Benzodiazepine Receptor Subtypes Research Articles

Postnatal development of proteins associated with different benzodiazepine receptors.

The postnatal development of several proteins irreversibly labeled by [3H]flunitrazepam in membranes from rat cerebral cortex was investigated. It was demonstrated that in the early postnatal days proteins with apparent molecular weights 55,000 and 59,000 were predominantly labeled whereas irreversible labeling of a protein with apparent molecular weight 51,000 started to predominate only in the second postnatal week. Irreversible labeling of another protein with apparent molecular weight 62,000 was weak throughout development. All these proteins seem to be associated with central benzodiazepine receptors. Irreversible labeling at various time points after birth seems to parallel the postnatal development of these proteins, and the different time course of development and different binding properties of the individual proteins support the hypothesis that these proteins are associated with separate and distinct benzodiazepine receptor subtypes. The pharmacological properties of the individual receptor subtypes seem to be fully developed in the early postnatal days, and therefore newborn animals seem to be a good model system for the investigation of properties and function of these various benzodiazepine receptor subtypes.

Autoradiographic demonstration of the selectivity of two 1-N-trifluoroethyl benzodiazepines for the BZD-1 receptors in the rat brain

Receptor autoradiographic techniques have been used to demonstrate the selectivity of two trifluoroethyl-containing benzodiazepines for one of the subtypes of benzodiazepine receptor. Indirect localization of the binding sites for quazepam and halazepam was accomplished by using the ability of these compounds to displace [ 3H]-flunitrazepam binding. The appropriate binding parameters were selected on the basis of initial studies aimed at identifying the binding characteristics of several benzodiazepine compounds in comparison with the triazolopyridine CL218,872. Autoradiographic analysis of the benzodiazepine sites displaceable with quazepam and halazepam revealed the two benzodiazepine compounds preferentially labeled receptor sites in regions of the brain dominated by the type 1 benzodiazepine receptor subtype. Thus, quazepam and halazepam preferentially bind to benzodiazepine type-1 receptors in lamina IV of the cerebral cortex, the zona incerta, substantia nigra and the cerebellum.

Neurotransmitter receptor alterations in Huntington's disease: autoradiographic and homogenate studies with special reference to benzodiazepine receptor complexes.

In vitro receptor autoradiography was used to construct semiquantitative maps of subtypes of muscarinic cholinergic (labeled with [3H]N-methylscopolamine), benzodiazepine ([3H]flunitrazepam), gamma-aminobutyric acid ([3H]muscimol), dopamine, and serotonin ([3H]spiperone) receptors in frontal cortex, parietal cortex, caudate, putamen, and globus pallidus in tissue sections from 5 patients with clinically well-evaluated Huntington's disease and 5 controls matched with respect to age, sex, and postmortem delay. Homogenates were prepared from the remaining cortical and striatal tissue and used to characterize pharmacologically these same receptors, as well as histamine, adenosine, and nitrendipine receptors. Neuronal loss and gliosis were assessed in the contralateral formalin-fixed caudate and putamen. All binding sites measured (except serotonin) were reduced relative to control values in striatum primarily because of changes in the number of receptors rather than in affinity. Autoradiographic studies generally revealed that these changes were greater in the caudate than the putamen, paralleling the more severe neuropathological changes present in the caudate. In addition, autoradiographic studies demonstrated an increase in gamma-aminobutyric acid-related receptors in the globus pallidus. In the cortex, receptor alterations were limited to an increase in the number of benzodiazepine receptors in the frontal cortex which was most prominent in superficial cortical layers.

Ontogenetic properties of benzodiazepine receptor subtypes in rat spinal cord

Ontogenetic properties of benzodiazepine receptor subtypes in rat spinal cord

Binding of Various Benzodiazepine Receptor Ligands to Different Benzodiazepine Receptor Subtypes

Binding of Various Benzodiazepine Receptor Ligands to Different Benzodiazepine Receptor Subtypes

Autoradiographic Studies on Benzodiazepine Receptor Subtypes in the Rat Brain

The technique of in vitro autoradiography which was developed by Kuhar and others was applied to the rat brain with use of 3H-flunitrazepam (flu) as a radioactive ligand. The cerebral cortex, hippocampus, substantia nigra and cerebellar cortex were rich in 3H-flu binding sites. To differentiate the benzodiazepine receptor (BZR) subtype, the authors used a type 1 specific ligand, either triazolopiridazine (Cl 218872) or methyl-β-carboline-caboxylate (β-CCM), as an unlabeled displacer. The preparations were exposed on a 3H-sensitive film and then the film was developed. Computer-analysis of thus obtained autoradiographic pictures revealed that type 2 binding sites were distributed evenly within the rat brain, but with slight predominance in the hippocampus. After adding β-CCM, no silver grains were noticed in the cerebellum and substantia nigra. These data meant that these two structures contained essentially type 1 BZR, while the hippocampus contained both type 1 and type 2 receptors. Autoradiographically, characteristic distribution of BZR represented by 3H-flu binding was considerably lost by adding a type 1 specific ligand, and this treatment caused the silver grains to be evenly distributed. These data suggest that the BZR which is directly associated with characteristic pharmacological actions such as anxiolytic and hypnotic effects is type 1, and type 2 binding sites have a less characteristic distribution pattern and might be pharmacologically less specific.

Benzodiazepines modulate the A2 adenosine binding sites on 108CC15 neuroblastoma X glioma hybrid cells.

We have demonstrated high affinity diazepam binding sites of the Ro5-4864 benzodiazepine receptor subtype on 108CC15 neuroblastoma X glioma hybrid cells. These cells were previously shown to have purinoceptors of the A2 adenosine subtype and we have now found that [3H]-adenosine can be displaced from this binding site by the benzodiazepines and related compounds that can also bind to the Ro5-4864 site. Diazepam was found to have no intrinsic activity at the A2-receptor as measured by the stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in this cell line. At concentrations sufficient to compete for the A2-receptor, diazepam was shown to facilitate, by approximately 2 fold, the stimulation of cyclic AMP by adenosine. These effects are not due to inhibition of adenosine uptake or phosphodiesterase activity, but are probably a consequence of modulation of the coupling of the A2-receptor to cyclic AMP production in this hybrid cell line.

The benzodiazepine receptor ligand CL218,872 has both anxiolytic and sedative properties in rodents

The triazolopyridazine, CL218,872, inhibited the binding of [ 3H]flunitrazepam in the cerebellum more potently than in cortex, both in vitro and in vivo. The relationship between this phenomenon and the selectivity of benzodiazepine receptor subtypes is considered. In a range of behavioural tests the overall profile of CL218,872 was similar to that of diazepam in the rat and mouse, although CL218,872 was half as potent as diazepam in the rat and some 20-fold weaker in the mouse. In particular contrast to published observations, CL218,872 reduced locomotor activity in rats and mice at doses only slightly larger than those required to inhibit conflict in rats and footshock-induced fighting in mice. The only qualitative difference between the compounds that could be detected was in the mouse where large doses of CL218,872 did not produce the marked degree of motor incoordination seen after diazepam. However, no such difference was observed in the rat. The presently held view than CL 218,872 is a non-sedative anxiolytic agent needs to be revised in the light of these observations.

Differential Postnatal Ontogeny of Opiate and Benzodiazepine Receptor Subtypes in Rat Cerebral Cortex: Binding Characteristics of Tifluadom and Brotizolam

The postnatal development of 3[H] ethylketocyclazocine (EKC) binding characteristics was examined using membranes from the cerebral cortex of rats at various ages. Binding site affinity did not vary significantly between postnatal days 1 and 90. However, the apparent density of cortical binding sites increased fivefold between birth and adulthood. These results were similar to another ontogenic study of brain opiate receptor binding. Whereas EKC was equally potent as a competitor for 3[H] EKC binding in cortex from neonatal and adult rats, tifluadom was three times more potent in neonatal cortex than in adult cortex as a displacer of specific EKC binding. Brotizolam, a new thienodiazepine and a potent sedative hypnotic, also was distinctly more potent as an inhibitor of 3H-diazepam binding in neonatal rat brain cortex than in adult rat brain cortex. These results suggest that subtypes of benzodiazepine receptors, as well as some opiate receptor subtypes, exhibit different rates of postnatal development.

Preferential protection of cortical type II benzodiazepine receptors by γ‐aminobutyric acid during heat inactivation

AbstractPharmacological and biochemical evidence supports the existence of a heterogeneous population of benzodiazepine (BDZ) receptors. One major tool which has been used to identify distinct receptor subtypes is a novel series of triazolopyridazines (TPZ) that recognize two subpopulations (types I and II) of BDZ binding sites. Earlier studies demonstrated that γ‐aminobutyric acid (GABA) enhanced specific BDZ binding and protected a fraction of BDZ binding sites against heat inactivation. GABA/BDZ interactions were further investigated by studying the properties of those BDZ binding sites that are thermostable in the presence of 1.0 mM GABA when cortical membrane fragments are heated at 60°C. Saturation isotherms of 3H‐flunitrazepam (3H‐Flu) binding revealed a 90% and 60% diminution of binding sites in the absence and presence of 1 mM GABA, respectively. In the presence of 1 mM GABA, displacement of 3H‐Flu binding with varied concentrations of CL 218,872, a TPZ, in heated cortical membrane preparations indicated a significant increase in the IC50 value, with no effect on the Hill coefficient when compared to parallel control experiments with unheated membranes. In contrast, no significant differences in either Hill coefficients or IC50 values were obtained when similar displacement experiments were performed utilizing diazepam, an agent that does not distinguish between BDZ receptor subtypes. Hofstee plots of CL 218,872 displacement of 3H‐Flu binding were curvilinear in both heated and unheated membranes and could be resolved into two components by computer analysis. The relative affinity of CL 218,872 at the high‐affinity (KD1) or lowaffinity (KD2) recognition sites was unchanged after heating the membrane preparations. However, after heating, the density of type I receptors (Bmax1) was reduced 75%, while the number of type II receptors (Bmax2) was decreased only 38%. These results are indicative of a preferential GABA‐mediated protective effect on the thermal inactivation of type II BDZ recognition sites.

Several new benzodiazepines selectively interact with a benzodiazepine receptor subtype

The potency of several new benzodiazepines as inhibitors of [ 3H]flunitrazepam binding was investigated in membranes from rat cerebellum or hippocampus. It was found that quazepam and two of its metabolites have a higher affinity for benzodiazepine receptors in cerebellum than for those in hippocampus, indicating a preferential interaction of these compounds with BZ 1-receptors. Other experiments indicated that these compounds have benzodiazepine agonistic properties similar to diazepam or flunitrazepam. Thus, for the first time, benzodiazepines have been identified which differentially interact with different benzodiazepine receptors.

Aging, diazepam exposure and benzodiazepine receptors in rat cortex

Densities of two benzodiazepine receptor subtypes (BDZ 1 and BDZ 2) in the frontal cortex of 10- and 27-month-old male Fischer-344 rats were nearly identical. Acute diazepam pretreatment produced a 73% increase in receptor density in the aged rats, mainly in the BDZ 2 sites, whereas with the normal adult rat the density increased 42%, primarily due to stimulation of BDZ 1 sites. Chronic exposure elicited similar increases of 28–29% in both normal and aged rats but there were subtle differences in the distribution of specific receptors. This study demonstrates a definite effect of age on the response of benzodiazepine receptors to diazepam pretreatment.

Photoaffinity labeling of benzodiazepine receptors in rat brain with flunitrazepam alters the affinity of benzodiazepine receptor agonist but not antagonist binding.

Recently, it was proposed that beta-carbolines interact with a subset of benzodiazepine (BZD) binding sites in mouse brain. This postulate was based upon evidence showing changes in binding properties of the BZD receptor following photoaffinity labeling of membranes with flunitrazepam (FLU). Under conditions in which 80% of specific [3H]diazepam binding was lost in photolabeled membranes, specific [3H]propyl beta-carboline-3-carboxylate [( 3H]PCC) binding was spared. In this study, the binding of the BZD antagonists [3H]PCC, [3H]Ro15 1788 and [3H]CGS 8216 was examined in rat brain membranes following photoaffinity labeling with FLU. No significant changes in the apparent KD and small reductions in the Bmax of 3H antagonist binding were observed. However, in the same membranes, up to 89% of specific [3H]FLU binding was lost. When [3H]PCC (0.05 nM) was used to label the receptors in control and photolabeled membranes, the ability of BZD receptor agonists to inhibit [3H]PCC binding was greatly diminished in the photolabeled membranes. In contrast, the potency of BZD antagonists remained the same in both control and treated membranes. Based upon PCC/[3H]Ro15 1788 competition experiments, the ability of PCC to discriminate between BZD receptor subtypes was unaffected by photoaffinity labeling of cortical membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

CL 218872 antagonism of diazepam induced loss of righting reflex: Evidence for partial agonistic activity at the benzodiazepine receptor

A recent hypothesis suggests that the “selective anxiolytic” activity of the triazolopyridazine, CL 218872, is a reflection of this compounds high affinity for a benzodiazepine (BZD) receptor subtype. Subsequent to this proposal, the observation was made that CL 218872 does not effectively discriminate BZD receptor subtypes in v vitro at physiological temperatures (37°C). Based upon this observation, a selective effect in vivo related to the high affinity of CL 218872 for a BZD receptor subtype appears unlikely. The present study provides evidence for an alternative hypothesis to explain the unique pharmacological properties of CL 218872. The ability of CL 218872 to antagonize diazepam induced loss of righting reflex and enhance the anticonvulsant effect of diazepam in mice suggests that this triazolopyridazine may act as a partial agonist at the BZD receptor. Compared to the pharmacologically active BZDs, the unique actions of CL 218872 may be related to the lower intrinsic activity of this compound.

Regional heterogeneity of benzodiazepine receptors at 37°C: An [formula omitted] study in various regions of the rat brain

The most compelling pharmacological evidence in support of benzodiazepine (BZD) receptor heterogeneity is derived from the study of the complex interactions of CL 218872 and propyl beta-carboline-3-carboxylate (PCC) with brain BZD receptors. In the present study, we provide evidence to support the hypothesis that intraregional BZD receptor heterogeneity in rat brain is a result of the different conformational states of a single receptor. This hypothesis is based upon the observation that CL 218872 and PCC lose the ability to effectively discriminate BZD receptor subtypes in rat cerebral cortex, hippocampus and pons-medulla at physiological temperature (37°C). Interestingly, both PCC and CL 218872 show higher affinity for BZD receptors in the cerebellum when compared to other brain regions at 37°C. This observation suggests that interregional BZD receptor heterogeneity occurs under physiologically relevant temperatures. We propose that distinct cerebellar and non-cerebellar type BZD receptors exist in vivo while marked differences in the affinity of the type I and type II BZD receptor subtypes postulated by Klepner et al. 1979 may only occur in vitro at 0°–4°C.

Neurotransmitters in anxiety.

The most predictable anxiolytic effects of neurotransmitters are linked to the activation of a gamma-aminobutyric acid (GABA)-ergic subsystem associated with specific benzodiazepine receptors. Recent studies have indicated that subtypes of benzodiazepine receptors may be associated specifically with anxiolytic actions. Animal studies suggest that some forms of anxiety are mediated through the noradrenergic system, but a recent study testing this hypothesis confirmed it only partially. Other data implicate the serotonergic system in at least some types of anxiety. Currently the role of other neurotransmitters, such as dopamine, histamine, acetylcholine, and peptides, appears to be minimal. Clinical responses to drugs suggest that existence of at least two types of anxiety disorders representing perhaps different psychobiologic mechanisms.

The influence of temperature and gamma-aminobutyric acid on benzodiazepine receptor subtypes in the hippocampus of the rat

The influence of GABA on the affinity of flunitrazepam (FLU) for benzodiazepine receptor subtypes (type I and II) was studied by measurement of the competitive inhibition of [ 3H]FLU and [ 3H]propyl beta-carboline-3-carboxylate ([ 3H]PCC) binding. When assays were carried out at 0°C using a low concentration (0.040 nM) of [ 3H]PCC so that the type I receptors were selectively labelled, no significant effect of GABA (10 −4 M) on the FLU [ 3H] PCC competition curve was detected. In contrast, when assays were carried out at 0°C using [ 3H]FLU or a high concentration of [ 3H]PCC to achieve [ 3H]ligand receptor occupancy of both type I and type II receptors, GABA (10 −4 M) caused a significant increase in the affinity of FLU as measured by FLU [ 3H] FLU and FLU [ 3H] PCC competition experiments. Collectively, these data suggest that the influence of GABA on benzodiazepine receptor binding is mediated, primarily, by the type II receptor. It was also noted that the PCC [ 3H] FLU competition curve had a Hill coefficient of approximately 1 at 37°C as compared to the results of experiments at 0°C during which a Hill coefficient of approximately 0.7 was calculated.

The effect of temperature on CL 218872 and propyl beta-carboline-3-carboxylate inhibition of [ 3H]-flunitrazepam binding in rat brain

The competitive inhibition of [ 3H]-flunitrazepam binding by CL 218872 and propyl beta-carboline-3-carboxylate (PCC), non-benzodiazepine compounds that show differential affinities for benzodiazepine (BZD) receptor subtypes, was studied in the rat cerebral cortex and hippocampus at different temperatures of incubation. The potency of both inhibitors was significantly greater at 0° than at 37°C. The magnitude of temperature induced enhancement of potency may correlate with the pharmacological efficacy of compounds that interact with BZD receptors. Hill slopes for CL 218872 shifted from 0.52 to 0.97 in the cerebral cortex when incubations were performed at 0° and 37°C, respectively. Hill values for PCC changed from 0.68 to 0.93 under similar temperature conditions. These observations suggest the presence of a homogenous population of benzodiazepine receptors at physiological temperatures or the inability of CL 218872 and PCC to distinguish between receptor subtypes at 37°C.