Benzodiazepine Receptor Sites Research Articles

Antagonistic action of pitrazepin on human and rat GABA(A) receptors.

Pitrazepin, 3-(piperazinyl-1)-9H-dibenz(c,f) triazolo(4,5-a)azepin is a piperazine antagonist of GABA in a variety of electrophysiological and in vitro binding studies involving GABA and glycine receptors. In the present study we have investigated the effects of pitrazepin, and the GABA(A) antagonist bicuculline, on membrane currents elicited by GABA in Xenopus oocytes injected with rat cerebral cortex mRNA or cDNAs encoding alpha1beta2 or alpha1beta2gamma2s human GABA(A) receptor subunits. The three types of GABA(A) receptors expressed were reversibly antagonized by bicuculline and pitrazepin in a concentration-dependent manner. GABA dose-current response curves for the three types of receptors were shifted to the right, in a parallel manner, by increasing concentrations of pitrazepin. Schild analyses gave pA2 values of 6.42+/-0.62, n = 4, 6.41+/-1.2, n = 5 and 6.21+/-1.24, n = 6, in oocytes expressing rat cerebral cortex, alpha1beta2 or alpha1beta2gamma2s human GABA(A) receptors respectively (values are given as means +/- s.e. mean), and the Hill coefficients were all close to unity. All this is consistent with the notion that pitrazepin acts as a competitive antagonist of these GABA(A) receptors; and that their antagonism by pitrazepin is not strongly dependent on the subunit composition of the receptors here studied. Since pitrazepin has been reported to act also at the benzodiazepine binding site, we studied the effect of the benzodiazepine antagonist Ro 15-1788 (flumazenil) on the inhibition of alpha1beta2gamma2s receptors by pitrazepin. Co-application of Ro 15-1788 did not alter the inhibiting effect of pitrazepin. Moreover, pitrazepin did not antagonize the potentiation of GABA-currents by flunitrazepam. All this suggests that pitrazepin does not affect the GABA receptor-chloride channel by interacting with the benzodiazepine receptor site.

Modulation of Long-term Potentiation in CA1 Region of Mouse Hippocampal Brain Slices by GABA A Receptor Benzodiazepine Site Ligands

Enhancement of GABA A receptor function with benzodiazepine (BZ) site agonists can disrupt memory formation and hippocampal synaptic plasticity. To investigate this further the effects of the agonist, flunitrazepam, were contrasted with that of the inverse agonist, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), on NMDA-dependent LTP induction in the CA1 region of mouse hippocampus. Under control conditions, a priming stimulus (10 stimuli at 100 Hz) potentiated e.p.s.p. slopes by 198%, and subsequent burst stimuli (4 × 10 events at 100 Hz every 20 sec) by 306%. This potentiation was blocked by the non-competitive NMDA receptor antagonist MK-801 and the glycine site antagonist L-701,324. Flunitrazepam (1 μM) alone caused a slight but significant reduction in e.p.s.p.s to 83% of control, suppressed LTP induced by priming stimuli (133%) and burst stimuli (188%), but not that induced by sustained high-frequency stimulation (2 × 100 events at 100 Hz, 20 sec apart). The suppression of LTP induction by flunitrazepam was blocked by the benzodiazepine site antagonist flumazenil. In contrast, the inverse agonist DMCM (100 nM) potentiated LTP formed by both priming (to 283%) and burst stimuli (to 477%). This was associated with an enhancement of paired pulse facilitation during the induction phase and the subsequent appearance of paroxysmal burst discharges. Therefore, in addition to improvements in learning and memory as a result of improved vigilance, benzodiazepine inverse agonists can have direct effects on synaptic processes thought to contribute to memory formation. © Elsevier Science Ltd.

NEUROSTEROID MODULATION OF THE BENZODIAZEPINE RECEPTOR IN DEVELOPING CHICK OPTIC LOBE

The modulation of [ 3H]flunitrazepam binding by 3α-hydroxy-5α-dihydroprogesterone (3α-OH-DHP) and 3α,5α-tetrahydrodeoxicorticosterone (3α-THDOC) was investigated in synaptic membranes isolated from chick optic lobe at three developmental stages, in order to evaluate the role of neurosteroids in central nervous system functional maturation. It was demonstrated that both steroids modulate [ 3H]flunitrazepam binding in a concentration-dependent manner at embryonic day 14, hatching and adult stage, producing maximal [ 3H]flunitrazepam binding enhancement at early stages of development and declining thereafter. EC 50 values for 3α-OH-DHP were lower than for 3α-THDOC at all stages examined. On the other hand, E max values were higher with 3α-OH-DHP than with 3α-THDOC. Scatchard analysis performed at embryonic day 14, hatching and adult stage showed that dissociation constants (K d) were of 22.23±0.15, 18.38±1.15 and 19.86±0.62 nM, and maximal number of binding sites (B max) were 1.95±0.15, 3.13±0.21 and 2.25±0.13 pmol/mg protein, respectively. By adding 4 μM of either 3α-OH-DHP or 3α-THDOC, K d values decreased significantly to 10.65±0.62, 9.71±0.85 and 13.25±0.74 nM or 9.54±0.65, 11.20±1.27 and 12.96±1.38 nM, at the above mentioned stages, respectively. Thus, either drug at the given concentration increased the affinity of benzodiazepine receptor sites for [ 3H]flunitrazepam, while the density of receptor sites remained unchanged. Our results suggest that these steroids display a positive allosteric modulation of benzodiazepine receptor sites which is inversely related to age.

Initial human studies with single-photon emission tomography using iodine-123 labelled 3-(5-cyclopropyl-1,2,4-oxadiazo-3-yl)- 7-iodo-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]-benzodiazepine (NNC 13-8241).

The iodine-123 labelled ligand 3-(5-cyclopropyl-1,2, 4-oxadiazo-3-yl)-7-iodo-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a][1,4]-benzodiazepine ([123I]NNC 13-8241) was evaluated as a probe for in vivo imaging of benzodiazepine receptor sites in the human brain. Four healthy volunteers were imaged with a high-resolution single-photon emission tomography (SPET) scanner. The metabolism of [123I]NNC 13-8241 in plasma was slow. The total brain uptake was about 1.5-fold higher than that of [123I]iomazenil. The specific binding in the cortical areas was high and less intense in the thalamus. The most intense uptake was seen in the occipital cortex. The peak cortical uptake of [123I]NNC 13-8241 was observed 6-10 h after the injection of tracer. The radiation burden to the patient was moderate, being 2.5 middle dot10(-2 )mSv/MBq (effective dose equivalent). A slow metabolism together with favourable kinetics indicates that [123I]NNC 13-8241 is a specific and promising SPET ligand for imaging benzodiazepine receptor sites in the living human brain.

Receptor binding sites and uptake activities mediating GABA neurotransmission in chronic alcoholics with Wernicke encephalopathy

Superior frontal cortex (SFC) and primary motor cortex tissue was obtained at autopsy from thirteen severe chronic alcoholics with neuropathologically confirmed Wernicke Encephalopathy (WE) and 22 controls. Cases with both WE and cirrhosis showed markedly fewer neurones in SFC than did WE cases without cirrhosis. The extent of the apparent neuronal loss corresponded to an increase in post-synaptic GABA A receptor sites, as assessed by the binding of [ 3H]muscimol to synaptic membranes. Increased [ 3H]muscimol binding was not accompanied by an increase in ‘central-type’ benzodiazepine binding sites: as assessed by [ 3H]flunitrazepam binding, these sites were apparently unaltered, while as assessed by [ 3H]diazepam binding, they were decreased. The affinities of the two benzodiazepine ligands varied differently with disease. These discrepancies between [ 3H]flunitrazepam and [ 3H]diazepam binding could not be accounted for, either by the presence of a second, diazepam-preferring, ‘central-type’ benzodiazepine binding site, or by loss of ‘peripheral-type’ sites. The changes in the post-synaptic GABA A benzodiazepine receptor sites did not reflect any regional, disease-related deficit of afferent GABAergic terminals, as assessed by synaptosomal high-affinity [ 3H]GABA uptake. On a number of indices, it appears most likely that the data reflect both a loss of receptor sites, and a change in the population of receptor sub-types.

Characterization of a benzodiazepine receptor site with exceptionally high affinity for Ro 15-4513 in the rat CNS

The binding of [ 3H]Ro 15-4513, [ 3H]flunitrazepam and [ 3H[Fumazenil to rat CNS membranes was studied at 2°C, 22°C and 37°C using ligand concentrations ranging from ≈ 0.06 nM to 10 μM. Analysis of the binding saturation data suggested the existence of high-affinity sites ( K d < 10nM) and low-affinity sites ( K d > 100nM) for each ligand. When binding was performed using very low ligand concentrations a benzodiazepine site with an exceptionally high affinity for Ro 15-4513 ( K d ≈ 0.1nM) was evident in all major regions of the CNS except the cerebellum. This site was most prevalent in the hippocampus, medulla and spinal cord where it accounted for ≈ 70% of the specific binding when [ 3H]Ro 15-4513 was ≈ 0.06 nM. The selectivity of Ro 15-4513 for this site as compared to other high-affinity sites was 20- to 60-fold depending on the incubation temperature and CNS region. The affinity for the very high-affinity site was decreased ≈ 3-fold as temperature was increased from 2°C to 37°C ( K d ≈ 0.1nM and ≈ 0.3, respectively), which was similar to the effect of temperature on other high-affinity sites ( K d ≈ 2.6nM at 2°C and ≈ 8 nM at 37°C). Flumazenil, flunitrazepam, and diazepam did not differentiate the very high-affinity [ 3H]Ro 15-4513 site from other BZ sites, but alpidem exhibited a low affinity for it (IC 50 ≈ 5 μM). GABA at 100 μM had little effect on the K d value for the very high-affinity site (GABA shift: ≈ 0.8 to 1.0), suggesting that Ro 15-4513 is a partial inverse agonist or an antagonist at this site. These findings provide further evidence for the pharmacologic diversity of BZ sites on different subtypes of GABA A receptors.

Chronological study of peripheral benzodiazepine binding sites in the rat brain stab wounds using [3H] PK-11195 as a marker for gliosis.

Chronological studies of the development of the peripheral benzodiazepine receptor sites were undertaken with the goal of evaluating the sensitivity of this marker for the study of the gliosis development in the injured brain. No significant increase in [3H] PK-11195 binding occurred in the rat brain stab wound one day following the puncture. A significant increase in the receptor density (Bmax) from the second day onward was observed. The Bmax reached its highest levels in the grey matter on the sixth day after a 23-gauge needle wound (8.75 +/- 0.09; pmol mg-tissue-1) and on the seventh day after an 18-gauge needle wound (8.98 +/- 0.31 pmol mg-tissue-1). In the white matter, the Bmax was greatest seven days after the wound (3.42 +/- 0.07; pmol mg-tissue-1; 23-gauge needle and 3.56 +/- 0.1 pmol mg-tissue-1 in the 18-gauge needle injury). Between 30 and 60 days after the wound, the Bmax was significantly lower than the Bmax observed between 6 and 14 days. The Bmax in the wound produced with needles was seven to eight times greater than the Bmax in the grey matter of the ipsilateral and contralateral cortices. Histological examination showed that there were no astrocytes or macrophages in the stab wound one day after the lesion. However, the glial fibrillary acidic protein positive cells and macrophages appeared on D3 after an injury. Gliosis, as measured by the PK-11195 binding, was also observed in the remote contralateral cortex. Data shows that PK-11195 binding is a very sensitive method of evaluating brain injury and could be of great value in studying progressive injuries in the living human brain in conjunction with positron emission tomography.

Abecarnil in Generalised Anxiety Disorder

Abecarnil is a β-carboline partial benzodiazepine agonist which may be selective for certain subpopulations of benzodiazepine receptors. It has greater affinity for central benzodiazepine receptor sites than diazepam, but low affinity for peripheral benzodiazepine, adrenergic, dopaminergic, opiate and serotonergic receptors. At present, virtually all published pharmacodynamic data for abecarnil are derived from animals studies. Abecarnil is active in a number of animal models of anxiety and is generally more effective than diazepam. It also attenuated some, but not all, chemically- or electrically-induced seizures in animals (diazepam antagonised most types of induced seizures) and was protective in genetic models of epilepsy. In most instances, its anticonvulsant activity did not appear to be attenuated during long term administration. Although some sedation occurs with abecarnil administration to animals, the drug is associated with less sedation, muscle relaxation, ataxia and impairment of memory than are several benzodiazepines. It also produces less potentiation of the effects of ethanol and hexobarbital than diazepam. In animals, the withdrawal syndrome after abecarnil discontinuation or administration of the benzodiazepine antagonist flumazenil is generally reduced or absent compared with benzodiazepines, suggesting that abecarnil has a low dependence potential. Abecarnil appears to have less propensity to induce tolerance than diazepam; it may also have a reduced abuse liability compared with benzodiazepines, as suggested by limited evidence in animals. The pharmacokinetic profile of abecarnil has yet to be investigated in patients with generalised anxiety disorder. Although pharmacokinetic parameters showed great intra- and interindividual variability in volunteers, the mean oral bioavailability of abecarnil 5 or lOmg was 39% in older volunteers and 55 to 65% in younger volunteers, and maximum plasma concentrations and AUC values after a single dose or at steady-state were dose-proportional. Abecarnil is predominantly metabolised, with less than about 10% of the drug being excreted unchanged. The main metabolite is the glucuronide conjugate of abecarnil, although 4 metabolites have been identified. Only one clinical trial assessing the efficacy of abecarnil in the treatment of generalised anxiety disorder has been published in full. Low dosages of the drug (3 to 9 mg/day) were significantly more effective than placebo and produced few serious adverse effects with no significant withdrawal syndrome upon discontinuation of 3 weeks of treatment. However, higher dosages of abecarnil (15 to 30 mg/day, and to a lesser extent 7.5 to 15 mg/day) produced a high incidence of adverse CNS effects [including drowsiness (71 and 51%), a loss of equilibrium (26 and 11 %), confusion (24 and 3%), amnesia (15 and 3%), fatigue (15 and 17%), lethargy (18 and 3%), insomnia (15 and 6%) and dizziness (18 and 11%)] which caused discontinuation of treatment in 29% of patients receiving abecarnil 15 to 30 mg/day. Higher drug dosages were also associated with some withdrawal signs upon discontinuation of therapy. In addition, there appeared to be some loss of efficacy with all tested dosages of abecarnil in comparison with placebo during the third week of treatment. Other trials of abecarnil, published collectively in abstract form with little detail, have suggested that 4 to 6 weeks of treatment with the drug had efficacy similar to benzodiazepines and produced a similar number of adverse effects or withdrawal symptoms as placebo when discontinued in a tapered fashion. In conclusion, experimental evidence to date suggests that abecarnil may be a valuable alternative to currently used agents for the treatment of generalised anxiety disorder, particularly if efficacy is maintained at lower dosages. However, clinical trials, including comparisons with benzodiazepines, will first need to demonstrate the potential advantages (reduced adverse effects, dependence and abuse potential) observed with abecarnil in animal models.

Receptor binding in Japanese quail selected for long or short tonic immobility

Japanese quail, selectively bred for long (LTI) and short (STI) tonic immobility (TI) responses, are thought to represent high and low fear groups, respectively. To study the neurochemical mechanisms underlying the behavioral distinctions, binding parameters were determined at the benzodiazepine, 5-HT 1A, 5-HT 3, α 2, and opioid receptor sites in the forebrains of the two lines. No differences were found in 5-HT 1A, 5-HT 3, α 2, μ- or κ-opioid receptor binding between the lines. The K D for the binding of [ 3H]-flunitrazepa at the benzodiazepine receptor was significantly greater in the LTI than in the STI birds, indicating lower affinity for benzodiazepine ligands. The lines did not differ in benzodiazepine receptor number. Using [ 3H]-naltrindole, the LTI line was found to have fewer δ-opioid receptors than the STI line; the birds did not vary with respect to the affinity of these receptors. Thus, the selective breeding of the two lines has resulted in differences in benzodiazepine and δ-opioid binding, and these could produce differences in activity levels, fear, and pain responses, all of which could contribute to the tonic immobility response.

Molecular determinants of recognition and activation at the cerebellar benzodiazepine receptor site

Semiempirical quantum mechanical and molecular mechanics calculations were carried out to identify and characterize the steric and electronic properties that modulate ligand recognition and activation of the cerebellar GABA A/benzodiazepine (BDZ) receptor. For this hypothesis development, thirteen compounds belonging to structurally diverse chemical families were selected for study. Among the compounds selected were nine that bind and four that do not bind with appreciable affinity to this receptor and some that are known agonists, antagonists and inverse agonists, as measured by their modulation of GABA (γ-aminobutyric acid) enhanced chloride ion flux in cerebellum. The stereoelectronic requirements for recognition deduced from commonalities among the ligands are the presence of at least two of three hydrogen bonding centers, and a lipophilic aromatic ring, in a specific spatial relationship. The results suggest that the selectivity for the cerebellar or Type I subtype, demonstrated by some of these ligands, could be failure to meet the requirements for binding at other receptors because of the absence of one of the proton accepting centers or the larger surface area and volume of these ligands. The requirement for activation, deduced from comparisons of agonist, antagonist, and inverse agonist properties is the presence of an electron accepting aromatic ring in a specific geometric arrangement with respect to the components of recognition. The validity of the ‘3D-Pharmacophore’ developed was probed by using it for predictions of the behavior of 11 additional compounds not used for its development.

Hippocampal benzodiazepine receptors in schizophrenia.

Benzodiazepine receptor sites were determined by saturable [3H]flunitrazepam binding in hippocampal tissue from schizophrenic and control subjects. No difference in receptor density or affinity between the two groups was observed, indicating that the previously identified GABAergic deficit in the hippocampus in schizophrenia does not apparently lead to up-regulation of these receptor sites. The implications of this finding are discussed.

11C]Ro 15-4513, a ligand for visualization of benzodiazepine receptor binding. Preparation, autoradiography and positron emission tomography.

Ro 15-4513, a partial inverse agonist at the benzodiazepine (BZ) receptor site was labelled with 11C and used for in vitro autoradiography on human post mortem brain sections and for positron emission tomography (PET) on Cynomolgus monkeys. The total radiochemical yield of [11C]Ro 15-4513 was 30-40% with an overall synthesis time of 40 min. The specific radioactivity was about 1000 Ci/mmol at end of synthesis. In vitro autoradiography showed that [11C]Ro 15-4513 bound specifically predominantly in the neocortex of the human brain. Specific binding was also demonstrated in the basal ganglia and the cerebellar cortex. Flumazenil (Ro 15-1788) and clonazepam inhibited the binding in cerebral regions, but a significant proportion in the cerebellum was not inhibited by these agents. This proportion may represent alpha 6-containing BZ receptors. PET examination of [11C]Ro 15-4513 binding in Cynomolgus monkeys demonstrated high uptake of radioactivity in neocortex. The uptake of radioactivity was markedly displaced by high doses of Ro 15-4513 or clonazepam. [11C]Ro 15-4513 should be a useful ligand to examine BZ receptor characteristics in the living human brain by PET.

Specific Inhibition of Binding to Benzodiazepine Receptors by 1, 2, 3‐Triazole Derivatives

AbstractCertain 1, 2, 3‐triazole derivatives were prepared and tested for their ability to displace [3H]diazepam that was bound to bovine brain membrane protein. All the tested compounds are essentially lacking in this ability, except for B.1, which inhibited binding of [3H]diazepam in 50% of the trials at 2.5 μM. The structure of B.1, with a 1, 2, 3‐triazole ring with acidic properties, supports the hypothesis proposed for binding to the benzodiazepine receptor site. Comparison of B.1 with 1, 2, 3‐triazole derivatives bearing a bicyclic substituent in position 1 of the heterocyclic ring suggests that a high steric hindrance increases the affinity of a compound for the benzodiazepine receptor.

Expression of GABA A receptor polypeptides in clonal rat cell lines

The neuronal-like cell lines, B35, B65, B103, and B104, previously reported to possess high affinity GABA binding, were analyzed for various cellular properties. They possessed peripheral but lacked central benzodiazepine binding. Only B65 cells possessed [ 3H-]muscimol binding; none bound [ 35S]TBPS. None of the cells exhibited GABA-stimulated chloride conductance with patch clamp recordings. By Western blots the cells possessed α subunits. Northern blot and polymerase chain reaction analysis that showed that out of α 1, α 4, β 1 and γ 2 subunits, only α 1 subunit mRNA was present. Thus, GABA A-receptor binding without associated central benzodiazepine receptor sites and without functional chloride channels appears to result from expression of an incomplete subunit composition.

The postnatal development of benzodiazepine receptor sites in the chick optic lobe is modulated by environmental lighting

The present paper describes the ability of benzodiazepin receptor sites to undergo light mediated-plastic changes during the early postnatal development of the chick optic lobe. The postnatal development pattern of these receptors was studied under different levels of light stimulation, i.e. normal-, light- and dark-rearing. At hatching the specific binding of [ 3H]Flunitrazepam was 0.23 ± 0.01 pmol/mg protein. The developmental profile shows a sharp and transient peak of receptor overexpression between the 1st and the 2nd postnatal day in the three experimental groups. Between the 2nd and the 6th day significant differences were found between the three groups, being this difference maximal during the peak of over-expression. In fact, on the 2nd day the specific [ 3H]Flunitrazepam binding showed an increase of 17% ( P < 0.0005) and a decrease of 34% ( P < 0.0005) for light- and dark-reared animals as compared with normally-reared ones. The changes in receptor density were transient since from the 6th day onward they gradually disappeared, being almost identical in the three groups by the day 15. At this moment the number of benzodiazepine receptor sites stabilized at the adult level. Scatchard analysis at the 2nd postnatal day revealed that the differences observed in the high affinity benzodiazepine binding sites between the three groups were due to modifications in the total number of binding sites while the affinity remained unchanged. The maximal number of binding sites were: 2.76 ± 0.03, 3.40 ± 0.01 and 1.46 ± 0.11 pmol/mg protein in normally-, light- and dark-reared chicks, respectively; while the apparent dissociation constants were unaffected. The present work shows that variations in the light intensity during the early postnatal life affect the postnatal developmental pattern of benzodiazepine receptor sites. This effect is maximal at the time of appearance of the peak of receptor overexpression (1–2 postnatal day) suggesting that the peak corresponds to a brief period of high plasticity for the receptor development.

Effects of diazepam on nociception in rats

Acute i.p. injection of diazepam (1 mg/kg) resulted in a moderate increase in the tail-flick latency in rats. Tolerance to this diazepam effect developed after 10 days of diazepam treatment (1 mg kg-1 day-1). The benzodiazepine antagonist Ro 15-3505 only partially reversed the effect of diazepam on nociception. Naloxone (5 mg/kg i.p.) failed to affect the effect of diazepam on nociception, while the kappa antagonist MR 2266 fully antagonized the diazepam-induced increase of the tail-flick latency. Diazepam injected intracerebroventricularly (1, 5, 20 micrograms/rat) did not alter basal nociceptive threshold, however, diazepam injected intrathecally (20 micrograms/rat) prolonged the tail-flick latency. Furthermore, intracerebroventricular injection of muscimol partially antagonized the i.p. diazepam-induced increase of the tail-flick latency. These results suggest that benzodiazepine receptor sites are partially involved in the effect of diazepam on nociception and indicate that an indirect kappa-opioid-receptor-mediated mechanism may be involved. The anatomical site of diazepam action on tail-flick latency seems to be at the spinal level. Descending axons to the spinal cord from brain areas reached by intracerebroventricular injection of muscimol seem to modulate the effect of diazepam effect on nociception.

X-ray crystal structures of three nonbenzodiazepinic ligands for the benzodiazepine receptor sites: SR95926, CMW1842, and L16317:nab initio MO study of the electronic properties

The crystal structures ofp-methoxyphenyl-3-triazolo [4,3-a] isoquinoline (SR95926),p-methoxyphenyl-3-triazolophtalazine (CMW1842), andp-methoxyphenyl-3-N-dimethoxyethylamino-6-triazolophtalazine (L16317) have been solved by direct methods from single-crystal X-ray diffraction data, and refined by full-matrix least squares. SR95926: monoclinic,P21/n,a=20.950(3),b=6.769(1),c=9.465(2) A,β=100.90(1)°. CMW1842: triclinic,P¯1,a=8.784(1),b=9.160(4),c=8.555(1) A,α=99.10(2),β=93.90(1), γ=106.77(1)°. L16317: monoclinic,P21/n,a=20.124(3),b=9.586(1),c=10.788(1) A,β=91.91(1)°. FinalR factors are 0.034, 0.037, and 0.053, respectively. Experimental geometries were used to perform STO-3Gab initio molecular-orbital calculations. A relationship between the electronic pattern within the molecules and the affinity of the benzodiazepine receptor sites is pointed out.

Drug modulation of GABA A-mediated transmission

Synaptic transmission mediated by GABA A receptors is a target for a variety of centrally acting anticonvulsant, anxiolytic, sedative-hypnotic and convulsant drugs. Picrotoxin, a non-competitive antagonist of GABA, binds to a distinct site on the oligomeric complex and is allosterically coupled to GABA, benzodiazepine, barbiturate and steroid sites. Recent studies have shown that the picrotoxin site can be modulated both positively and negatively, in a manner analogous to the benzodiazepine receptor sites.

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

To investigate the interaction between gamma-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 IC50s 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.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA-benzodiazepine receptors: demonstration of pharmacological subtypes in the brain.

The GABAA receptor is a ligand-gated chloride ion channel that mediates the majority of rapid-acting inhibitory synapses in the central nervous system (Olsen and Venter, 1986). The GABAA receptors are also the target of numerous clinically important depressant and excitatory drugs (Olsen, 1981; Tallman and Gallager, 1985; Biggio and Costa, 1988). The convulsant drug bicuculline acts as a competitive antagonist at the GABA recognition site, beta-carbolines block GABA function as ’ inverse agonists’ at the benzodiazepine recognition site, and picrotoxin and cage convulsants inhibit the chloride channel function at a site on the receptor complex distinct from the GABA and benzodiazepine receptor sites. Clinically important depressant benzodiazepines enhance GABA-mediated inhibition via their own binding sites on the receptor complex. Still additional sites on the receptor-ion channel complex mediate the action of barbiturates, steroid anesthetics, and possibly ethanol to enhance GABAA receptor function at the membrane level (Olsen and Venter, 1986; Biggio and Costa, 1988).