Identification of a high-affinity peripheral-type benzodiazepine binding site in rat aortic smooth muscle membranes

The existence of a benzodiazepine binding site in rat aortic smooth muscle membranes was explored employing [3H]Ro5-4864 as radioligand. The binding site was concentrated in the mitochondrial fraction enriched with cytochrome c oxidase and semicarbazide-insensitive monoamine oxidase. [3H]Ro5-4864 binds to the membranes in the mitochondrial fraction with high affinity. The dissociation constant (KD) determined by saturation binding was 2.8 +/- 0.7 nM (n = 5). The association rate constant (k1) was 4.7 +/- 0.8 x 10(6) M1 min-1, and the dissociation rate constant (k-1) was 0.028 +/- 0.005 min-1 (n = 3). The kinetically determined KD was 6.0 +/- 0.8 nM (n = 3) at 0.5 nM [3H]Ro5-4864. The density of binding determined from saturation binding experiments was 14.0 +/- 1.2 pmol/mg protein (n = 5). The Hill coefficient of binding was 0.94 +/- 0.02 (n = 5) indicating that [3H] Ro5-4864 binds to a single site. The [3H]Ro5-4864 binding was inhibited by Ro5-4864 (Ki = 6.1 +/- 1.9 nM), PK 11195 (Ki = 8.9 +/- 1.8 nM), diazepam (Ki = 87.3 +/- 3.4 nM), flunitrazepam (Ki = 94.6 +/- 1.8 nM), clonazepam (Ki = 6.3 +/- 1.3 microM) and Ro15-1788 (Ki = 16.8 +/- 1.5 microM). The rank order of potency of the competitive inhibition of [3H]Ro5-4864 binding (Ro5-4864 = PK 11195 greater than diazepam = flunitrazepam much greater than clonazepam greater than Ro15-1788) is characteristic of the peripheral-type benzodiazepine binding site. The data indicate an abundant high affinity peripheral-type benzodiazepine binding site of unknown function in rat aortic smooth muscle cells.     

AHN 683: a fluorescent ligand for peripheral-type benzodiazepine receptors.

AHN 683 [1-(2-fluoro-5-N[1,3-dihydrol-1,1-bis(4-hydroxyphenyl)-3-oxo- 5-isobenzofurancarboxamide]-phenyl)-N-methyl-N-(1-methylpropyl)-3- isoquinoline carboxamide] is a fluorescein-derived ligand at peripheral-type benzodiazepine receptors structurally related to the isoquinoline carboxamide, PK 14105. The binding of AHN 683 to rat renal membranes measured by fluorescence techniques was saturable with a maximum number of binding sites of 2.3 +/- 0.3 pmol/mg of protein. The KD (40.4 +/- 2.2 nM) estimated by fluorescence was in good agreement with the Ki (77.4 +/- 13.5 nM) obtained in competition studies with [3H] Ro 5-4864. AHN 683 exhibited rapid and reversible binding which was significantly reduced by the histidine modifying reagent, diethylpyrocarbonate. The potencies of a pair of isoquinoline carboxamide enantiomers as well as other structurally diverse peripheral-type benzodiazepine receptor ligands estimated by inhibition of AHN 683 binding were in good agreement with values obtained using radioligand binding techniques. AHN 683 binding was unaffected by compounds that do not recognize peripheral-type benzodiazepine receptors. Moreover, a significant increase in the maximum number of binding sites of AHN 683 to rat renal membranes after chronic furosemide treatment (29.2%, P less than .02) was comparable to the increase measured using [3H]PK 11195 (35.6%, P less than .001). These findings demonstrate the feasibility of using fluorescent ligand binding techniques to quantitatively characterize peripheral-type benzodiazepine receptors.     

Peripheral type benzodiazepine receptors in human and guinea pig lung: characterization and autoradiographic mapping

Binding of [3H]Ro5-4864, a ligand selective for peripheral-type benzodiazepine receptors, to human and guinea pig lung membranes was characterized and the binding sites localized in lung sections by light microscopic autoradiography. Inhibition of [3H]Ro5-4864 binding by unlabeled Ro5-4864, PK11195, midazolam, diazepam, clonazepam and flumazenil indicated that [3H]Ro5-4864 bound to specific receptors and binding was reversible. Scatchard analysis indicated a single class of binding site, with a dissociation constant (Kd) of 12.8 +/- 2.1 nM with maximum binding capacity of 2.6 +/- 0.6 pmol/mg of protein for human lung and Kd of 8.4 +/- 1.5 nM and maximum binding capacity of 3.5 +/- 0.3 pmol/mg of protein for guinea pig lung. Autoradiograms showed specific labeling over discrete structures in both human and guinea pig lung. Labeling was particularly dense over submucosal glands in intrapulmonary bronchi of human. Airways were also labeled, with epithelium having a higher grain density than smooth muscle. Labeling over smooth muscle was denser in small compared to large airways. Vascular smooth muscle was not labeled in either human and guinea pig. The alveolar walls were uniformly labeled in both species. The functional significance of pulmonary peripheral-type benzodiazepine receptors remains to be determined.     

Withdrawal from diazepam substitutes for the discriminative stimulus properties of pentylenetetrazol

Rats were trained to discriminate pentylenetetrazol (PTZ, an anxiogenic drug), 20.0 mg/kg, from saline using a food-maintained two-lever-choice task. When treated chronically with diazepam (DZP) and tested with the benzodiazepine-receptor antagonist Ro 15-1788, withdrawal from DZP produced a PTZ-like stimulus in these subjects that was related directly to the dose of DZP given every 8 hr for 6 days. In contrast, only the highest dose of DZP (80 mg/kg/8 hr) given chronically produced even minimal physical signs of precipitated abstinence after Ro 15-1788. In a separate experiment, Ro 15-1788 produced a PTZ-like stimulus when given at 2-day intervals during chronic administration of DZP. In this experiment, rats were maintained on DZP, 40.0 mg/kg/6 hr for 14 days. These subjects were tested with Ro 15-1788, 40.0 mg/kg, every 2 days during days 6 through 14 of chronic DZP, and Ro 15-1788 substituted for PTZ on 4 of these 5 tests. Because these experiments involved periods of nontraining on the discrimination task, a final experiment was performed to test the stability of stimulus control in rats trained to detect PTZ. DZP was administered for up to 20 days, withdrawal was precipitated by Ro 15-1788 and after an additional 16 to 40 days of nontraining, stimulus control was tested. There was no significant decline in stimulus control over this period. These results suggest that PTZ discrimination provides a sensitive, stable assay for the detection of withdrawal from benzodiazepine dependence.     

Inhibitory action of peripheral-type benzodiazepines on dopamine release from PC12 pheochromocytoma cells.

Characteristics of the benzodiazepine inhibition of dopamine (DA) release in PC12 cells were investigated. Diazepam inhibited DA release evoked by high concentrations of extracellular K+ in a dose-dependent manner (IC50, 10 microM). Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine- 2-one], a peripheral-type benzodiazepine, also inhibited DA release effectively. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide], a benzodiazepine generally considered a peripheral-type benzodiazepine receptor antagonist, did not antagonize the inhibition induced by diazepam, but rather inhibited DA release itself. On the other hand, the central-type benzodiazepines, clonazepam and Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) did not affect the DA release. Diazepam, Ro 5-4864 and PK 11195 also inhibited a Ba(++)-current carried by voltage-gated Ca++ channels, and diazepam suppressed an increase in intracellular Ca++ evoked by 80 mM extracellular K+ as measured by the fura-2 method. These results suggest that the inhibitory action of diazepam and other benzodiazepines on DA release from PC12 cells may be mediated through one type of peripheral-type benzodiazepine receptors which are coupled to voltage-gated Ca++ channels and that these receptors may not necessarily be the same as those in other tissues.     

Effects of Ro15-4513 and other benzodiazepine receptor inverse agonists on alcohol-induced intoxication in the rat

The ability of the imidazobenzodiazepine Ro15-4513 to antagonize the behavioral intoxication produced by ethanol and related short-chain alcohols was examined in the rat. Ro15-4513 dose dependently (0.5-10 mg/kg i.p.: IC50, 1.5 mg/kg) inhibited the intoxication induced by ethanol (2 g/kg), as well as t-amyl alcohol (0.36 g/kg) and methanol (4.66 g/kg). The effects of Ro15-4513 in blocking ethanol-induced intoxication were blocked by the benzodiazepine receptor antagonists Ro15-1788 and CGS-8216. However, Ro15-4513 was ineffective in antagonizing the intoxication observed after higher doses of ethanol (4 g/kg). In contrast, ethanol-induced intoxication was not antagonized by the benzodiazepine receptor antagonists Ro15-1788 (10 mg/kg) or CGS-8216 (20 mg/kg), nor by the inverse agonists FG-7142 (10-30 mg/kg) or beta CCE (10 mg/kg). When administered after ethanol, Ro15-4513 also reversed ethanol-induced intoxication in a dose-dependent manner (2.5-10 mg/kg i.p.: IC50, 5 mg/kg), an effect which was also blocked by Ro15-1788 and CGS-8216. However, neither beta CCE (10 mg/kg) or FG-7142 (less than or equal to 30 mg/kg) alone reversed ethanol-induced intoxication. Moreover, beta CCE (10 mg/kg), when administered just before Ro15-4513, completely antagonized the actions of Ro15-4513 in reversing ethanol-induced intoxication. These data suggest that the ability of Ro15-4513 to antagonize, and to reverse, ethanol-induced intoxication is mediated via central benzodiazepine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

QUANTITATIVE AUTORADIOGRAPHIC DETERMINATION OF BINDING SITES FOR A PERIPHERAL BENZODIAZEPINE LIGAND ([3H]PK 11195) IN HUMAN IRIS

Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 micron) labelled with [3H]PK 11195 (1 nM) at 25 degrees C. Nonspecific binding was determined with PK 11211 (5 microM) or Ro 5-4864 (5 microM). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris, and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively, were: 42.7 +/- 0.2, 30.1 +/- 0.5, and 37.4 +/- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.     

Pharmacologic characterization of acute chlordiazepoxide dependence in the rat

A single intoxicating dose of chlordiazepoxide HCl (p.o.) in the rat can induce quantifiable manifestations of physical dependence. Dependence was revealed by antagonist precipitation (Ro 15-1788, CGS-8216) as well as spontaneous emergence of neurobehavioral signs of withdrawal observed by multiple raters blind to treatments. Ro 15-1788 was 45% more effective than CGS-8216 in both reversing chlordiazepoxide intoxication and expressing withdrawal signs. The severity of Ro 15-1788-precipitated withdrawal varied with chlordiazepoxide dose, Ro 15-1788 dose and the agonist-antagonist dose interval. Maximal precipitated dependence was evoked 3 days after chlordiazepoxide HCl (450 mg/kg) by Ro 15-1788 (25 mg/kg i.p.). The precipitated syndrome consisted of tail erection, reduced motor activity, high step, curled claw, arched back, muscle hypertonus and piloerection. Ro 15-1788-precipitated dependence emerged between 28 and 52 hr, peaked at 76 hr and disappeared by 124 hr. Spontaneous withdrawal had emerged from 100 to 124 hr and then faded gradually. The neurobehavioral expression of central nervous system depression and its reversal were necessary but not sufficient conditions for the induction and expression of acute chlordiazepoxide dependence. These results suggest caution in reviving acute benzodiazepine-overdosed patients to avoid iatrogenic withdrawal analogous to naloxone for opiates.     

Ro 19-8022, a nonbenzodiazepine partial agonist at benzodiazepine receptors: neuropharmacological profile of a potential anxiolytic.

The partial agonist at benzodiazepine receptors, Ro 19-8022, has been characterized as a putative anxiolytic drug with an improved side effect profile. This orally active compound is a representative of a quinolizinone structure class and shows potent anticonflict activity in mice and rats. It protects rodents from convulsions induced by pentylenetetrazol, N-methyl-D-aspartic acid and maximal electroshock, as well as against audiogenic seizures, with an efficacy comparable to that of the full agonist alprazolam. No appreciable sedative or motor-impairing effects could be detected up to a very high dose (100 mg/kg) in the horizontal wire test or the rotarod performance test in mice and rats and in spontaneous behavior in monkeys. Consistent with its characterization as a partial agonist, Ro 19-8022 antagonized the motor impairment induced by the full agonists diazepam or meclonazepam measured in horizontal wire and rotarod tests in rodents, and reduced flunitrazepam-induced effects in squirrel monkeys, with an efficacy comparable to that of the benzodiazepine receptor antagonist flumazenil. After subchronic administration of Ro 19-8022 to mice, antagonist-precipitated withdrawal syndrome was dramatically weaker than after alprazolam treatment, which is indicative of a lower physical dependence liability of Ro 19-8022. Pharmacodynamic effects recorded in convulsion and reversal of motor impairment tests after i.v. administration suggest a long duration of action of this compound. Taken together, such preclinical data suggest that benzodiazepine receptor partial agonists with a neurological and behavioral profile such as that of Ro 19-8022 may offer an innovative therapeutic approach to the treatment of anxiety disorders.     

Electrophysiological actions of Ro5-4864 on cerebellar Purkinje neurons: evidence for "peripheral" benzodiazepine receptor-mediated depression

The effects of Ro 5-4864 (4'-chlorodiazepam; the archetypic peripheral benzodiazepine receptor ligand) were examined on the electrophysiological responses of rat cerebellar Purkinje neurons maintained in vitro. Ro 5-4864 produced a biphasic response (consisting of an increase followed by a decrease in spontaneous firing) in approximately 50% of the neurons studied. The remaining neurons responded to Ro 5-4864 application with decreased spontaneous activity. The EC50 and IC50 values for the excitatory and depressant responses to Ro 5-4864 were 490 and 450 nM, respectively. Preincubation with PK 11195 [N-methyl-N-(methyl-1-propyl)chloro-2-phenyl-1-isoquinoline-3-carboxamid e; a peripheral benzodiazepine receptor antagonist] reduced the potency of Ro 5-4864 to inhibit cell firing in a competitive fashion, but did not alter Ro 5-4864-elicited excitations. A similar effect was observed with the monocarboxylic acid anion transport inhibitor UK 5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate]. In contrast, the increases in cell firing elicited by t-butylbicyclophosphorothionate (a GABA-gated chloride channel blocker) were enhanced 6-fold by coperfusion with both Ro 5-4864 and PK 11195. These findings suggest that the Ro 5-4864 induced depressions of Purkinje neuron spontaneous activity is mediated by peripheral benzodiazepine receptors, whereas the excitation may result from modulation of the gamma-aminobutyric acid-gated chloride ionophore.     

Involvement of peripheral-type benzodiazepine receptors in the proconvulsant actions of pyrethroid insecticides

It has been demonstrated previously that select Type II pyrethroids are potent proconvulsants in the rat and that the proconvulsant actions of deltamethrin are blocked by administration of PK 11195, an antagonist of the peripheral-type benzodiazepine receptor (PTBR). The present investigation has extended these findings to include various Type I pyrethroids as proconvulsants. Additionally, the proconvulsant activity of cismethrin was reversed by administration of PK 11195. Pyrethroid displacement of specific [3H]Ro5-4864 binding to rat brain membranes was investigated to further define the interaction of pyrethroids with the PTBR. Both Type I and Type II pyrethroids potently inhibited [3H]Ro5-4864 binding with affinities ranging from nanomolar to micromolar. The ED50 values for the proconvulsant effects of both Type I and Type II pyrethroids were significantly correlated with their respective IC50 values as inhibitors of [3H]Ro5-4864 binding. [3H]Ro5-4864 saturation isotherms performed in the presence of fixed concentrations of deltamethrin or cismethrin showed that these pyrethroids increased the observed Kd values for [3H]Ro5-4864 with no change in the maximum number of binding sites. However, Schild plot analysis of the effect of deltamethrin on [3H]Ro5-4864 affinity was nonlinear with the Kd shift approaching a limiting value. Considered together these results suggest an allosteric effect of pyrethroids on [3H]Ro5-4864 binding, and provide additional support for the involvement of the PTBR in the proconvulsant actions of pyrethroids.     

Peripheral-type benzodiazepine receptors: autoradiographic localization in whole-body sections of neonatal rats

We have developed a procedure that allows the autoradiographic localization of benzodiazepine receptors in whole-body sections of neonatal rats. Central-type benzodiazepine receptors, visualized with [3H]methylclonazepam, are restricted to nervous tissue. In contrast, peripheral-type benzodiazepine receptors, visualized with [3H]Ro5-4864, occur widely, but with discrete localizations throughout the body. Peripheral-type benzodiazepine receptors are most concentrated in the adrenal cortex and the skin. Substantial levels of these receptors are also evident in the heart, the salivary glands, discrete regions of the kidney, the epithelium of the lung, the nasal and lingual epithelia, the lining of the pulmonary arteries, the thymus, the hair follicles of the vibrissae, the tooth buds and the bone marrow. Considerable binding of [3H]Ro5-4864 is observed in the brown fat pads, the liver and the spleen, but high levels of nonspecific binding preclude accurate evaluation of the actual specific binding in these organs. Only low levels of [3H]Ro5-4864 binding sites are found in the brain and they are virtually undetectable in the skeletal muscle, the eye, the inner ear and the gastrointestinal tract. High levels of peripheral-type benzodiazepine receptor appear present in tissues that derive their metabolic energy primarily from oxidative phosphorylation, whereas only low levels are present in tissues that can derive their metabolic energy largely from glycogenolysis. Association of these receptors with mitochondria and a possible role in modulation of energy metabolism is suggested further by the observation that the histochemically visualized distribution of cytochrome oxidase activity overlaps the autoradiographic pattern of [3H]Ro5-4864 binding sites.     

Relaxation of rat vascular muscle by peripheral benzodiazepine modulators.

Effects of peripheral benzodiazepine receptor modulating drugs, Ro 5-4864 and PK 11195, on tension induced by K+ and the calcium agonist SDZ 202 791 (S isomer), were studied in rat caudal arteries. A significant reduction of tonic phase tension occurred with 30 nM PK 11195 or 3 microM Ro 5-4864, but decreases of the initial (first 3 min), phasic contraction were detected only at the highest concentrations of Ro 5-4864 and PK 11195. Protoporphyrin IX, the putative endogenous ligand of the peripheral benzodiazepine receptor, (at 10-100 nM) markedly increased the effectiveness of Ro 5-4864 and PK 11195 in reducing phasic contraction. Intracellular calcium localization and distribution in fura-2 loaded single vascular cells were quantitated using a high sensitivity, two-stage microchannel plate, photon-counting (PMI-VIM) camera. Peripheral benzodiazepines reduced intracellular calcium release from centrally located calcium pools, and this decrease of calcium release was potentiated by protoporphyrin IX. The decrease in intracellular calcium activity, which was more pronounced in the central regions where sarcoplasmic reticular elements are numerous, was probably the major mechanism of these vasodilator properties. Measurements of soluble guanylate cyclase activity also supported the intracellular Ca2+ release mechanism. Under conditions where protoporphyrin IX did not significantly stimulate guanylate cyclase, Ro 5-4864 alone or more effectively in combination with protoporphyrin IX stimulated cGMP production and caused relaxation. Guanylate cyclase forms a possible target for these benzodiazepine modulators, a hypothesis that merits further investigation.     

Reversal of alcohol dependence and tolerance by a single administration of flumazenil.

Chronic exposure to ethanol is associated with the development of tolerance to the acute effects of ethanol and a withdrawal syndrome characterized by anxiety and seizure susceptibility. In the present study we examined the ability of flumazenil (Ro15-1788), a benzodiazepine receptor antagonist, to reverse neuronal and behavioral manifestations of ethanol tolerance and dependence. A single injection of flumazenil (10 mg/kg, 14 hr before withdrawal) to mice administered a liquid diet containing ethanol for 10 days, reduced seizure severity during withdrawal from ethanol. Acute tolerance to ethanol-induced hypothermia was not sensitive to flumazenil treatment, but tolerance and diazepam-induced cross-tolerance to the ataxic effects of ethanol were reversed by a single injection of flumazenil given 2 to 26 hr before evaluation of tolerance. At a biochemical level, the ability of benzodiazepine inverse agonists (e.g., Ro15-4513) to reduce the activity of gamma-aminobutyric acid (GABA) receptor-operated chloride channels may represent a neuronal manifestation of ethanol dependence (Buck and Harris, 1990). Flumazenil treatment of ethanol-dependent mice 14 hr before isolation of brain membrane vesicles partially reversed the augmentation of Ro15-4513 inhibition of muscimol-stimulated 36Cl- uptake in vitro. These results demonstrate that brief occupation of benzodiazepine receptors by an antagonist may reset the cellular mechanisms responsible for the development of ethanol tolerance and dependence, and support the hypothesis that increased sensitivity to benzodiazepine inverse agonists is involved in the development of ethanol dependence.     

地黄苏合香合剂对早期肝性脑病大鼠脑内外周型苯二氮(革)受体的影响

目的 观察中药地黄、苏合香(地苏合剂)对早期肝性脑病大鼠脑皮层线粒体外周型苯二氮(革)受体(PBRs)活性的影响.方法 采用CCl4致化学性肝损伤大鼠模型,阳性药组给予乳果糖6g/kg,治疗组分别给予不同剂量的地苏合剂,同位素法检测PBRs.结果 模型组PBRs结合活性较对照组明显升高(P＜0.01),中、高剂量组PBRs结合活性较模型组降低(P＜0.05)；模型组脑皮层线粒体PBRs的最大结合容量(Bmax)值较对照组明显升高(P＜0.01),高剂量组较模型组明显降低(P＜0.01).结论地苏合剂可能通过降低早期肝性脑病大鼠脑皮层线粒体PBRs结合活性,减少神经损伤,从而延缓肝性脑病的发生、发展.     

Effects of a benzodiazepine antagonist, Ro 15-1788, on hippocampal field potentials in freely moving rats.

Effects of a benzodiazepine receptor agonist (diazepam) and an antagonist (Ro 15-1788, flumazenil) administered separately or in combination on field potentials recorded from the hippocampal dentate area were examined in unanesthetized, unrestrained rats. Population excitatory postsynaptic potentials (EPSPs) evoked by stimulation of the perforant path were depressed significantly by diazepam (4 mg/kg, i.p.). However, diazepam did not affect the firing (spike) threshold of dentate granule cells. The injection of Ro 15-1788 (4 mg/kg, i.p.) alone affected neither excitatory synaptic transmission nor population spike threshold. Strength of gamma-amino butyric acid-mediated recurrent inhibition as measured by the paired-pulse technique was potentiated by diazepam but unaffected by Ro 15-1788. However, the diazepam-enhanced inhibition was reversed by a subsequent administration of Ro 15-1788. Previous studies indicate that Ro 15-1788 acts not only as a selective benzodiazepine antagonist but also as a partial agonist-antagonist or an inverse agonist depending probably on doses. The present study demonstrated that Ro 15-1788 acted as a pure antagonist at low doses. These data suggest that the clinical use of Ro 15-1788 at high doses against comas induced by unidentified drugs could worsen the conditions and that low doses are recommendable for initial treatments because of its pure antagonist action.     

In vivo modeling of the pharmacodynamic interaction between benzodiazepines which differ in intrinsic efficacy.

The pharmacokinetic and pharmacodynamic interactions between the benzodiazepine agonist midazolam, on one hand, and the partial agonist bretazenil and inverse agonist Ro 19-4603, on the other, were characterized in vivo in rats using effect parameters derived from quantitative EEG analysis. Male Wistar-derived rats received an i.v. infusion of 3 mg/kg bretazenil or 3 mg/kg Ro 19-4603 in 5 min during a steady-state infusion of midazolam at a rate of 2.8 mg/kg/hr. EEG signals were continuously measured and quantified by aperiodic EEG analysis and the change in amplitudes in the 11.5 to 30 Hz (beta) frequency band was used as measure of drug effect on the central nervous system. Frequent arterial blood samples were taken to determine the pharmacokinetics of the drugs. Both bretazenil and Ro 19-4603 did not influence the steady-state plasma concentrations of midazolam, but markedly antagonized the midazolam-induced increase in EEG effect measure to the extent of their own intrinsic maximal effects. The changes in EEG effect as a function of drug concentrations were described by a competitive interaction model, allowing the estimation of the pharmacodynamic parameters (means +/- S.E.) of the partial agonist, Emax = 15 +/- 5 microV/sec and EC50 = 19 +/- 5 ng/ml, and inverse agonist, Emax = -5.8 +/- 1.6 microV/sec and EC50 = 7.8 +/- 2.5 ng/ml. Similar values of the pharmacodynamic parameters were obtained after a single i.v. administration of these drugs, which provides evidence for the validity of the proposed interaction model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Group II metabotropic and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate glutamate receptors regulate the deficit in brain reward function associated with nicotine withdrawal in rats

This study investigated the role of ionotropic and metabotropic glutamate receptors in the deficits in brain reward function, as measured by elevations in intracranial self-stimulation (ICSS) reward thresholds, associated with nicotine withdrawal. The group II metabotropic glutamate (mGluII) receptor agonist LY314582 [a racemic mixture of LY354740 ([+]-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid])] (2.5-7.5 mg/kg) precipitated withdrawal-like elevations in ICSS thresholds, a sensitive measure of reward function, in nicotine-dependent but not control rats. LY314582 did not affect response latencies, a measure of performance in the ICSS paradigm. Bilateral microinfusion of LY314582 (10-100 ng/side) into the ventral tegmental area likewise precipitated dose-dependent threshold elevations in nicotine-dependent rats. Furthermore, a single injection of the mGluII receptor antagonist LY341495 (2S-2-amino-2-[1S,2S-2-carboxycyclopropan-1-yl]-3-[xanth-9-yl]propionic acid) (1 mg/kg) attenuated the threshold elevations observed in rats undergoing spontaneous nicotine withdrawal. mGluII receptors are primarily located on glutamatergic terminals throughout the mesocorticolimbic system, where they act as inhibitory autoreceptors. To investigate whether mGluII receptors contributed to nicotine withdrawal by decreasing glutamatergic transmission, we next examined whether direct blockade of postsynaptic glutamate receptors precipitated withdrawal-like reward deficits in nicotine-dependent rats. The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX; 0.01-1 mg/kg) precipitated withdrawal-like threshold elevations in nicotine-dependent but not control rats, whereas 6-methyl-2-[phenylethynyl]-pyridine (MPEP; 0.01-3 mg/kg) and dizocilpine (MK-801; 0.01-0.2 mg/kg), antagonists at metabotropic glutamate 5 and N-methyl-d-aspartate receptors, respectively, did not. Overall, these data demonstrate that mGluII receptors play an important role in the reward deficits associated with nicotine withdrawal. Furthermore, it is likely that mGluII receptors generate this reward deficit, at least in part, by decreasing glutamate transmission at AMPA/kainate receptors.     

Pharmacologic actions of Ro 24-5913, a novel antagonist of leukotriene D4.

Ro 24-5913, (E)-4-[3-[2-(4-cyclobutyl-2- thiazolyl)ethenyl]phenylamino]-2,2-diethyl-4-oxobutanoic acid, has been identified as a chemically unique, potent and selective LTD4 antagonist. In vitro, Ro 24-5913 competes with [3H]LTD4 for its binding site on guinea pig lung membranes with an IC50 of 6.4 +/- 2.2 nM. In isolated guinea pig tracheal smooth muscle, Ro 24-5913 produces concentration-dependent rightward shifts of LTD4-induced contraction curves (pA2 value of 9.6 +/- 0.2). The slope of the Schild plot is not significantly different from 1, indicating that the antagonism is of a competitive nature. In the human bronchus, Ro 24-5913 is an effective antagonist of LTD4-induced contractions (pKB of 9.3 +/- 0.1). In vivo, Ro 24-5913 dose-dependently inhibits LTD4-induced bronchoconstriction in guinea pigs by the i.v. (ID50 0.13 mg/kg), oral (ID50 0.12 mg/kg) and aerosol (IC50 0.008%) routes of administration. This in vivo activity is specific as evidenced by the inability of Ro 24-5913 to inhibit bronchoconstriction induced by LTB4, PAF or histamine. In comparison with other LTD4 antagonists evaluated in this guinea pig model, Ro 24-5913 is markedly superior in terms of oral potency, bioavailability and oral duration of action. Ro 24-5913 also blocks allergic bronchospasm mediated by endogenously generated leukotrienes in guinea pigs; the potency and duration of action is nearly equivalent to that seen as an antagonist of bronchoconstrictions produced by exogenous LTD4. In summary, Ro 24-5913 is representative of a novel chemical class of LTD4 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)