Regional heterogeneity of benzodiazepine receptors at 37 degrees C: an in vitro study in various regions of the rat brain

The most compelling pharmacological evidence in support of benzo-diazepine (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 degrees C). Interestingly, both PCC and CL 218872 show higher affinity for BZD receptors in the cerebellum when compared to other brain regions at 37 degrees 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 degree--4 degree C.     

Identification and structure-activity studies of novel ultrashort-acting benzodiazepine receptor agonists

The synthesis and evaluation of novel ultrashort-acting benzodiazepine (USA BZD) agonists is described. A BZD scaffold was modified by incorporation of amino acids and derivatives. The propionate side chain of glutamic acid tethers an enzymatically labile functionality where the metabolite carboxylic acid displays markedly reduced BZD receptor affinity. The USA BZDs were characterized by full agonism profiles. Copyright2000 Elsevier Science Ltd.     

Heterogeneity in the Allosteric Interaction Between the γ-Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System

In the present communication we have investigated the allosteric coupling between the gamma-aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high- and low-affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very-low-affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high- and low-affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very-low-affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very-low-affinity binding site for zolpidem.     

Photoaffinity Labeling of Benzodiazepine Receptors in Rat Brain with Flunitrazepam Alters the Affinity of Benzodiazepine Receptor Agonist But Not Antagonist Binding

Abstract: Recently, it was proposed that β-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 β-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 K_D and small reductions in the B_max 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. Overall, these findings suggest that β-carbolines do not interact with a subset of BZD binding sites per se, but may be a consequence of the differential interaction of BZD agonists and antagonists with BZD binding sites that have been photoaffinity labeled with FLU. A possible mechanism underlying this phenomenon is discussed. The ability of photolabeled membranes to differentiate between BZD agonists and antagonists provides a potential screen for agonist and antagonist activity in compounds that interact with the BZD receptor.     

Inhibitors of peripheral-type benzodiazepine receptors present in human urine and plasma ultrafiltrates

Several endogenous substances that inhibit central-type benzodiazepine (BZD) receptor binding have recently been identified. We have found that ultrafiltrates of human uremic plasma, normal plasma, and urine contain competitive inhibitors of peripheral-type benzodiazepine receptors. Using urine as source, we have partially purified a peripheral-type BZD receptor inhibitor(s) by adsorption to and selective elution from small octadecyl-silane (Sep-pak) columns and thin layer chromatography. The inhibitor has a 125-fold greater affinity for peripheral-type than central-type BZD receptors and has been purified 8000-fold from urine.     

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.     

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 vitro at physiological temperatures (37 degrees 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.     

Isoliquiritigenin, a chalcone compound, is a positive allosteric modulator of GABAA receptors and shows hypnotic effects

Isoliquiritigenin (ILTG) is a chalcone compound and has valuable pharmacological properties such as antioxidant, anti-inflammatory, anticancer, and antiallergic activities. Recently, the anxiolytic effect of ILTG has been reported; however, its action mechanism and hypnotic activity have not yet been demonstrated. Therefore, we investigated the hypnotic effect and action mechanism of ILTG. ILTG significantly potentiated the pentobarbital-induced sleep in mice at doses of 25 and 50 mg/kg. The hypnotic activity of ILTG was fully inhibited by flumazenil (FLU), a specific gamma-aminobutyric acid type A (GABA_A)–benzodiazepine (BZD) receptor antagonist. The binding affinity of ILTG was 0.453 μM and was found to be higher than that of the reference compound, diazepam (DZP, 0.012 μM). ILTG (10⁻⁵ M) potentiated GABA-evoked currents to 151% of the control level on isolated dorsal raphe neurons. ILTG has 65 times higher affinity for GABA_A–BZD receptors than DZP, and the dissociation constant for ILTG was 4.0 × 10⁻¹⁰ M. The effect of ILTG on GABA currents was blocked by 10⁻⁷ M FLU and ZK-93426. These results suggest that ILTG produces hypnotic effects by positive allosteric modulation of GABA_A–BZD receptors.     

Heterogeneity of γ-Aminobutyric Acid/Benzodiazepine/β-Carboline Receptor Complex in Rat Spinal Cord

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly less than 1; the potency of the different BCs in displacing [3H]flumazenil is 20-50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5-4864 (4'-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of less than 1 (nH = 0.4) by CL 218,872 (3-methyl-6-(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b] pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (approximately 80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominantly of the central-type, BZD1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel spiro[indene-1,2′-quinazolin]-4′(3′H)-one derivatives as potent anticonvulsant agents: One-pot synthesis,in vivo biological evaluation,and molecular docking studies

A new series of spiro[indene-1,2′-quinazolin]-4′(3′H)-one derivatives 4a–m were synthesized via a one-pot method and evaluated for anticonvulsant activities using pentylenetetrazole (PTZ) and maximal electroshock (MES)-induced seizures. Obtained results demonstrated that these compounds have not anticonvulsant activity in PTZ test while are active in the MES test. Among the synthesized compounds, the best anticonvulsant activity was obtained with compound 4h . This compound also was not neurotoxic. Given that the title new compounds have the pharmacophore requirement for benzodiazepine (BZD) receptor agonist, the most potent compound was assayed in vivo and in silico as BZD receptor agonist. After treatment with flumazenil as a standard BZD receptor antagonist, anticonvulsant activity of compound 4h decreased. Therefore, the involvement of BZD receptors in anticonvulsant activity of this compound confirmed. Furthermore, docking study of compound 4h in the BZD-binding site of GABA_A receptor confirmed that this compound interacted with the important residues.     

Occurrence of "natural" benzodiazepines

There is accumulating evidence that benzodiazepines (BZD)--agents widely used as anxiolytics and hypnotics-could be regarded as "natural" drugs since they have been found in trace amounts also in plants, various tissues of different animal species and even humans. The biosynthesis of such BZD is still unknown and the hypothesis is favoured that they may be of plant origin. Besides diazepam (D) and its major metabolite desmethyldiazepam (DD) several other BZD (e.g. delorazepam, deschloro-diazepam, delormetazepam, isodiazepam, lormetazepam, oxazepam) could be detected. In some cases identification of these compounds was accomplished by specific mass spectrometry (GC-MS) and for quantification various methods have been applied resulting in different concentrations which range for D from about 0.005 to 1 ng/g and for DD from 0.01 to 0.5 ng/g. It is very unlikely that these trace amounts exert any direct pharmacological effects and at the moment only speculations upon their physiological/biological role are possible. Recently BZD-receptor binding activity equivalent to surprisingly high levels of more than 900 ng/ml was found in cerebrospinal fluid of patients with advanced hepatic encephalopathy. As long as the structure of this binding activity has not been elucidated no firm conclusions can be drawn from these findings. If pertinent analytical problems (e.g. drug-free biological material; exact quantification by internal standard techniques) are solved and if the source(s) of BZD are established it might be possible to answer also the critical question whether "endogenous" or "natural" BZD play any (in-) direct role in the regulation of CNS activity.     

Potential anxiolytic agents. Part 4: novel orally-active N(5)-substituted pyrido[1,2-a]benzimidazoles with high GABA-A receptor affinity

A series of pyrido[1,2-a]benzimidazoles (PBIs) with substitution on the N(5)-nitrogen has been synthesized and found to possess high affinity for the benzodiazepine (BZD) site on the GABA-A receptor. The compounds evaluated include those bearing a heteroalkyl group and heterocyclic rings. The most promising of these compounds is ethoxymethyl analogue 24, which has an IC(50) of 0.1 nM for the BZD site on the GABA-A receptor and has been advanced to human clinical trials.     

The Role of Marital Status in the Association between Benzodiazepines,Psychotropics and Injurious Road Traffic Crashes: A Register-Based Nationwide Study of Senior Drivers in Sweden

Background

Among senior drivers, benzodiazepines (BZDs) have a documented effect on the risk of road traffic crashes (RTCs). It remains unclear however if BZDs play the same role when considering marital status. Therefore, we aimed to investigate the role of marital status in the association between BZD use and injurious RTCs among senior drivers.

Methods

Matched case-control study based on five national Swedish registers (n = 154 225). Cases comprised the first non-alcohol-related injurious RTC sustained by drivers aged 50–80 years from July 2005 to December 2009 and controls included registered residents with a valid license who did not crash during that period. Four controls were matched to each case by sex, age and place of residence. Conditional logistic regression analysis for injurious RTC was performed with adjustment for occupation and number of medications. The main exposure was dispensation of BZDs, alone or in combination with other psychotropic medications, 1–30 days prior to the crash date stratified by marital status.

Results

BZD use, alone or in combination with other psychotropic medications, increased the risk of being involved in an RTC (BZD only: adjusted OR: 1.26, 95% CI: 1.17–1.36; BZDs and other psychotropics: adjusted OR: 1.25, 95% CI: 1.12–1.41). Compared to married drivers, those divorced (1.48, 1.43–1.53) and widowed (1.54; 1.45–1.63) had higher adjusted ORs. Marital status modified the association between BZDs and RTCs, particularly among younger male drivers.

Conclusions

Both BZDs and marital status independently affect the risk for senior drivers to be involved in an RTC. However, marital status plays a role in the association between BZD use and RTCs and this may have implications for targeting risk populations for RTCs among senior drivers.     

Diazepam binding inhibitor (DBI) reduces testosterone and estradiol levels in vivo

Diazepam binding inhibitor (DBI) is a putative endogenous ligand capable of binding to the central type benzodiazepine (BZD) receptor located on the GABAA receptor and the peripheral type BZD receptor on the mitochondrial outer membrane. We examined the effects of an intracerebroventricular injection of DBI on the serum levels of the gonadal hormones, testosterone and estradiol, respectively, in male and female mice. DBI (0.3-10 nmol/mouse, i.c.v.) significantly reduced the levels of both gonadal hormones in a dose-dependent manner. The decrease in the gonadal hormone levels became evident at 1 hr and lasted for at least 4 hrs after the DBI injection. The effects of DBI (3 nmol/mouse, i.c.v.) in male and female mice were completely attenuated by the coadministration of flumazenil (66 nmol/mouse), a selective antagonist for the central type BZD receptor. These results suggest that DBI acts as an endogenous modulator to regulate the levels of gonadal hormones in vivo, and that the DBI-induced decrease in gonadal hormone levels is mediated by down regulation of the GABAergic system, implicated in gonadotropin-releasing systems and/or the hypothalamic-pituitary-gonadal axis.     

Involvement of diazepam binding inhibitor and its fragment octadecaneuropeptide in social isolation stress-induced decrease in pentobarbital sleep in mice.

Diazepam binding inhibitor (DBI) and its fragment, octadecaneuropeptide (ODN), are putative endogenous ligands for benzodiazepine (BZD) receptors and have been shown to act as an inverse BZD receptor agonist in the brain. A previous study suggested that the social isolation stress-induced decrease in pentobarbital sleep in mice was partly due to endogenous substances with an inverse BZD receptor agonist-like property. In this study, we examined the effects of DBI and ODN on pentobarbital sleep in group-housed and socially isolated mice to test the possible involvement of DBI and ODN in a social isolation-induced decrease in pentobarbital sleep. The socially isolated mice showed significantly shorter durations of pentobarbital (50 mg/kg, intraperitoneally, i. p.) sleep compared to the group-housed animals. When injected intracerebroventricularly (i.c.v.), DBI and ODN (3 and 10 nmol) dose-dependently shortened the pentobarbital-induced sleeping time in group-housed mice at the same dose range, but these peptides had no effect on the sleeping time in socially isolated animals. In contrast, flumazenil (16.5-33 nmol, i.c.v.), a BZD receptor antagonist, reversed the pentobarbital sleeping time in socially isolated mice to the level of group-housed animals without affecting the sleeping time in group-housed animals. The effects of DBI and ODN in group-housed mice were significantly blocked by flumazenil (33 nmol, i.c.v.). Moreover, the effect of flumazenil in socially isolated mice was significantly attenuated by DBI and ODN (10 nmol, i.c.v.). These results suggest that the changes in the activity of DBI and/or ODN are partly involved in the social isolation-induced decrease in the hypnotic action of pentobarbital in mice.     

Drug transport mechanisms in helminth parasites: passive diffusion of benzimidazole anthelmintics

Anthelmintic molecules must reach their receptors inside target parasites to exert the pharmacological effect. Available data suggest that the main route of entry of antiparasitic drugs into helminth parasites would be through their external surface. However, it is unclear if trans-tegumental/cuticular penetration is the most important way of entry of benzimidazole (BZD) anthelmintics into their target parasites compared to oral ingestion. The relative involvement of active and passive transport mechanisms has not been defined. The goal of the work reported here was to determine the main processes involved in the entry of BZD anthelmintic molecules into the three main classes of helminth parasites. Adult specimens of Moniezia benedeni (cestode), Fasciola hepatica (trematode) and Ascaris suum (nematode) were incubated in Kreb's Ringer Tris buffer (pH 7.4, 37 degrees C) (1g parasite/10 ml incubation medium) for 15, 45, and 90 min, respectively, in the presence of a concentration gradient of either fenbendazole (FBZ), oxfendazole or triclabendazole sulphoxide (TCBZSO) (1-30 mol/ml, n=4). Dead helminth specimens were also incubated with the same drug concentration gradient. Specimens of F. hepatica with the oral route closed off by ligation were incubated with TCBZSO in the presence or absence of bovine serum albumin. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography analysis to measure drug/metabolite concentrations. Equivalent drug concentrations were measured within ligated and non-ligated liver flukes, demonstrating that BZD do mainly penetrate by trans-tegumental diffusion. The higher the concentration of BZD molecules in the incubation medium, the greater their concentration recovered within the helminth parasites. High correlation coefficients (>0.98) were obtained between initial drug concentration in the incubation medium and those measured inside the nematode, cestode, and trematode parasites. FBZ concentrations recovered from tissues of dead cestodes/nematodes over time were significantly greater compared to those measured in living parasites. These differences in drug diffusion may be related to the morphological/functional properties of the parasite's external surfaces. The outcome of the work reported here indicates that passive drug transfer through the external helminth surface is the main transport mechanism accounting for BZD accumulation into target parasites.     

Benzodiazepine receptor sites in the chick optic lobe: Development and pharmacological characterization

To investigate the, interaction between -aminobutyric acid (GABA) and benzodiazepine (BZD) receptor sites during development, the time-course of appearance of flunitrazepam (FNZ) binding sites and their pharmacological characterization were studied in developing chick optic lobe. At the earliest stage examined, embryonic day (Ed) 12, the receptor density was 30.9 % (0.05±0.01 pmol/mg protein) of that found in the chick optic lobes of adult chicks. The adult value was achieved on Ed 16 (0.16±0.01 pmol/mg protein). After this stage there was a sharp and transient increase in specific [³H]FNZ binding of about two-fold reaching a maximal value between hatching and the postnatal day (pnd) 2 (0.33±0.01 pmol/mg protein). Scatchard analysis at different stages of development revealed the presence of a single population of specific FNZ binding sites. The increase in [³H]FNZ binding during development was due to a large number of binding sites while their affinity remained unchanged. Competition experiments in the chick optic lobe revealed that the order of potency for displacement of specific [³H]FNZ binding paralleled the pharmacological potency of the BZDs tested. The IC₅₀ s for clonazepam, flunitrazepam, Ro 15-1788 and chlordiazepoxide were 3.02, 4.30, 0.32, and 4778.64 nM respectively. Ro 5-4864, a potent inhibitor of BZD binding to peripheral tissues, had no effect on specific [³H]FNZ binding indicating that only central BZD binding sites are present in the chick optic lobe. The peak of maximal expression of BZD receptor sites precedes in 5–6 days the peak of GABA receptor sites indicating a precocious development of BZD receptor sites. The different appearance of both peaks may represent important events during development probably related to synaptogenesis.     

A novel pyrazoloquinoline that interacts with brain benzodiazepine receptors: characterization of some in vitro and in vivo properties of CGS 9896

The novel pyrazoloquinoline, CGS, 9896, was a potent inhibitor of specific [3H]-flunitrazepam binding in several brain regions with subnanomolar KI values. The inhibition of [3H] propyl beta-carboline-3-carboxylate ([3H]-PCC-) binding by CGS 9896 was enhanced by gamma-aminobutyric acid (GABA) but not by chloride ion. GABA enhancement of CGS 9896 inhibition of [3H]-PCC binding predicts this compound has benzodiazepine (BZD) agonist-type activity. Behavioral studies support this prediction. CGS 9896 was found to protect mice against bicuculline and metrazol induced seizure at doses that did not induce ataxia or sedation. CGS 9896 may represent a class of compounds with potential therapeutic value. The high affinity of this non-BZD compound suggests that CGS 9896 may also be of value as a high affinity ligand for the continued study of BZD receptors.     

Evidence that the Peripheral-Type Benzodiazepine Receptor Ligand Ro 5–4864 Inhibits β-Endorphin Release from AtT-20 Cells by Blockade of Voltage-Dependent Calcium Channels

The demonstrations that Ro 5-4864, a ligand selective for the peripheral-type benzodiazepine (BZD) binding site, inhibited cellular differentiation and proliferation and that occupancy of the peripheral-type BZD binding site likely mediated the observed BZD effects on diverse endocrine tissues suggested that Ro 5-4864 disrupted a common cellular regulatory event. Using a well-characterized anterior pituitary-derived tumor cell line (AtT-20 cells), which synthesizes and secretes adrenocorticotropic hormone (ACTH), beta-lipotropin hormone (beta-LPH), and beta-endorphin (BE), we have investigated the molecular mechanism of action of Ro 5-4864's capacity to alter BE secretion. Ro 5-4864 inhibits basal and induced BE release from AtT-20 cells, through a cyclic AMP-independent mechanism. Ro 5-4864 completely blocked the corticotropin-releasing hormone and forskolin-induced release of BE without altering the concomitant production of cyclic AMP. The addition to AtT-20 cells of CGP 28392, a dihydropyridine that has been demonstrated in other systems to specifically activate voltage-dependent Ca2+ channels, resulted in a cyclic AMP-independent, dose-related increase in BE secretion. This CGP-induced BE release was blocked by increasing concentrations of Ro 5-4864. In contrast to the capacity of Ro 5-4864 to block CGP-induced BE release, Ro 5-4864 lacked the capacity to block enhanced BE secretion due to the calcium ionophore A23187, which increases intracellular Ca2+ levels independent of the voltage-dependent Ca2+ channels. Our findings suggest that Ro 5-4864 inhibits BE secretion from AtT-20 cells through a blockade of the voltage-dependent membrane Ca2+ channels and this mechanism of action may be responsible for Ro 5-4864's diverse effects observed on other cell types.