affinity for the GABA A -R were tested regarding their anxiolytic properties in Wistar adult male rats using the Elevated Plus Maze (EPM). Thus, PQs with a p-methoxy phenyl group at N-1 (7b-ii and 7c-ii) displayed a remarkable anxiolytic activity at low doses (0.5-1.0 mg/kg). Meanwhile, PQs featuring an unsubstituted phenyl (7b-i) or p-fluoro phenyl group (7b-iii) at the N-1 showed anxiogenic effects in the EPM test., (Copyright © 2018 Elsevier Ltd. All rights reserved.)