Protection against anthramycin-induced toxicity in mice by coenzyme Q10.

Pretreatment of Swiss Webster mice with coenzyme Q10 (CoQ) markedly reduced the lethality of the antitumor antibiotic anthramycin as well as its ability to decrease ventricular weights. In tumor-bearing mice CoQ pretreatment did not produce any consistent alteration of radioactivity levels in blood, heart, tumor, lungs, kidneys, liver, muscles, brain, or spleen after [15-3H]anthramycin administration. Gross alterations in anthramycin distribution is probably not the mechanism by which CoQ alters the cardiotoxicity and lethality of anthramycin.

Cibacron blue-induced enhancement of agonist binding to cholecystokinin (CCK) receptors in solubilized pancreatic membranes.

The pancreatic receptor for cholecystokinin (CCK) typifies many G protein-coupled receptors in that its ability to bind agonist can be reduced by GTP or the solubilization of membranes. We found, however, that a dye, cibacron blue, caused up to a 6-fold increase in binding of the CCK receptor agonist, 125I-CCK-8, to rat pancreatic membranes solubilized with digitonin. Binding optimally enhanced in this manner was comparable to binding of 125I-CCK-8 to native membranes with respect to time-course, maximal amount bound, reversibility, and sensitivity to inhibition by various CCK receptor ligands. Increases in affinity of the CCK receptor for CCK-8 accounted fully for the enhancement of binding of 125I-CCK-8. Cibacron blue did not enhance binding of 125I-CCK-8 to native membranes, and also failed to enhance binding of the CCK receptor antagonist, [3H]L-364,718, to solubilized or native membranes. The ability of cibacron blue to enhance binding of agonist but not that of antagonist suggests that this dye may mimic or perhaps stimulate the effects of G protein on CCK receptors. Such a phenomenon may provide new insights into the mechanisms by which receptors distinguish agonists from antagonists.

Stimulatory effects of cholecystokinin on isolated perifused islets inhibited by potent and specific antagonist L 364718 [corrected].

The influence of L 364718 on islet responsiveness to sulfated cholecystokinin (CCK-8S) was investigated. In islets whose inositol-containing phospholipids were prelabeled during a 2-h incubation period, subsequent exposure to L 364718 (1 nM) significantly impaired the secretion of insulin usually noted in response to 200 nM CCK-8S in the simultaneous presence of 7 mM glucose. A higher level of the antagonist (10 nM) completely abolished insulin secretion. L 364718 (1-10 nM) reduced the efflux of 3H from myo-[2-3H]-inositol prelabeled islets in parallel with the reduction in secretion. L 364718 (10 nM) significantly reduced the accumulation of 3H-containing inositol phosphates usually noted with CCK-8S addition. L 364718, at levels 10- to 100-fold greater than those necessary to attenuate CCK-8S-induced insulin secretion, had no adverse effect on the insulin secretory response of freshly isolated islets to 10 mM glucose alone, 5 mM D-glyceraldehyde, 15 mM alpha-ketoisocaproate, or 50 ng/ml gastric inhibitory polypeptide. L 364718 (1000 nM) had no adverse influence on carbamylcholine (1 mM)-induced phosphoinositide hydrolysis. These results establish L 364718 as a potent and highly selective antagonist of cholecystokinin's stimulatory actions on beta-cells. Because of its potency, selectivity, and oral effectiveness, in vivo studies with L 364718, aimed at unraveling the pleiotropic effects of CCK-8S on glucose and insulin homeostasis, seem feasible.

Endogenous inhibitors of 4'-[3H]chlorodiazepam (Ro 5-4864) binding to 'peripheral' sites for benzodiazepines.

'Peripheral' binding sites for benzodiazepines are under neural or homonal control in the pineal gland, olfactory bulb, and kidney. These observations prompted a search for an endogenous substance which could modulate these sites under physiological conditions. Acidified methanol extracts from several tissues (e.g. stomach, kidney, lung) were found to inhibit the binding of [3H]Ro 5-4864 to 'peripheral' binding sites, but did not significantly affect the binding of [3H]diazepam to 'brain' benzodiazepine receptors. Fractionation of a crude extract prepared from antral stomach by either ultrafiltration or gel filtration chromatography yielded high (Mr greater than 10 000) and low (Mr less than 1000) Mr fractions which competitively inhibited [3H]Ro 5-4864 binding to 'peripheral' sites. These observations suggest the presence of endogenous substances in several rat tissues which may represent physiologically important ligands for 'peripheral' binding sites for benzodiazepines.

Impairment of stress adaptive behaviours in rats by the CCKA receptor antagonist, devazepide.

1. Cholecystokinin (CCK) is released during stress both in limbic and hypothalamic areas suggesting that CCK could participate in modulating neuroendocrine as well as behavioural responses to stress. 2. In this study we have examined the effect of CCK receptor antagonists on the retention of the immobility response to a forced-swim stress in rats. In this test, rats are forced to swim during 15 min (conditioning period) and 24 h later, the duration of immobility is measured during a period of 5 min (re-test period). During the conditioning period rats display a period of vigorous activity, followed by progressive inactivity. During the re-test period rats remain 70-80% of the time in an immobile posture. 3. The CCKA receptor antagonist, devazepide (MK-329) but not the CCKB receptor antagonist, L-365,260, administered s.c. immediately before the conditioning period, decreased the duration of acquired immobility during the re-test period. The effect of devazepide was prevented by cholecystokinin octapeptide (CCK-8; 40 micrograms kg-1, s.c) as well as by the selective glucocorticosteroid GII receptor agonist, dexamethasone (30 micrograms kg-1, s.c.) 4. Neither corticosterone nor ACTH plasma levels measured both after the re-test period and after the conditioning period were modified by devazepide treatment. 5. The results suggest a role for CCK in the behavioural adaptation to stress and indicate a relationship between CCK systems and glucocorticoids in the neuronal mechanisms involved in the acquisition of adaptive behaviours to stress.

Interactions of the imidazodiazepine Ro 15-4513 with chemical convulsants.

1. The proconvulsant effects of the imidazodiazepine Ro 15-4513, were investigated in mice by use of intravenous infusion of a variety of convulsant drugs. 2. Dose-response and time course studies of Ro 15-4513 against gamma-aminobutyric acid (GABA) antagonists were performed. On the basis of these studies a maximally effective dose of 5 mg kg-1 was administered 5 min before the determination of seizure thresholds in subsequent experiments. 3. Ro 15-4513 (5 mg kg-1) significantly lowered seizure thresholds to pentylenetetrazole, bicuculline and the convulsant benzodiazepine Ro 5-3663, but failed to alter seizure thresholds to picrotoxin, strychnine, caffeine and quipazine. 4. Ro 15-4513 significantly raised seizure threshold to the benzodiazepine receptor inverse agonist methyl 6,7-dimethoxy-4 ethyl-beta-carboline-3-carboxylate (DMCM). 5. These results are discussed in relation to other studies investigating the proconvulsant and alcohol-antagonizing effects of Ro 15-4513.

Cueing effects of anxiolytic benzodiazepines (DZP and Ro 11-3128): stereospecificity and antagonism by the convulsant benzodiazepine Ro 5-3663.

Two groups of gerbils were trained in a T-maze to discriminate between the vehicle condition (4 ml/kg) and either of the benzodiazepine (BDZ) agonists diazepam (DZP) and Ro 11-3128; administration (5.6 mg/kg) was IP 5 min prior to training onset. Once trained, novel doses and drugs were assessed in test sessions interposed between the regular training days. A dose-related generalization effect occurred with both compounds (range 0.1-5.6 mg/kg), the effect being similar at both the 5 and 15 min test intervals; the two intervals were evaluated after a single injection. The lack of generalization of Ro 11-3624 (range 5.6-56 mg/kg) indicates a stereoisomeric separation of BDZ agonist activity. Ro 5-4864 (range 17.5-56 mg/kg), an agent chemically/structurally related to DZP, did not produce DZP responding at either of the two test inverals; clear-cut convulsant activity occurred at the 15 min interval. The convulsant BDZ compound Ro 5-3663 (3 and 10 mg/kg) antagonized the DZP stimulus irrespective of whether Ro 5-3663 was given either prior to, simultaneously with, or shortly after the DZP injection.

The peripheral benzodiazepine receptor ligand Ro 5-4864 induces supraspinal convulsions in rabbits. Reversal by the central benzodiazepine antagonist Ro 15-1788.

The peripheral BDZ receptor ligand Ro 5-4864 was administered to rabbits in doses ranging from 0.2 to 7 mg/kg IV. Changes in electrocortical activity appeared within 1 min after administration, characterized by trains of slow waves in the posterior sensorimotor and optic cortices (0.6-2 mg/kg) and by grand mal seizures (2-10 mg/kg). The low doses also induced alterations in the basic rhythms both of the hippocampus (reduced amplitude and spike-like waves) and of the nucleus ventralis of thalamus (trains of slow waves), not associated with observable behavioural changes. The paroxysmal EEG activity observed at higher doses of the drug was first recorded in the cortical areas and then spread to the subcortical structures. No change in electrical activity could be observed in the spinal cord. The paroxysmal activity was associated with tonic-clonic convulsions and scialorrea. The EEG and behavioural manifestations were inhibited by administration of Ro 15-1788. This drug at doses of 0.6 and 6 mg/kg antagonized the effects of Ro 5-4864 at doses of 0.6-5 mg/kg and 6-7 mg/kg, respectively. This effect began 1-3 min after administration of the antagonist, and led to EEG synchronization. These data suggest that in rabbits the convulsant effect of Ro 5-4864 is due to interference of the drug at the GABA-BDZ-picrotoxin receptor oligomeric complex. Such an effect seems to be mediated at least in part by central BDZ receptors.

RO5-4864, a ligand for benzodiazepine micromolar and peripheral binding sites: antagonism and enhancement of behavioural effects.

RO5-4864, a ligand for both the peripheral and for the central nervous system micromolar benzodiazepine binding sites, was investigated in the holeboard, alone and in combination with several other drugs. RO5-4864 alone caused a marked reduction in rears and motor activity and reduced head-dipping when objects were placed under the holes. All these reductions were enhanced by picrotoxin (2 and 4 mg/kg) and by CGS 8216 (3 mg/kg). RO15-1788 (10 mg/kg) reversed the reduction in rears and PK11195 (30 mg/kg), a putative antagonist for the peripheral binding site, reversed the reduction in head-dipping. The results are discussed in terms of the various benzodiazepine binding sites and possible non-specific drug effects.

Nootropic nefiracetam inhibits proconvulsant action of peripheral-type benzodiazepines in epileptic mutant EL mice.

Piracetam and structurally related nootropics are known to potentiate the anticonvulsant effects of antiepileptic drugs. It remains to be seen, however, whether these nootropics inhibit proconvulsant actions of many toxic agents including Ro 5-4864, a specific agonist for peripheral-type benzodiazepine receptors (PBR). The present study was designed to address this issue using EL mice, an animal model of epilepsy. In behavioral pharmacological experiments, EL mice were highly susceptible to convulsions induced by Ro 5-4864 (i.p.) in comparison with nonepileptic DDY mice. Nefiracetam administered orally to EL mice inhibited spontaneous seizures. In DDY mice, convulsions induced by Ro 5-4864 were prevented by nefiracetam when administered by i.v. injection. Aniracetam (i.v.) was partially effective, but piracetam and oxiracetam were ineffective as anticonvulsants. Binding assay for brain tissues revealed a higher density of mitochondrial PBR in EL mice compared with DDY mice. Binding of the PBR ligands Ro 5-4864 to either EL or DDY mouse brain was inhibited by micromolar concentrations of these nootropic agents in the sequence of nefiracetam > aniracetam >> oxiracetam, piracetam. This rank order is identical to potency as anticonvulsants. These data suggest that nefiracetam may prevent toxic effects of PBR agonists through interacting with PBR.

The anxiogenic action of Ro 15-1788 is reversed by chronic, but not by acute, treatment with chlordiazepoxide.

The benzodiazepine receptor antagonist, Ro 15-1788 (10 mg/kg) is anxiogenic in the social interaction test. Acute administration of chlordiazepoxide (5 or 10 mg/kg) did not reverse the anxiogenic effects of Ro 15-1788; however, in animals pretreated with chlordiazepoxide (5 mg/kg) for 5 days prior to testing, there was a reduction in the anxiety produced by Ro 15-1788.

A partial agonist at the anticonvulsant benzodiazepine receptor: reversal of the anticonvulsant effects of Ro 15-1788 with CGS-8216.

A benzodiazepine antagonist (Ro 15-1788) prevents the development of kindled seizures. CGS-8216, another benzodiazepine antagonist, prevents DMCM-induced seizures (indicating that CGS-8216 acts at a benzodiazepine receptor) but has no effect on kindling or kindled seizures. CGS-8216 prevents the anticonvulsant actions of Ro 15-1788 suggesting that Ro 15-1788 is a partial agonist at an anticonvulsant benzodiazepine receptor. These results support that idea of of distinct, separate receptors for anticonvulsant and sedative effects of benzodiazepines.

The Ro 15-1788 cue: evidence for benzodiazepine agonist and inverse agonist properties.

Rats discriminating Ro 15-1788 (10 mg/kg, i.p.) from vehicle completely generalized this cue to typical benzodiazepines, and partially generalized it to barbiturates, pentylenetetrazol, CGS 8216, beta-CCM and PK 8165. CL 218872, Ro 5-4864, phenytoine, progabide, propranolol, yohimbine and various CNS stimulants predominantly induced vehicle-appropriate responding. Chlordiazepoxide, pentobarbital, CL 218 872, PK 8165, pentylenetetrazol, CGS 8216 and beta-CCM failed to block the Ro 15-1788 cue. Generalization to both anxiolytics/anticonvulsants and anxiogenics/(pro) convulsants suggests that Ro 15-1788 has benzodiazepine agonist and inverse agonist properties.

Effect of benzodiazepine and beta-carboline antagonists and partial agonists on loprazolam- and ZK 93423-induced hypothermia.

The pharmacology of the hypothermia induced by benzodiazepine and beta-carboline full agonists in mice has been investigated using partial agonists and antagonists from both chemical series. The benzodiazepine antagonist flumazenil (10 mg/kg i.p.) blocked the hypothermia induced by loprazolam (3 mg/kg i.p.) but not that induced by the beta-carboline ZK 93423 (3 mg/kg i.p.). Both hypothermic responses were reduced by the beta-carboline antagonist ZK 93426 (3 mg/kg i.p.) and the benzodiazepine partial agonist Ro 17-1812 (10 mg/kg i.p.). On the other hand, the beta-carboline partial agonist ZK 91296 (30 mg/kg i.p.) blocked ZK 93423-hypothermia but not that induced by loprazolam. Thus, the hypothermic actions of benzodiazepine and beta-carboline agonists appear to be differentially antagonised by antagonists and partial agonists of the two chemical classes suggesting receptor subtype interactions which are non-uniformly related to the chemical class. These results cannot be explained simply in terms of pharmacokinetics or thermoregulatory effects of the compounds themselves. They suggest that, at least in the hypothalamus, different subtypes of benzodiazepine receptor may exist.

Modulatory mechanisms of cyclic AMP-stimulated steroid content in rat brain cortex.

The modulation of cyclic AMP dependent neurosteroidogenesis was studied in minces prepared from the cerebral cortex of adult rat. Forskolin or dibutyryl-cyclic AMP enhanced pregnenolone and progesterone production in a time and dose-dependent manner. The forskolin effect was mimicked by the cyclic AMP phosphodiesterase inhibitor isobutyl-methyl-xanthine, but not by the adenylate cyclase inactive forskolin analogue 1,9,dideoxy-forskolin. 4'-Chloro-diazepam, a high affinity ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor, also elicited a time dependent increase in steroidogenesis. The forskolin and the 4'-chloro-diazepam stimulated pregnenolone increase was prevented by preexposing the rat brain cortical minces to 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide (PK 11195), a high affinity ligand for the mitochondrial DBI receptor endowed with antagonistic properties. The protein synthesis inhibitor cycloheximide prevented the forskolin and 4'-chloro-diazepam stimulation of pregnenolone formation. In brain cortical minces of adrenalectomised/orchiectomised rats dibutyryl-cyclic AMP increased both pregnenolone and progesterone formation, while forskolin only increased progesterone. These data show that cyclic AMP enhances brain steroidogenesis by acting on a labile protein substrate which interacts with the mitochondrial DBI receptor.

The anxiogenic action of Ro 5-4864 is reversed by phenytoin.

Ro 5-4864, a selective ligand for the benzodiazepine micromolar and peripheral receptors, had a significant dose-related (5-20 mg/kg) anxiogenic action in the social interaction test of anxiety. This anxiogenic effect was reversed by phenytoin (10 mg/kg), a ligand for the micromolar receptors, but unaffected by the isoquinoline PK 11195 (10 mg/kg), a ligand for the peripheral receptors.

Physostigmine antagonizes benzodiazepine-induced myoclonus in the baboon, Papio papio.

The antagonism of some benzodiazepine (Bz) actions by physostigmine was investigated in 4 Papio papio baboons. As a model of these actions, the myoclonus induced in this species by clonazepam i.m. administration was used. The baboon develops, 20-30 min after Bz i.m. injection, a non-epileptic myoclonus characterized by clinical symptomatology (jerks involving mainly the neck and the trunk bilaterally), by the absence of any correlative EEG discharge, and by its facilitation during movement. This Bz-induced myoclonus resembles the intention myoclonus of human patients, as seen for example after anoxia. In the present series, the effect of physostigmine i.v. injection on the frequency of clonazepam-induced myoclonus was tested. Physostigmine produces a rapid and total abolition of the myoclonus, and this effect lasts for a period which corresponds to the pharmacological activity of physostigmine. On the contrary, atropine i.v. injection considerably increases the amount of Bz-induced myoclonus. These results allow the existence of an anticholinergic action of benzodiazepines, reversed by physostigmine, and the theory that the myoclonus would be the consequence of a cholinergic system depression to be hypothesized.

Convulsant effect of Ro 5-4864, a peripheral type benzodiazepine, on the baboon (Papio papio).

The effects of Ro 5-4864, a 1,4-benzodiazepine with a high affinity for the peripheral-type benzodiazepine (Bz) binding site, were investigated in the baboon (Papio papio), which is genetically predisposed to epilepsy. A proconvulsant effect of low doses (1-3 mg/kg, i.v.) of Ro 5-4864 was observed by studying its effect on the photic responses induced by intermittent light stimulation in non-photosensitive baboons. Higher doses of Ro 5-4864 (10 mg/kg, i.v.) were overtly convulsant. The Bzs clonazepam and diazepam blocked these convulsant actions of Ro 5-4864 whereas neither Ro 15-1788, an antagonist of central Bz binding sites, nor PK 11 195, an antagonist of peripheral Bz binding sites, had any effect. It thus appeared that the convulsant effect of Ro 5-4864 was not mediated by Bz binding sites of either the central or the peripheral type. It is possible that Ro 5-4864 exerts its convulsant action at the picrotoxin site of the central Bz receptor - gamma-aminobutyric acid receptor-chloride ionophore complex.

RO 15-1788 selectively reverses antagonism of pentylenetetrazol-induced discriminative stimuli by benzodiazepines but not by barbiturates.

The specificity of ethyl 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-a) (1,4) benzodiazepine-3-carboxylate (RO 15-1788) in reversing the effectiveness of diazepam and des-methylclobazam, but not of pentobarbital, in antagonizing discriminative stimuli produced by pentylenetetrazol is described. Male hooded rats were trained to discriminate pentylenetetrazol-induced interoceptive discriminative-stimuli (IDS) in a two-lever choice paradigm on an FR10 schedule of food reinforcement. These IDS pharmacologically model verbal report of anxiogenic activity in humans. Diazepam (1,4 benzodiazepine), des-methylclobazam (1,5 benzo-diazepine), and pentobarbital antagonized pentylenetetrazol-IDS. RO 15-1788 neither generalized to nor antagonized pentylenetetrazol-IDS. It also did not cause convulsions in pentylenetetrazol sensitized rats at doses up to 40 mg/kg. It did, however, antagonize the action of diazepam (10 mg/kg) as well as that of des-methylclobazam (160 mg/kg) but not that of pentobarbital. These data suggest that RO 15-1788 is not an anxiomimetic, anxiolytic or a convulsant drug, but it is a specific and effective antagonist of anxiolytic action of benzodiazepines.

Opposite effects of an agonist, RO5-4864, and an antagonist, PK 11195, of the peripheral type benzodiazepine binding sites on audiogenic seizures in DBA/2J mice.

Two compounds with high affinity for the "peripheral type" benzodiazepine binding sites, PK 11195 (an isoquinoline derivative) and RO5-4864 (a benzodiazepine derivative) can modify the sensitivity of DBA/2J mice to audiogenic seizures. RO5-4864 (1-15 mg/kg) facilitates in a dose-dependent manner the audiogenic seizures and PK 11195 (2-5 mg/kg) antagonizes the RO5-4864 effects. At these doses PK 11195 alone does not modify the sensitivity to audiogenic seizures, but at doses between 20-80 mg/kg it protects DBA/2J mice against audiogenic seizures. By contrast PK 11195 is inactive against the facilitation of audiogenic seizures by ethyl-beta-carboline-3-carboxylate (a brain benzodiazepine receptor inverse agonist) and against the seizure elicited in absence of noise stimuli by RO5-4864 at doses between 20-40 mg/kg. These results suggest that facilitation by RO5-4864 of the audiogenic seizures and its antagonism by PK 11195 are mediated by the peripheral type benzodiazepine binding sites and agree with the thermodynamic analysis of the binding data which suggested that RO5-4864 might be an agonist and PK 11195 an antagonist. The good correlation between pharmacological effects and the occupancy degree of the binding sites as measured by the displacement of the "in vivo" [3H]-PK 11195 binding give an additional support to binding sites mediated effects.