Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles

The effects of 1,4-diazepines with two annelated heterocycles [brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008] on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA (30 microM)-stimulated 36Cl- influx (146.1% of control), while ciclotizolam and WE 1008 showed only a small enhancement (119.3% and 119.1%, respectively) of GABA-stimulated 36Cl- uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA (10 microM), while at higher concentrations of GABA (1 mM), brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated 36Cl- uptake by brotizolam (0.1 microM) was antagonized by Ro 15-1788. At higher concentration of GABA (300 microM), brotizolam inhibited GABA-stimulated 36Cl- uptake in a dose dependent manner and Ro15-1788 failed to antagonize this effect. These results suggest that 1) brotizolam produces an enhancement of GABA (30 microM)-stimulated chloride influx through the benzodiazepine receptor. 2) brotizolam inhibition of GABA (300 microM)-stimulated chloride influx involves an additional mechanism, and 3) the sedative-hypnotic action of brotizolam may be related to its high efficacy at the benzodiazepine/GABA-gated chloride channel.     

The effect of benzodiazepines and beta-carbolines on GABA-stimulated chloride influx by membrane vesicles from the rat cerebral cortex

Benzodiazepine agonists such as diazepam, flunitrazepam and clonazepam enhanced GABA (30 microM)-stimulated 36Cl- uptake in membrane vesicles from the rat cerebral cortex. The rank order of potencies was flunitrazepam greater than diazepam = clonazepam. beta-Carboline-3-carboxylate esters beta-CCM, beta-CCE and DMCM inhibited GABA-stimulated 36Cl- uptake. The rank order of inhibitory potencies was DMCM greater than beta-CCM greater than beta-CCE. The benzodiazepine antagonist Ro15-1788 antagonized the enhancement of flunitrazepam and the inhibition of DMCM on GABA-stimulated 36Cl- uptake in a competitive inhibitory manner. These results suggest that benzodiazepine receptors regulate GABA-stimulated 36Cl- uptake and there is a functional coupling between the GABA and benzodiazepine receptors, and chloride channels in membrane vesicles from the rat cerebral cortex.     

Amoxapine inhibition of GABA-stimulated chloride conductance: investigations of potential sites of activity

Amoxapine inhibits GABA-stimulated chloride conductance by acting on the GABAA-receptor chloride-ionophore complex which can be studied using membrane vesicles prepared from rat cerebral cortex. Amoxapine produces a right shift in the GABA concentration-response curve for the stimulation of 36Cl- uptake into these vesicles with no apparent change in the maximum response. Schild analysis of these data gave a pA2 value of 5.52 with a slope of 0.79. Amoxapine inhibits the binding of the GABAA receptor selective antagonist [3H]SR 95531 with an IC50 value of 3.45 microM and a pseudo Hill coefficient of 0.83. In contrast, 10 microM amoxapine inhibits [3H]flunitrazepam binding by less than 25% while the benzodiazepine antagonist Ro 15-1788 reduces the amoxapine inhibition of GABA-stimulated chloride conductance only at high concentrations. These data suggest that amoxapine does not inhibit chloride conductance by acting as a benzodiazepine inverse agonist and either acts directly on the GABAA receptor as an antagonist or blocks GABA activity at a site closely coupled to it. The ability of amoxapine to inhibit GABA-stimulated chloride conductance is a likely explanation for its proconvulsant activity observed at high doses.     

Heterotropic Cooperativity Between Putative Recognition Sites for Progesterone Metabolites and the Atypical Benzodiazepine Ro 5–4864

The binding of the cage convulsant t-butylbicyclophosphorothionate (TBPS) and 36Cl- uptake by synaptoneurosomes were used to test the ability of progesterone metabolites to modulate allosterically the Ro 5-4864 (4'-chlorodiazepam) binding site that is functionally coupled to the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex (GBRC) in rat brain. Dose-dependent enhancement of [35S]TBPS binding by Ro 5-4864 occurs in rat cerebral cortex in the presence of the progesterone metabolites 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha, 20 alpha-diol (pregnanediol). The pregnanediol effect is completely GABA dependent, whereas that of 3 alpha-OH-DHP is not. Conversely, Ro 5-4864 opposed the action of 3 alpha-OH-DHP by increasing the IC50 for 3 alpha-OH-DHP inhibition of [35S]TBPS binding. In cortical synaptoneurosomes, Ro 5-4864 antagonized both 3 alpha-OH-DHP and pregnanediol enhancement of GABA-stimulated 36Cl- uptake. In both binding and functional studies, pregnanediol showed limited efficacy relative to 3 alpha-OH-DHP, as previously reported. These findings provide the initial evidence that the GBRC-linked Ro 5-4864 binding site is allosterically coupled to the putative progesterone metabolite recognition site and confirm the GABA-mimetic properties of 3 alpha-OH-DHP and pregnanediol.     

Benzodiazepine Enhancement of γ-Aminobutyric Acid-Mediated Chloride Ion Flux in Rat Brain Synaptoneurosomes

Benzodiazepine agonists such as Ro 11-6896 [B10(+)], diazepam, clonazepam, and flurazepam were found to enhance muscimol-stimulated 36Cl- uptake into rat cerebral cortical synaptoneurosomes. The rank order of potentiation was B10(+) greater than diazepam greater than clonazepam greater than flurazepam. These benzodiazepines had no effect on 36Cl-uptake in the absence of muscimol. Further, the inactive enantiomer, Ro 11-6893 [B10(-)], and the peripheral benzodiazepine receptor ligand Ro 5-4864 did not potentiate muscimol-stimulated 36Cl- uptake at concentrations up to 10 microM. In contrast, the benzodiazepine receptor inverse agonists ethyl-beta-carboline-3-carboxylate and 6,7-dimethoxy-4-ethyl-beta- carboline-3-carboxylic acid methyl ester inhibited muscimol stimulated 36Cl- uptake. Benzodiazepines and beta-carbolines altered the apparent K0.5 of muscimol-stimulated 36Cl- uptake, without affecting the Vmax. The effects of both benzodiazepine receptor agonists and inverse agonists were reversed by the benzodiazepine antagonists Ro 15-1788 and CGS-8216. These data further confirm that central benzodiazepine receptors modulate the capacity of gamma-aminobutyric acid receptor agonists to enhance chloride transport and provide a biochemical technique for studying benzodiazepine receptor function in vitro.     

Triazolam, an Anomalous Benzodiazepine Receptor Ligand: In Vitro Characterization of Alprazolam and Triazolam Binding

Both alprazolam and triazolam displaced clonazepam (but not Ro 5-4864) from rat brain membranes with high affinity, showing them to act at central but not peripheral benzodiazepine receptors. At 0 degrees C, 10 microM gamma-aminobutyric acid (GABA) increased the ability of alprazolam, but not of triazolam, to displace ethyl-beta-carboline-3-carboxylate (beta-CCE) and Ro 15-1788 from these receptors. At 37 degrees C, GABA increased the affinity of the receptors for both drugs, with a +GABA/-GABA ratio of 1.5 for each in promoting Ro 15-1788 binding displacement. As both triazolam and alprazolam act as anxiolytics in vivo, the results at 37 degrees C would be compatible with the hypothesis that GABA causes an increase in affinity of drugs that act in this way, but the results at 0 degrees C would not be compatible. At 37 degrees C, alprazolam had a higher IC50 for the benzodiazepine receptor than at 0 degrees C, whereas triazolam showed the reverse effect. The relative IC50 values in vitro at 37 degrees C correlated better with the potency in vivo than those obtained at 0 degrees C. At 0 degrees C, both drugs showed Hill plots with slopes of 0.9-1 with beta-CCE and Ro 15-1788. At 37 degrees C, the slopes with triazolam were much reduced, indicating that the drug may have a selective action on a subclass of central benzodiazepine receptors. In the studies reported here, alprazolam behaved like other benzodiazepines, whereas triazolam showed several anomalous properties. It would be of interest if these properties could be related either to the drug's use as a hypnotic or to the side effects it sometimes induces.     

3H]Ro 15-1788 binding sites to brain membrane of the saltwater Mugil cephalus

The equilibrium binding parameters of the benzodiazepine antagonist [3H]Ro 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a]-1,4 benzodiazepine) were evaluated in brain membranes of the saltwater teleost fish, Mugil cephalus. To test receptor subtype specificity, displacement studies were carried out by competitive binding of [3H]Ro 15-1788 against six benzodiazepine receptor ligands, flunitrazepam [5-(2-fluoro-phenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one], alpidem [N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-acetamide], zolpidem [N,N-6 trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate], and beta-CCM (methyl beta-carboline-3-carboxylate). Saturation studies showed that [3H]Ro 15-1788 bound saturatably, reversibly and with a high affinity to a single class of binding sites (Kd value of 1.18-1.5 nM and Bmax values of 124-1671 fmol/mg of protein, depending on brain regions). The highest concentration of benzodiazepine recognition sites labeled with [3H]Ro 15-1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. The rank order of displacement efficacy of unlabelled ligands observed suggested that central-type benzodiazepine receptors are present in one class of binding sites (Type I-like) in brain membranes of Mugil cephalus. Moreover, the uptake of 36Cl- into M. cephalus brain membrane vesicles was only marginally stimulated by concentrations of GABA that significantly enhanced the 36Cl- uptake into mammalian brain membrane vesicles. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.     

Benzodiazepine antagonist Ro 15-1788: binding characteristics and interaction with drug-induced changes in dopamine turnover and cerebellar cGMP levels

The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50 = 2.3 +/- 0.6 nmol/liter). [3H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation (KD) of 1.0 +/- 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15-1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels

Abstract: The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [³H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [³H]diazepam binding in vitro (IC₅₀= 2.3 ± 0.6 nmol/liter). [³H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant ( K _D) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [³H]Ro 15-1788 from its binding site was of the same rank order as found previously in [³H]diazepam binding. Autoradiograms of [³H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [³H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Differences in the mechanism of the antihypoxic action of benzodiazepine receptor agonists and muscimol

Neuropharmacological analysis of previously revealed antihypoxic activity of benzodiazepines (BDZ) has been performed in experiments on mice exposed to hypoxia. Antihypoxic effect of diazepam is shown to be antagonized by the central BDZ receptor blocker, Ro 15-1788. A certain degree of antihypoxic activity also abolished by Ro 15-1788 is exhibited by hypothetical ligands of BDZ receptors: inosin, nicotinamide, ethyl-beta-carboline-3-carboxylate. The effect of dipyridamole, a drug with high affinity for BDZ receptors of the peripheral type is not antagonized by Ro 15-1788, another evidence of Ro 15-1788 affinity precisely to the central BDZ receptors. GABA-mimetics (muscimol and GABA cetyl ester) were also found to have marked antihypoxic activity. Unlike BDZ receptor agonists, this effect is reduced by bicuculline and not by Ro 15-1788. The data obtained suggest that antihypoxic activity of BDZ is caused by their direct interaction with the central BDZ receptors, probably with the type which is not modulated by GABAA receptors.     

Prolonged exposure to GABA activates GABA-gated chloride channels in the presence of channel-blocking convulsants.

1. In assays of 36Cl- uptake into mouse brain vesicles, 100 microM GABA markedly increased both the initial rate of 36Cl- uptake and the total amount of chloride taken up over a 120-sec incubation period. Specific GABA-dependent 36Cl- uptake (the difference between total and background uptake) was essentially complete within 15 sec of incubation. 2. Incubation with GABA following preincubation with 10 microM endrin, a polychlorocycloalkane insecticide and established blocker of GABA-gated chloride channels, showed a stimulation of uptake over background levels that was much slower in onset than that observed with GABA alone but nevertheless achieved virtually the same level of stimulation above background levels after 90 sec of incubation with GABA. 3. In electrophysiological assays of GABA receptors expressed in Xenopus oocytes following injection with rat brain mRNA, endrin (20 microM) effectively blocked the transient currents elicited by brief exposure of oocytes to GABA (200 microM). However, prolonged exposure to GABA in the absence of perfusion produced a large, slowly-developing inward current. 4. The actions of several known GABA antagonists were also compared as inhibitors of GABA-dependent 36Cl- uptake into mouse brain vesicles at short (4 sec) and long (120 sec) incubation times using concentrations of inhibitors known to produce approximately 70-90% inhibition of GABA-dependent chloride uptake in 4-sec incubations. Picrotoxinin and TBPS, like endrin, were completely ineffective as inhibitors in 120-sec incubations. In contrast, bicuculline was almost as effective at 120 sec as at 4 sec, and avermectin Bla produced approximately 50% inhibition of the GABA response after 120 sec.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of benzodiazepine receptors in the discriminative stimulus properties of delta-9-tetrahydrocannabinol

Twenty male Sprague-Dawley rats were trained to discriminate 3.0 mg/kg delta-9-tetrahydrocannabinol (THC) from its vehicle. Following acquisition of this discrimination animals were tested for generalization to 3.0 mg/kg diazepam. Thirteen animals showed a generalization from THC to diazepam, whereas the remaining seven animals did not. The generalization curve for diazepam was dose-dependent from 0.1 to 10.0 mg/kg in the first group; the latter group showed no generalization from THC at any dose of diazepam in this range. No differences were found between these groups in the generalization curve for THC. The benzodiazepine antagonist Ro 15-1788 (2.0 mg/kg) antagonized the generalization to diazepam in the group that discriminated diazepam as THC. In contrast, Ro 15-1788 increased THC lever responding of 10 mg/kg diazepam in the group which did not generalize from THC. Ro 15-1788 did not alter the discriminability of THC in either group. THC also showed partial generalization to pentobarbital (1 to 10 mg/kg). The generalization was again complete in one subgroup and absent in another, but there was only a 43 percent overlap between the subgroups found with testing for generalization to diazepam. The percent THC lever responding with 3.0 mg/kg pentobarbital was increased by Ro 15-1788 in the group which generalized to diazepam, but not the other group. These data suggest that the discriminative stimulus properties of THC may have some commonality with the effects of diazepam in a subpopulation of rats trained to discriminate THC. These THC-like effects of diazepam are probably mediated by benzodiazepine receptors since they are antagonized by a specific benzodiazepine receptor antagonist.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Reversal of the antinociceptive effects of centrally-administered morphine by the benzodiazepine receptor antagonist Ro 15-1788

The effects of the benzodiazepine receptor antagonist, Ro 15-1788, were examined on analgesia induced by morphine after central (intracerebroventricular, i.c.v., or intrathecal, i.t.) and systemic administration. Analgesia was assessed in squirrel monkeys trained to respond under an electric shock tiltration procedure and in mice using the radiant heat tail-flick test. Central and systemic administration of morphine produced antinociceptive effects that were antagonized by 0.1 mg/kg of naloxone in both species. Ro 15-1788 antagonized the effects of morphine after central (i.c.v. or i.t.) administration but did not alter the effects of morphine given by the systemic route. This novel interaction suggests that Ro 15-1788 may be useful in pharmacologically separating neural substrates subserving opiate analgesia.     

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Attenuating effect of diazepam on stress-induced increases in noradrenaline turnover in specific brain regions of rats: antagonism by Ro 15-1788

One-hour immobilization stress increased levels of the major metabolite of brain noradrenaline (NA), 3-methoxy-4-hydroxyphenyl-ethyleneglycol sulfate (MHPG-SO4), in nine brain regions of rats. Diazepam at 5 mg/kg attenuated the stress-induced increases in MHPG-SO4 levels in the hypothalamus, amygdala, hippocampus, cerebral cortex and locus coeruleus (LC) region, but not in the thalamus, pons plus medulla oblongata excluding the LC region and basal ganglia. The attenuating effects of the drug on stress-induced increases in metabolite levels in the above regions were completely antagonized by pretreatment with Ro 15-1788 at 5 or 10 mg/kg, a potent and specific benzodiazepine (BDZ) receptor antagonist. When given alone, Ro 15-1788 did not affect the increases in MHPG-SO4 levels. Behavioral changes observed during immobilization stress such as vocalization and defecation, were also attenuated by diazepam at 5 mg/kg and this action of diazepam was antagonized by Ro 15-1788 at 10 mg/kg, which by itself had no effects on these behavioral measurements. These findings suggest: (1) that diazepam acts via BDZ receptors to attenuate stress-induced increases in NA turnover selectively in the hypothalamus, amygdala, hippocampus, cerebral cortex and LC region and (2) that this decreased noradrenergic activity might be closely related to relief of distress-evoked hyperemotionality, i.e., fear and/or anxiety in animals.     

GABA-stimulated 36Cl- influx into reconstituted vesicles with purified GABAA/benzodiazepine receptor complex

Solubilized and Purified gamma-aminobutyric acid (GABA)A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl- influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl- influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABAA/benzodiazepine receptor complex and Cl- channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl- channel also exhibited GABA-stimulated 36Cl- influx. This GABA-stimulated influx of 36Cl- was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABAA receptor antagonist. These results strongly suggest that GABAA receptor is directly coupled with Cl- channel, whereas benzodiazepine receptor may be functionally coupled with GABAA receptor and modulates the GABA-stimulated Cl- influx through GABAA receptor. The present results also indicate that the purified GABAA receptor complex is coupled with Cl- channel and possesses functional characteristics as GABAA receptor.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Benzodiazepine Receptor Binding of Triazolobenzodiazepines In Vivo: Increased Receptor Number with Low-Dose Alprazolam

Abstract: Triazolobenzodiazepines are in clinical use as hypnotics and anxiolytics. We analyzed in vivo receptor binding and brain concentrations of alprazolam, triazolam, and estazolam. Drug concentrations measured in the cerebral cortex 1 h after administration were directly proportional to dose for all three compounds. In vivo receptor binding, as defined by the specific uptake of [³H]Ro 15–1788, decreased with increasing doses of estazolam and triazolam, a finding indicating dose-related increases in receptor occupancy due to these compounds. Triazolam was substantially more potent, with an IC₅₀ value of 16 ng/g, compared with 117 ng/g for estazolam. At higher doses of alprazolam (>0.2 mg/kg), receptor binding by [³H]Ro 15–1788 likewise decreased with increasing dose of the former drug. However, at lower doses of alprazolam (0.02–0.05 mg/kg), which resulted in cortex concentrations of 2–7 ng/g, receptor binding was increased above control values in cortex, hypothalamus, and hippocampus but not in several other brain regions. Binding returned to control values at doses of ≤0.01 mg/kg. Similar results were obtained in time course studies. At 8 and 10 h after a dose of 1 mg/kg i.p., corresponding to cortex concentrations of 2.7–7 ng/g, receptor binding was increased compared with controls. Similarly, at 1, 2, and 3 h after a single dose of 0.05 mg/kg, corresponding to cortex concentrations of 3.7–5.8 ng/g, receptor binding was also increased. The apparent affinity of benzodiazepine receptors for clonazepam in mice receiving alprazolam (0.05 mg/kg) was unchanged from that in untreated control mice, an observation suggesting that low doses of alprazolam increased receptor number. The brain concentration vs. receptor occupancy relationships for triazolam and estazolam resemble those for other benzodiaze-pines, but alprazolam appears to be anomalous in that low brain concentrations increase benzodiazepine receptor number.     

Ultrasonic vocalization in response to unavoidable aversive stimuli in rats: effects of benzodiazepines

The effects of two benzodiazepine derivatives (diazepam, 0.5-1 mg/kg; alprazolam, 1.25-2.5 mg/kg) on ultrasonic calling elicited in adult rats by unavoidable aversive stimuli (footshocks) were investigated. The results show that either diazepam or alprazolam affected the duration of ultrasonic calls. In particular, a significant decrease in the length of ultrasounds was found in the group of animals treated with these benzodiazepines. The effects of diazepam were counteracted by the benzodiazepine-antagonist Ro 15-1788. On the other hand, neither a neuroleptic agent, such as haloperidol (0.5-1 mg/kg), nor an antidepressant, such as desipramine (5-10 mg/kg) influenced the parameters of ultrasonic emission in this experimental situation. The present results suggest that ultrasonic vocalization in response to unavoidable aversive stimuli could be considered as a potential new tool for studying drugs with antianxiety properties.