Binding of flunitrazepam to differentiating neurons cultured in a chemically defined,hormone-supplemented medium

[³H]Flunitrazepam (FNZ) binding to cortical neurons from fetal rat brain was investigated in vitro. The use of a synthetic medium specific for neurons made it possible to plot a developmental curve of³H-FNZ binding in an almost pure neuronal culture. Detectable specific binding was present in vitro at time 0 (that is, the 16th gestational day). A progressive increase of binding, due to an increment in the number of recognition sites, was observed on the subsequent days. The affinity of the specific binding sites to³H-FNZ was enhanced by the addition of exogenous GABA, whereas the density was not affected.     

Autoradiographic study on [3H]flunitrazepam binding in rat cortex and hippocampus after chronic ethanol treatment

Ethanol alters almost all membrane functions, but it behaves essentially like a benzodiazepine-type GABAergic agonist. The mechanism by which ethanol affects the GABA/benzodiazepine complex is not clear. We studied the possible changes in [³H]flunitrazepam binding induced by chronic ethanol treatment, using light microscopic autoradiography, to try to elucidate the controversy underlying this topic. This technique allows us to measure densities of benzodiazepine receptors in different anatomical brain areas—visual cortex and hippocampus—which seem to constitute the anatomical support for the behavioral and physiological responses affected by ethanol. Autoradiographic studies on the visual cortex and hippocampus from rats chronically treated with ethanol do not show statistically significant differences in the binding of, [³H]flunitrazepam with respect to control animals. Furthermore, we did not find either rostro-caudal or medio-lateral differences, in benzodiazepine receptor densities in each layer of the visual cortex.     

Decreased High-Affinity Binding of [3H]Muscimol to Cerebral Synaptic Membranes of Scrapie-Infected Mice

Scrapie is a transmissible disease that results in progressive degeneration of the central nervous system and death. Although scrapie has been studied histopathologically, relatively little is known concerning neurotransmitter alterations. Specific [3H]muscimol binding to whole brain crude synaptic membranes (CSM) from mice clinically affected with scrapie was significantly (p less than 0.01) reduced, to approximately 73% of that of the controls. Of the brain regions examined, binding to only cerebral CSM was significantly (p less than 0.0001) decreased. Scatchard analyses of saturation curves revealed that the high-affinity (KD = 8 +/- 3 nM) site for muscimol was abolished in cerebral CSM from scrapie-infected mice, while the low-affinity site was unaffected. Binding of [3H]flunitrazepam to cerebral CSM was unaffected by scrapie and was stimulated by GABA to the same extent in both scrapie and control mice. These results suggest that scrapie agent 139A in C57BL/6J mice manifests a portion of its CNS pathology via a high-affinity GABA binding site that is unassociated with the benzodiazepine receptor.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam Binding in Rat Brain by the Prolonged Ventricular Infusion of Transformed Ginsenosides

Ameliorating effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. Ginseng saponins are transformed by intestinal microflora and the transformants would be absorbed from intestine. In the present study, we have investigated the effects of transformed ginsenoside Rg3, Rh2 and compound K on the modulation of NMDA receptor and GABA_A receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using [³H]MK-801 binding, and GABA_A receptor bindings were analyzed by using [³H]muscimol and [³H]flunitrazepam binding in rat brain slices. Ginsenoside Rg3, Rh2 and compound K were infused (10 g/10 l/h) into rat brain lateral ventricle for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]MK-801 binding were highly decreased in almost all regions of frontal cortex and hippocampus by ginsenoside Rh2 and compound K. The levels of [³H]muscimol binding were elevated in part of frontal cortex and granule layer of cerebellum by the treatment of ginsenoside Rh2 and compound K. However, the [³H]flunitrazepam binding was not modulated by any tested ginsenosides. Ginsenoside Rh2 and compound K induced the downregulation of the [³H]MK-801 binding as well as upregulation of the and [³H]muscimol binding in a region-specific manner after prolonged infusion into lateral ventricle. However, ginsenoside Rg3 did not show the significant changes of ligand bindings. In addition, ginsenoside Rh2 decreased the expression of nNOS in the hippocampus although Rg3 decreased the expression in the cortex. These results suggest that biotransformed ginsenoside Rh2 and compound K could play an important role in the biological activities in the central nervous systems and neurodegenerative disease.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Characterization of [3H]flunitrazepam binding to melanin.

In both clinical and forensic toxicology, the analysis of hair for drugs is an important tool to determine drug use in the past or to verify abstinence from illegal drugs during extended periods. Melanin is proposed as one of the factors that influences drug incorporation to hair and we have characterized the binding of the drug flunitrazepam to melanin in vitro. The drug was 3H labeled and melanin granules from cuttlefish, Sepia officinalis, were used according to the suggested standard for melanin studies. We observed a rapid Langmuir-like binding followed by a slower diffusion-limited binding that may be interpreted as an initial surface binding followed by deeper bulk binding. From three concentrations of melanin, with a 60-min incubation time, a mean saturation value of 180 +/- 20 pmol/mg was calculated. The binding of a group of benzodiazepines and tranquilizers was compared to the binding of [3H]flunitrazepam by means of displacement experiments. These drugs showed binding characteristics similar to [3H]flunitrazepam except phenobarbital, which had a lower affinity to melanin. The method presented in this study allowed measurements with low melanin and drug concentrations and it has the strength of directly measuring the amount of drug bound to melanin, in contrast to previous indirect methods.     

Decreased density of [3H]TCP binding following antipsychotic drug withdrawal in rats

Antipsychotic drugs have been reported to increase the expression of subunits of the NMDA receptor at the level of mRNA but it is not clear whether such effects are apparent at the level of the radioligand binding or receptor protein. Therefore, we examined the effect of treatment of, and withdrawal from, haloperidol, chlorpromazine, olanzapine or clozapine on the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP ) to the open ion channel of the NMDA receptor in rat caudate-putamen, hippocampus and frontal cortex. [3H]TCP binding was not significantly different in the caudate-putamen, hippocampus and cortex after three months of treatment with any antipsychotic drug. There were significant decreases in [3H]TCP binding in rat caudate-putamen and cortex, but not hippocampus, one month after ceasing treatment. Decreases in the caudate-putamen were detected in rats previously treated with chlorpromazine (0.1 mg/kg/day) and clozapine (0.1 and 1.0 mg/kg/day). In the cortex, decreases in [3H]TCP binding were also detected in rats previously treated with olanzapine (0.1 mg/kg/day) for three months. These data suggest that changes in the NMDA receptor associated ion channels occur following antipsychotic drug withdrawal.     

γ-Aminobutyric Acid and Pentobarbital Enhance 2-[3H]Oxoquazepam Binding to Type I Benzodiazepine Recognition Sites in Rat and Human Brain

2-Oxoquazepam (2oxoquaz) is a novel benzodiazepine which shows preferential affinity for type I benzodiazepine recognition sites. In the present study, we analyzed the effect of gamma-aminobutyric acid (GABA), pentobarbital, and chloride ions on [3H]2oxoquaz and [3H]flunitrazepam ( [3H]FNT) binding to membrane preparations from rat and human brain. GABA stimulated [3H]-2oxoquaz and [3H]FNT binding in a concentration-dependent manner. The maximal enhancement produced by GABA on [3H]2oxoquaz binding was higher than that produced on [3H]FNT binding in both rat and human tissues. In the rat brain, the effect of GABA on [3H]2oxoquaz was similar throughout different brain areas, whereas the effect on [3H]FNT binding was lower in the cerebral cortex and hippocampus than in the cerebellum. Moreover, both [3H]2oxoquaz and [3H]FNT binding were stimulated by chloride ions and pentobarbital. The results are consistent with the hypothesis that type I benzodiazepine recognition sites are linked functionally to the GABA recognition site and the chloride ionophore.     

Chemical integrity of [3H]GABA used in binding studies

A method which is claimed to be able to determine the proportion of true GABA within radiolabeled GABA used in binding studies was tested using [³H]GABA. The method was found to be unsuitable for³H-labeled GABA and, furthermore, both theoretical considerations and the present experimental data indicated that it could also produce misleading results with [¹⁴C]GABA.     

Presence of Two Benzodiazepine Binding Sites in the Rat Hippocampus

Abstract: Characteristics of receptor binding of diazepam and flunitrazepam in three brain areas were compared. It was found that in the cerebral cortex and cerebellum the number of sites was similar for both ligands and that the affinity of diazepam was four times lower than the affinity of flunitrazepam. In contrast, when binding in the hippocampus was analyzed (assuming the presence of homogenous binding sites), it was found that the number of binding sites was higher and that the affinity was 17 times lower for diazepam than for flunitrazepam. This difference is due to the presence of two diazepam binding sites in this brain area, as demonstrated by a Scatchard analysis.     

Modulation of [3H]Nitrobenzylthioinosine Binding Kinetics

Abstract

Inhibitors and substrates of the nucleoside transporter were tested for their effects on the kinetics of [³H]nitrobenzylthioinosine binding. Results are discussed in terms of a distinct site mediating the allosteric modulation of [³H]nitrobenzylthioinosine binding affinity.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

[3H]Ketanserin Binding in Human Brain Postmortem

This study aimed at comparing the binding characteristics of [³H]ketanserin, a high-affinity serotonin 2A (5-HT_2A) receptor antagonist, in the prefrontal cortex, hippocampus and striatum of human brain post-mortem. The results indicated the presence of a single population of binding sites in all the regions investigated, with no statistical difference in maximum binding capacity (B_max) or dissociation constant (K_d) values. The pharmacological profile of [³H]ketanserin binding was consistent with the labeling of the 5-HT_2A receptor, since it revealed a competing drug potency ranking of ketanserin = spiperone > clozapine = haloperidol > methysergide > mesulergine > 5-HT. In conclusion, the 5-HT_2A receptor, as labeled by [³H]ketanserin, would seem to consist of a homogenous population of binding sites and to be equally distributed in human prefronto-cortical, limbic and extrapyramidal structures.     

Increased binding of [3H]GABA to striatal membranes following ischemia

Sodium-independent binding of [3H]gamma-aminobutyric acid ([3H]GABA) to membranes prepared from ischemic-damaged rat striatum was studied by kinetic and time-course analysis. Three days after 40 min of ischemia, [3H]GABA binding increased fourfold over control values. Scatchard analysis of the binding showed that ischemia significantly increased the affinity (KD) and the total number of binding sites (Bmax) for the high-affinity GABA receptor. These results support the conclusion that transient forebrain ischemia damages striatal GABAergic neurons.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

[3H]muscimol and [3H]flunitrazepam binding sites in the developing cerebellum of mice treated with methylazoxymethanol at different postnatal ages

Two models of perturbed cerebellar ontogenesis were obtained by a single administration of methylazoxymethanol (MAM), a potent antimitotic agent, to mouse pups either on the day of birth (MAM₀ mice) or at postnatal day 5 (MAM₅ mice). The alterations of the cerebellar GABAergic system were studied by measuring glutamic acid decarboxylase activity, [³H]muscimol binding sites, which are known to be concentrated in the GABA_A receptors in the internal granular layer, and [³H]flunitrazepam binding sites, which are more abundant in the molecular layer. The primary target of the antimitotic agent are the precursors of the glutamatergic and GABAceptive granule cells. In both models GABAergic structures, as revealed by GAD activity measurements, appear to be relatively spared, and recovery of granule cell numbers occurs during development in MAM₅ mice. In MAM treated mice the number of [³H]muscimol binding sites (on a per cerebellum basis) decrease as the number of granule cells decrease, although some recovery occurred in MAM₅ mice, but not in MAM₀ mice. In MAM₅ mice, [³H]flunitrazepam binding sites (on a per cerebellum basis) were relatively unaffected, while they were decreased significantly, but to a lesser extent than [³H]muscimol binding sites, in MAM₀ animals. The more significant reduction of granule cell numbers and the cytoarchitectural disruption resultant from the more precocious application of the antimitotic appear responsible for the significant alteration and lack of recovery in MAM₀ mice.     

High-performance liquid chromatography determination of flunitrazepam and its metabolites in plasma by use of column-switching technique: comparison of two extraction columns

A study, using on-line column-switching high-performance liquid chromatography, evaluated two different extraction columns for the determination of flunitrazepam and its major metabolites: 7-aminoflunitrazepam, 7-acetamidoflunitrazepam and desmethylflunitrazepam. The procedure was based on the enrichment of benzodiazepines on the extraction column, followed by transfer of the compounds to the analytical column. The two extraction columns were compared: the first column was a BioTrap 500 MS (hydrophobic polymer), 20×4 mm I.D., and the second was a LiChrospher RP-18 ADS, 25×4 mm I.D. The analytical column used was a LiChrospher select B RP-8, 125×3 mm I.D. with 5 μm particle size. The extraction conditions for the two pre-concentration columns, such as extraction temperature, buffer concentration, buffer pH, acetonitrile percentage and flow-rate, were studied for the extraction from plasma of flunitrazepam and its metabolites mentioned above. The mobile phase of the analytical column was isocratic and composed of acetonitrile–20 mM phosphate buffer at pH 2.1 (35:65, v/v) and at a flow-rate of 0.3 ml/min.     

Irreversible Binding of [3H]Flunitrazepam to Different Proteins in Various Brain Regions

Irreversible photolabeling by [3H]flunitrazepam of four proteins with apparent molecular weights 51,000 (P51), 53,000 (P53), 55,000 (P55), and 59,000 (P59) was investigated in various rat brain regions by SDS-polyacrylamide gel electrophoresis, fluorography, and quantitative determination of radioactivity bound to proteins. On maximal labeling of these proteins, only 15-25% of [3H]flunitrazepam reversibly bound to membranes becomes irreversibly attached to proteins. Results presented indicate that for every [3H]flunitrazepam molecule irreversibly bound to membranes, three molecules dissociate from reversible benzodiazepine binding sites. This seems to indicate that these proteins are either closely associated or identical with reversible benzodiazepine binding sites, and supports the hypothesis that four benzodiazepine binding sites are associated with one benzodiazepine receptor. When irreversible labeling profiles of proteins P51, P53, P55, and P59 were compared in different brain regions, it was found that labeling of individual proteins varied independently, supporting previous evidence that these proteins are associated with distinct benzodiazepine receptors.