BZ Binding Sites Research Articles

Differential effects of lorazepam on sleep and activity in C57BL/6J and BALB/cJ strain mice

Compared to C57BL/6 mice, BALB/c mice exhibit greater 'anxiousness' on behavioural tests of anxiety, and can show significantly longer sleep disruptions after exposure to anxiogenic situations. Relative to C57BL/6 mice, BALB/c mice also have reduced benzodiazepine (BZ) receptor densities in the brain and fivefold less BZ receptor density in the amygdala, a region important in anxiety and in the control of arousal. Lorazepam is a BZ receptor full agonist and has been used to treat both anxiety and insomnia. Differences between C57BL/6 and BALB/c mice raise the question of whether BZ agonists would impact sleep and activity differentially in the two strains. We examined the effects of two doses of lorazepam (0.5 and 1.5 mg kg(-1)) or saline alone (0.2 mL) on sleep and activity in C57BL/6 (n = 8) and BALB/c (n = 7) mice. Compared to saline, both doses of lorazepam significantly increased non-rapid eye movement (NREM) and reduced activity in both strains. In C57BL/6 mice, rapid eye movement (REM) was increased at both doses. In BALB/c mice, the 0.5 mg kg(-1) dose had no significant influence on REM, whereas REM was reduced significantly after the 1.5 mg kg(-1) dose. The results demonstrate significant differences between C57BL/6 and BALB/c mice in the effects of lorazepam on REM, whereas the effects on NREM and activity were similar. Strain differences in the number of BZ receptors in the amygdala, but not other brain regions, suggests possible site specificity in the effects of lorazepam on REM. These differences in BZ-binding sites in the amygdala could be a significant factor in differences in the sleep response between C57 and BALB/c mice.

The pharmacology and mechanisms of action of new generation, non-benzodiazepine hypnotic agents.

The new generation hypnotic drugs, zolpidem, zopiclone and zaleplon, are at least as efficacious in the clinic as benzodiazepines and may offer advantages in terms of safety. These drugs act through the BZ binding sites associated with GABAA receptors, but show some differences from benzodiazepines in pharmacological effects and mechanisms of action. Of particular interest is the finding that zolpidem shows a wide separation between doses producing sedative effects and those giving rise to other behavioural actions, and induces less tolerance and dependence than benzodiazepines. Zolpidem also demonstrates selectivity for GABAA receptors containing alpha1 subunits. Recent studies using genetically modified mice have confirmed that receptors containing alpha1 subunits play a particularly important role in mediating sedative activity, thus providing an explanation for the pharmacological profile of zolpidem.

Substituted imidazo[1,2-b]pyridazines: New compounds with activity at central and peripheral benzodiazepine receptors

A large range of substituted imidazo(1,2- b]pyridazines have been synthesized, and a number of potent ligands at central benzodiazepine (Bz) receptors on rat brain membranes have been identified in initial binding screens using [ 3H]diazepam. For those tested more extensively, binding studies conducted in the presence and absence of γ-aminobutyric acid suggest that they were full receptor agonists. Some preliminary evidence was found suggesting some species selectivity, i.e. several of the compounds were more active in in vivo tests in rats than in mice. The agonist activity of these 2-phenyl (and substituted phenyl) imidazo[1,2- b]pyridazines is consistent with the model of Bz receptor ligands as proposed by Fryer [Raven Press, 1983, pp. 7–20]. Several compounds were identified which had more selective activity at peripheral-type (mitochondrial) Bz binding sites. Thus, substituted imidazo[1,2- b]pyridazines represent yet another class of low molecular mass compounds which have activity at Bz receptor sites.

Pre- and postnatal development of GABA receptors in Macaca monkey visual cortex

GABA is a putative inhibitory neurotransmitter in adult mammalian visual cortex but also has been implicated as playing a crucial role in cortical information processing during development. In order to understand better the role of GABA during primate visual cortex development, we have examined the time course of GABAA and GABAB receptor ontogenesis in 18 Macaca nemestrina monkeys ranging from fetal day 61 (F61d) to adulthood. The GABA and benzodiazepine binding sites of the GABAA receptor were detected by 3H-muscimol (3H-MS) and 3H-flunitrazepam (3H-FZ), respectively. GABAB receptors were detected by 3H-baclofen (3H-BA). All ligands were visualized by in vitro autoradiography. Quantitative analysis of film density was done to compare laminar changes during pre- and postnatal development. Saturation binding experiments were done for MS and FZ binding sites to determine receptor number (Bmax) and affinity (Kd) at selected pre- and postnatal ages. Both MS and FZ binding sites were present at F61d-72d throughout the cortical plate and marginal zone. FZ binding sites were more dense than MS binding sites over the cortical plate at young ages and were especially dense over the marginal zone. FZ binding sites also were present in lesser amounts over the subplate and intermediate zone, but not over the subventricular zone. By F119d-126d, layer 4 could be distinguished by its higher density for both ligands. The basic adult laminar pattern was established for both MS and BZ binding sites by birth (birth = F165d-170d). After birth, MS density increases dramatically in all layers, but layer 4C remains most dense to adulthood. FZ labeling is heavy in both layers 4 and 3 at birth but after 4 weeks after birth (P4 wk) it declines somewhat in the supragranular layers so that layer 4C now predominates. Labeling in layers 5/6 virtually disappears after birth. BA binding sites were present at F126d, at which time layer 4 was slightly lighter than the remainder of striate cortex; this laminar pattern remained basically the same throughout our series to adulthood. Competitive binding of agonist and antagonists for the GABAA receptor showed that MS binding characteristics were similar at F126d and P8.5 years (yr). MS binding site Bmax was about 8% of adult values at F72d, 24% by F126d, and 56% at F152d. Bmax then rose rapidly after birth to peak at P18wk at 169% of adult values, and then declined to P1yr. A second peak of 143% was found around P3.5yr, with adult values reached by P8.5yr.(ABSTRACT TRUNCATED AT 400 WORDS)

Chronic ethanol treatment alters the behavioral effects of Ro 15-4513, a partially negative ligand for benzodiazepine binding sites

Pentylenetetrazol (PTZ)-induced convulsion were studied in control, chronic ethanol-maintained, and ethanol-withdrawal rats. The convulsive doses of PTZ varied among the different groups of rats. Ethanol-maintained rats required higher doses of PTZ to produce convulsions, compared to control and ethanol-withdrawal rats. The partially negative ligands for benzodiazepine binding sites, Ro 15-4513 (2 mg/kg, i.p.) and FG 7142 (20 mg/kg, i.p.) produced proconvulsant effect in saline (control) and ethanol-withdrawal rats as they potentiated the effect of subconvulsive dose of PTZ. A higher dose of Ro 15-4513 (4 mg/kg, i.p.), but not FG 7142 (up to 80 mg/kg, i.p.), also produced proconvulsant effect in ethanol-maintained rats. Furthermore, Ro 15-4513 (5, 10 mg/kg, i.p.), but not FG 7142 (up to 80 mg/kg, i.p.), produced clonic-tonic seizures of short duration in ethanol-withdrawal rats. These effects of Ro 15-4513 and FG 7142 were reversed by diazepam (2 mg/kg, i.p.), as well as by the GABA-neutral Ro 15-1788 (10 mg/kg, i.p.), thereby, indicating the involvement of central benzodiazepine receptors in the action of Ro 15-4513 and FG 7142. These observations suggest that chronic ethanol treatment selectively alters the receptor sensitivity to Ro 15-4513, an ethanol antagonist and partially negative ligand for BZ sites, and this observation supports the notion that ethanol effects are more susceptible to reversal by the imidazobenzodiazepine as compared to other negative ligand for BZ binding sites.

Selective affinity of the benzodiazepines quazepam and 2-oxo-quazepam for BZ1 binding site and demonstration of 3H-2-oxo-quazepam as a BZ1 selective radioligand

Quazepam and 2-oxo-quazepam are novel benzodiazepines containing a trifluoroethyl substituent on the ring nitrogen at position #1. Detailed competition binding experiments (25 to 30 concs.) at 4°C were undertaken with these compounds versus 3H-flunitrazepam using synaptic membranes from rat cortex or cerebellum. Unlike other benzodiazepines, both quazepam and 2-oxo-quazepam distinguished two populations of 3H-flunitrazepam binding sites in rat cortex which were present in roughly equal proportions and for which the compounds displayed a greater than 20-fold difference in affinity. In cerebellum, no such discrimination of sites was noted for 2-oxo-quazepam, but quazepam did distinguish a small, low affinity (15% of total) population of sites. 3H-2-oxo-quazepam was prepared and used in competition studies to substantiate the conclusion that these compounds discriminate two populations of benzodiazepine sites in rat cortex. This new radioligand was shown to specifically label BZ binding sites with high affinity in a saturable manner. The competition experiments were then conducted using 3H-2-oxo-quazepam at a radioligand concentration sufficiently low (0.5 nM) to ensure that only the higher affinity binding sites which 2-oxo-quazepam discriminates would be occupied. Competition experiments in both cortex and cerebellum under these conditions indicated single site binding for unlabelled quazepam and 2-oxo-quazepam in every instance. This suggests that 3H-2-oxo-quazepam should be a useful new tool for selectively labeling and studying the BZ1 population of benzodiazepine binding sites.

Muscarinic, benzodiazepine, gaba, chloride channel and other binding sites in frontal cortex in hepatic coma in man

1. 1. Alterations in several neurotransmitter systems in brain have been implicated in the pathophysiology of hepatic coma (HC). Studies on human autopsy material are few. We investigated H-quinuclidlnylbenzllate (QNB), 3H-spiperone, 3H-imipramine, 3H-PN-200-110, 3naloxone, 3H-Flunitrazepam, 3H-muscimol, 35S-t-butylbicyclophosphothionate and 3H-cyclohexyladenosine binding sites In frontal cortex from seven patients with HC and five controls. 2. 2. The density of 3H-QNB binding sites was significantly decreased and the affinity slightly increased in HC. The functional significance of these selective changes in muscarinic receptor binding sites Is unclear. Further studies evaluating cholinergic function In HC are Indicated. 3. 3. Acute studies in animals point to an Increase In GABA and BZ binding sites in HC. The present results show that the BZ/GABA-receptor-chloride-Ionophore complex is unchanged in HC in man. 4. 4. Serotonergic (5HT-2), adenosine (A-l), imipramlne (5HT uptake sites), opiate (naloxone) and calcium channel antagonist binding sites are unchanged in HC.

Octadecaneuropeptide (ODN; ‘anxiety peptide’) displaces diazepam more potently from astrocytic than from neuronal binding sites

The existence of specific binding sites for benzodiazepines (BZs) in the brain has prompted the search for endogenous BZ receptor ligands designated by the generic term « endozepines ». This has led to the identification of an 86-amino acid polypeptide capable of displacing [3H]diazepam binding to brain membranes, thus called diazepam-binding inhibitor (DBI). It was subsequently found that the sequence of DBI is identical to that of a lipid carrier protein termed acyl-CoA-binding protein (ACBP). The primary structure of DBI/ACBP has been well preserved, suggesting that endozepines exert vital functions. The DBI/ACBP gene is expressed by astroglial cells in the central nervous system, and by various cell types in peripheral organs. Endoproteolytic cleavage of DBI/ACBP generates several bioactive peptides including a triakontatetraneuropeptide that acts as a selective ligand of peripheral BZ receptors/translocator protein, and an octadecaneuropeptide that activates a G protein-coupled receptor and behaves as an allosteric modulator of the GABAAR. Although DBI/ACBP is devoid of a signal peptide, endozepines are released by astrocytes in a regulated manner. Consistent with the diversity and wide distribution of BZ-binding sites, endozepines appear to exert a large array of biological functions and pharmacological effects. Thus, intracerebroventricular administration of DBI or derived peptides induces proconflict and anxiety-like behaviors, and reduces food intake. Reciprocally, the expression of DBI/ACBP mRNA is regulated by stress and metabolic signals. In vitro, endozepines stimulate astrocyte proliferation and protect neurons and astrocytes from apoptotic cell death. Endozepines also regulate neurosteroid biosynthesis and neuropeptide expression, and promote neurogenesis. In peripheral organs, endozepines activate steroid hormone production, stimulate acyl chain ceramide synthesis and trigger pro-inflammatory cytokine secretion. The expression of the DBI/ACBP gene is enhanced in addiction/withdrawal animal models, in patients with neurodegenerative disorders and in various types of tumors. We review herein the current knowledge concerning the various actions of endozepines and discuss the physiopathological implications of these regulatory gliopeptides.

Peripheral-type benzodiazepine-binding sites in platelets of schizophrenics with and without tardive dyskinesia

The density of peripheral-type benzodiazepine (BZ)-binding sites was studied in platelets of 10 medicated chronic schizophrenics with tardive dyskinesia (TD), 10 medicated chronic schizophrenics without TD, 7 drug-free schizophrenics, and 10 normal controls. The age range of the study population was 36–60 years. Age and sex distribution were similar in all 4 groups. The unmedicated schizophrenics did not differ in their maximal binding capacity from the healthy controls. A significant decrease in the density of peripheral-type BZ-binding sites in platelets was observed in treated schizophrenics both with and without TD in comparison to controls and untreated schizophrenics. The reduction in [ 3H]PK 11195 binding was more pronounced in TD patients (31.3% of controls) than in patients without TD (21.1% of controls). However, this parameter failed to discriminate statistically between TD and non-TD medicated schizophrenics.

Convulsant effect of Ro 5-4864, a peripheral type benzodiazepine, on the baboon ( Papio papio)

The effects of Ro 5-4864, a 1,4-benzodiazepine with a high affinity for the peripheral-type benzodiazepine (Bz) binding site, were investigated in the baboon ( Papio papio), which is genetically predisposed to epilepsy. A proconvulsant effect of low doses (1–3 mg/kg, i.v.) of Ro 5-4864 was observed by studying its effect on the photic responses induced by intermittent light stimulation in non-photosensitive baboons. Higher doses of Ro 5-4864 (10 mg/kg, i.v.) were overtly convulsant. The Bzs clonazepam and diazepam blocked these convulsant actions of Ro 5-4864 whereas neither Ro 15-1788, an antagonist of central Bz binding sites, nor PK 11 195, an antagonist of peripheral Bz binding sites, had any effect. It thus appeared that the convulsant effect of Ro 5-4864 was not mediated by Bz binding sites of either the central or the peripheral type. It is possible that Ro 5-4864 exerts its convulsant action at the picrotoxin site of the central Bz receptor - γ-aminobutyric acid receptor-chloride ionophore complex.

Benzodiazepine binding sites in rat submaxillary gland: Absence of markers of GABA system

1. Specific binding of [ 3H]diazepam to the nuclear, mitochondrial and microsomal subcellular fractions of the rat submaxillary gland is reported. Apparent K d values were similar in the three fractions studied (around 80 nM). 2. The nuclear and mitochondrial fractions contain 95% of the total sites detected (20 pmoles/mg prot.). 3. The sites exhibit a much higher affinity for Ro 5-4864 than for clonazepam or Ro 15-1788, and are not modified by 10 −6 to 10 −4 GABA or 10 −5M bicuculline. 4. Neither sympathetic denervation or parasympathetic decentralization modified [ 3H]Dz binding, which is found to be considerably lower in the early post-natal state as well as following duct ligation of the gland of adult rats. 5. The results suggest that these peripheral Bz binding sites are mainly located in the acini. 6. No specific GABA binding or GAD activity was detected either in the presence or absence of NaCl. 7. Very low levels of endogenous GABA (58 ± 3 nmoles/g tissue) were found in homogenates of the gland.

The benzodiazepine/GABA receptor complex: molecular size in brain synaptic membranes and in solution.

The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated gamma-aminobutyric acid (GABA) receptor and for the component binding beta-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration ( approximately 230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [3H]flunitrazepam in the membrane, and contains a 51,000 Mr polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000-220,000 MW carries a class of BZ-binding sites and an associated class of GABAA sites.

Tofizopam selectively increases the affinity of benzodiazepine binding sites for [ 3H]flunitrazepam but not for β [ 3H] carboline-3-carboxylic acid ethyl ester

Summary The psychoactive properties of tofizopam (TP), a 3,4-benzodiazepine (BZ), differ from those of 1,4-BZ's. TP does not cause sedation or muscle relaxation, although it is claimed to be anxiolytic. TP does not act by occupying BZ binding sites, although similarly to 1,4-BZ's, it increases binding of GABA receptors. In this study the mechanism of the neurochemical action of TP was further studied. TP (300 mg/kg) enhanced the binding of [3H]flunitrazepam (3H-FP) to BZ binding sites in the mouse brain by 13.7 – 22.9 % in vivo, but failed to alter the binding of β [3H]carboline-3-carboxylic acid ethyl ester (3H-BCCE). 3H-BCCE is a selective ligand for a subclass of BZ binding sites that is most abundantly present in the cerebellum. In vitro binding of 3H-BCCE was not affected by TP; but diazepam (DP) decreased 3H-BCCE binding yielding IC50 of 16 nM. In vivo DP (50 mg/kg) also competed with bound 3H-FP and 3H-BCCE, decreasing binding of 3H-FP by 85.4–86.9 % and that of 3H-BCCE by 45.0–51.0 %. The psychoactive effect of TP seems to be mediated by influencing GABAergic transmission. TP is more selective in its action than DP, a 1,4-BZ; TP enhances binding of the GABA receptors and the binding of one or more subclasses of BZ binding sites, but does not affect the cerebellar type of BZ binding sites.

Tofizopam affects binding of [ 3H]muscimol to gammaamino-butyric acid receptors in rat and mouse brains

Summary Tofizopam (TP), a 3,4-benzodiazepine (BZ), does not bind to BZ binding sites, but increases their affinity for 1,4-BZ's, e.g. [3H]flunitrazepam. Some BZ binding sites and some of gamma-aminobutyric acid (GABA) receptors are coupled to each other; occupation of BZ binding sites increases the specific binding of GABA. In this study TP (1 μM) was shown to increase the binding of [3H]muscimol (3H-M), a GABA-agonist, to rat forebrain GABA receptors in vitro by ca. 17 %. Diazepam (DP) at 10 μM enhanced binding similarly. The stimulative effect of both TP and DP was diminished, when frozen, thawed and Triton X-100 treated brain tissue homogenate was used. After 0.92 ug/kg 3H-M was injected i.v. into mice, the in vivo binding of 3H-M was assessed. Pretreatment with TP (300 mg/kg) increased the binding by 115.5 %; DP (50 mg/kg) treatment increased binding by 97.4 %. Both in vitro and in vivo, the effects of TP and DP were more evident in the forebrain than in the cerebellum. Although TP does not bind to BZ or GABA receptors, it affects both. The results of this study suggest that TP counteracts the effects of inhibitory proteins present in the GABA/BZ receptor complex.