No changes in rat benzodiazepine receptors after withdrawal from continuous treatment with lorazepam and diazepam.

Five and 11 days after withdrawal from 8 weeks of treatment with 90 mg/kg/day of diazepam p.o. or 60 mg/kg/day of lorazepam p.o. there were no consistent changes in the number of benzodiazepine receptors or apparent affinity $\text{in vitro}$ for ³H-diazepam at 0°C in rat forebrain membranes. Daily exposure of rats from 10 days before birth until 7 days after birth was also without gross effects on the benzodiazepine receptor. Abstinence and tolerance to benzodiazepines were thus not attributable to changes in brain benzodiazepine receptors.     

Benzodiazepines stimulate sodium ion transport in frog skin epithelium

Benzodiazepine binding sites are present in a variety of non-neuronal tissues including the kidney where they are localized to distal nephron segments. It is postulated that renal binding sites are involved in modulating ion transport. This study examined the effects of two benzodiazepines on sodium transport in frog skin epithelium, a model system for sodium transport in renal collecting duct. Treatment of short-circuited frog skin with diazepam (a non-selective benzodiazepine agonist) stimulated amiloride-sensitive short-circuit current, reflecting stimulation of active sodium transport. The diazepam response was equally effective with either serosal or mucosal application of the drug. Maximal stimulation of the current (42 +/- 8%) was achieved with 10 microM diazepam (serosal). Short-circuit current was similarly augmented by serosal or mucosal addition of Ro5-4864, a benzodiazepine agonist with selective activity at peripheral (non-neuronal) receptors. The natriferic response to diazepam was additive to that of vasopressin or cyclic AMP suggesting that the mode of action of benzodiazepines is probably distinct from the cyclic AMP pathway. Thus, frog skin appears to be a useful model to examine the epithelial effects of benzodiazepines. Whether stimulation of sodium transport, however, involves peripheral-type benzodiazepine receptors in this tissue requires further studies.     

Diazepam Increases γ-Aminobutyric Acid in Human Cerebrospinal Fluid

In 11 neurological patients, levels of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) were determined in cerebrospinal fluid (CSF) before and 1, 3, 5, and 8 min after intravenous injection of diazepam (2 or 5 mg). GABA levels increased progressively after intravenous injection of 5 but not 2 mg of the benzodiazepine, the differences from preinjection values being significant at 3, 5, and 8 min. Furthermore, when relative CSF GABA alterations determined after injection of diazepam were compared to those determined in sequential CSF aliquots of 10 patients without diazepam injection, mean GABA increases after diazepam were significantly different from controls in all CSF fractions. The data suggest that, in addition to its well-known effects on postsynaptic GABA function, diazepam may exert effects on endogenous GABA concentrations and/or on GABA release in the human CNS as reflected by elevation of GABA levels in human CSF.     

Increased expression of peripheral benzodiazepine receptors and diazepam binding inhibitor in human tumors sited in the liver

The peripheral benzodiazepine receptor system triggers intracellular metabolic events and has been associated with cell proliferation. Its endogenous ligand, the diazepam binding inhibitor, contributes to steroidogenesis by promoting cholesterol delivery to the inner mitochondrial membrane. The present study was undertaken to verify whether this system is altered in tumors sited in the liver. Peripheral benzodiazepine receptors and diazepam binding inhibitor were studied using immunocytochemistry and in situ hybridization in 9 human tumors sited in the liver, in liver hyperplasia, cirrhotic nodular regeneration, intestinal adenocarcinoma and in surrounding non-tumoral tissue. Immunocytochemical staining and in situ hybridization demonstrated that peripheral benzodiazepine receptors and diazepam binding inhibitor were more prominently expressed in neoplastic cells than in non-tumoral tissue. They were present in the same cells, suggesting that diazepam binding inhibitor may act in an intracrine manner in these cells. Higher peripheral benzodiazepine receptors and diazepam binding inhibitor expression in tumor cells suggest an implication of this system in the metabolism of neoplastic cells. Furthermore the evaluation of peripheral benzodiazepine receptor and diazepam binding inhibitor expression might be useful in evaluating malignancy and in diagnostic approaches of tumors in liver tissue.     

Effect of multiple daily administration of fenibut and diazepam on GABA and benzodiazepine receptors in the mouse brain

It was shown in experiments on mice that 25 hours after chronic treatment with fenibut (100 mg/kg, twice daily for 10 days) was discontinued the number of benzodiazepine and GABAA (bicucullin-sensitive) receptor sites was increased and 48 hours after treatment discontinuation the number of GABAB (bicucullin nonsensitive) sites was decreased. The enhanced binding to GABAA and GABAB receptor sites and the decreased binding to benzodiazepine receptors was observed 24 hours after discontinuation of chronic treatment with diazepam (5 mg/kg, twice daily). Forty-eight hours after diazepam chronic treatment was discontinued the number of benzodiazepine receptor sites was increased. The involvement of the increased benzodiazepine receptor sensitivity in the mechanism of therapeutic activity of fenibut is suggested.     

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.     

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.     

Inactivation of Benzodiazepine Binding Sites by N-Ethylmaleimide

Abstract: The benzodiazepine receptors of bovine brain membranes have been identified by the specific binding of radiolabeled [³H]diazepam. Pretreatment of membranes with N -ethylmdleimide causes a dose- and time-dependent decrease of 45 to 60% in the number of binding sites. No decrease occurs when membranes are pretreated with N -ethylmaleimide before administration, or in the presence, of diazepam. Binding of [³H]diazepam to the remaining sites occurs with the same characteristics as binding to the untreated receptor population.     

Tofizopam enhances the action of diazepam against tremor and convulsions

Tofizopam, an anxiolytic 3,4-benzodiazepine, increases the affinity of benzodiazepine receptors for 1,4-benzodiazepines. In this study we investigated whether this increased affinity of the receptors alters the sensitivity of mice to tremor and to convulsions. Convulsions induced by harmane were not affected by tofizopam (50-300 mg/kg), but diazepam (15 mg/kg) increased the ED50 of harmane from 9.9 to 25.1 mg/kg. Tofizopam did not alter the threshold for electroshock-induced convulsions, while a dose of 10 mg/kg diazepam protected mice from convulsions. Low doses of tofizopam (12.5-25 mg/kg) sensitized mice to the tremorogenic effect of harmaline. Diazepam inhibited tremor: the ED50 of harmaline increased by 153% after 50 mg/kg of diazepam. In contrast to 1,4-benzodiazepines, tofizopam has no anticonvulsive effect. It sensitises mice to the tremor induced by harmaline. In combination with diazepam, however, tofizopam enhanced the anticonvulsive and antitremorogenic actions of this 1,4-benzodiazepine by 12-65%. This effect probably results from a tofizopam-induced increase in the occupation of benzodiazepine receptors.     

Brain benzodiazepine levels following intravenous administration of [34]diazepam: relationship to the potentiation of purinergic depression of central nervous system neurons

Levels of [3H]benzodiazepine were measured in rat cerebral cortex following intravenous injection of [3H]diazepam using a dose and time schedule reported to elicit a marked potentiation of the depressant effects of iontophoretically applied 5'-AMP to rat cerebral cortical neurons. The levels of [3H]benzodiazepine obtained strongly suggest (i) that blockade of adenosine uptake as a mechanism for this potentiation is not consistent with the potency of diazepam as an inhibitor of adenosine uptake in vitro, and (ii) that a potentiative interaction of adenosine and diazepam may reflect the binding of these compounds to benzodiazepine receptors.     

The Function of Male Agonistic Displays in Ursine Colobus Monkeys (Colobus vellerosus): Male Competition,Female Mate Choice or Sexual Coercion?

Male agonistic displays may allow males to assess competitors, females to assess mates, or could be directed at cycling females to sexually coerce them. We analysed the display output of 26 male ursine colobus monkeys (Colobus vellerosus) in four groups over 13‐mo at the Boabeng‐Fiema Monkey Sanctuary, Ghana. Display indices (including three behaviours, loud calls, stiff‐legs, and jump‐displays) were calculated for males in each group. Males vary in their expression of these behaviours suggesting they are sexually selected signals. We investigated the target of displays and whether display indices varied in relation to male dominance rank, eviction of other males, copulation rate, and proceptive behaviours received from females, to assess the primary function of these behaviours. Male displays decreased in vigour over time and were targeted to other groups and males. High‐ranking males displayed more than low‐ranking males. Alpha male display indices correlated with the number of other males evicted from the group. Display rates were generally higher when cycling females were present in the group. However, neither male display index nor rank correlated with copulation rates. Alpha and non‐alpha males gave cycling females equal rates of sexual solicitations; likewise cycling females showed no difference in the rates of proceptive behaviours directed towards alpha and non‐alpha males. Females mated promiscuously and did not seem to base mating decisions on male display output, although data on female hormones is needed to determine if they mate with strongly displaying males more in the periovulatory period. The male–male competition hypothesis received the greatest support, with some support for the female mate choice hypothesis. Although behaviours that appeared sexually coercive were observed, the function of male displays did not seem to be sexual coercion. Displays were rarely directed at females and males that displayed more did not have greater mating success.     

The F-loop of the GABA A receptor gamma2 subunit contributes to benzodiazepine modulation

GABA(A) receptors can be modulated by benzodiazepines, although these compounds do not directly activate or inhibit the receptors. The prototypic benzodiazepine, diazepam, potentiates responses to GABA in GABA(A) receptors that contain a gamma subunit. Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of the F-loop residues Asp(192)-Arg(197) in the GABA(A) receptor gamma(2) subunit in diazepam potentiation of the GABA response. Substitution of all of these residues with Ala and/or a residue with similar chemical properties to the wild type residue decreased the level of diazepam potentiation, and one mutation (D192A) resulted in its complete ablation. None of the mutations changed the GABA EC(50) or the [(3)H]flumazenil binding affinity, suggesting they do not affect GABA or benzodiazepine binding characteristics; we therefore propose that they are involved in the diazepam-mediated conformational change that results in an increased response to GABA. Homology models of the receptor binding pocket in agonist-bound and unbound states suggest that the F-loop is flexible and has different orientations in the two states. Considering our data in relation to these models, we find that the F-loop residues could contribute to hydrogen bond networks and hydrophobic interactions with neighboring residues that change during receptor activation.     

Effects of chronic sympathectomy on locally mediated cutaneous vasodilation in humans.

In human skin, the vasodilator response to local heating includes a sensory nerve-dependent peak followed by a nadir and then a slower, nitric oxide-mediated, endothelium-dependent vasodilation. To investigate whether chronic sympathectomy diminishes this endothelium-dependent vasodilation, we studied individuals who had previously undergone surgical T(2) sympathectomy (n = 9) and a group of healthy controls (n = 8). We assessed the cutaneous vascular response (laser-Doppler) to 30 min of local warming to 42.5 degrees C on the ventral forearm (no sympathetic innervation) and the lower legs (sympathetic nerves intact). Lower body negative pressure (LBNP) was measured to confirm sympathetic denervation. During local warming in sympathectomized individuals, vascular conductance reached an initial peak at both sites [achieving 1.73 +/- 0.22 laser-Doppler units (LDU)/mmHg in the forearm and 1.92 +/- 0.21 LDU/mmHg in the leg]. It then decreased to a nadir in the innervated leg [to 1.77 +/- 0.23 LDU/mmHg (P < 0.05)] but not in the sympathectomized arm (1.69 +/- 0.21 LDU/mmHg; P > 0.10). The maximal vasodilation seen during the slower phase was not different between limbs or between groups. Furthermore, LBNP caused a 44% reduction in forearm vascular conductance (FVC) in control subjects, but FVC did not decrease significantly in sympathectomized individuals, confirming sympathetic denervation. These data indicate that endothelial function in human skin is largely preserved after sympathectomy. The altered pattern of the response suggests that the nitric oxide-dependent portion may be accelerated in sympathectomized limbs.     

Covalent modification of GABAA receptor isoforms by a diazepam analogue provides evidence for a novel benzodiazepine binding site that prevents modulation by these drugs

Classical benzodiazepines, for example diazepam, interact with alpha(x)beta(2)gamma(2) GABA(A) receptors, x = 1, 2, 3, 5. Little is known about effects of alpha subunits on the structure of the binding pocket. We studied here the interaction of the covalently reacting diazepam analog 7-Isothiocyanato-5-phenyl-1,3-dihydro-2H-1,4-benzodiazepin-2-one (NCS compound) with alpha(1)H101Cbeta(2)gamma(2) and with receptors containing the homologous mutation, alpha(2)H101Cbeta(2)gamma(2), alpha(3)H126Cbeta(2)gamma(2) and alpha(5)H105Cbeta(2)gamma(2). This comparison was extended to alpha(6)R100Cbeta(2)gamma(2) receptors as this mutation conveys to these receptors high affinity towards classical benzodiazepines. The interaction was studied at the ligand binding level and at the functional level using electrophysiological techniques. Results indicate that the geometry of alpha(6)R100Cbeta(2)gamma(2) enables best interaction with NCS compound, followed by alpha(3)H126Cbeta(2)gamma(2), alpha(1)H101Cbeta(2)gamma(2) and alpha(2)H101Cbeta(2)gamma(2), while alpha(5)H105Cbeta(2)gamma(2) receptors show little interaction. Our results allow conclusions about the relative apposition of alpha(1)H101 and homologous positions in alpha(2), alpha(3), alpha(5) and alpha(6) with the position occupied by -Cl in diazepam. During this study we found evidence for the presence of a novel site for benzodiazepines that prevents modulation of GABA(A) receptors via the classical benzodiazepine site. The novel site potentially contributes to the high degree of safety to some of these drugs. Our results indicate that this site may be located at the alpha/beta subunit interface pseudo-symmetrically to the site for classical benzodiazepines located at the alpha/gamma interface.     

Day-old chicks categorised on latency to peck, exhibit a stable fear pattern until 15 days of age

Day-old chicks of both sexes were individually categorised on the latency to peck pebbles and termed as high latency (HL), moderate latency (ML) or low latency (LL). Anxiolytic doses of diazepam diminished the latency only in the HL category, suggesting that it is the most anxious category. At 15 days of age, the LL category showed the lowest latency to ambulate in the open-field test, the lowest immobility duration in the tonic immobility test and insensitivity to anxiolytic doses of diazepam in both behavioural tests suggesting that it is the less anxious category. The increase of the central benzodiazepine receptor density induced by acute stressors was the highest in the most anxious and/or fearful HL category. There were more females than males in the LL category and inversely in the HL category there were more males. The results suggest that the fear pattern depends on the sex and inter-individual differences within a same sex which are stable across life. This could be used as a test for fear and/or anxiety state, and useful to choose fowls with the best performance later in life.     

Tricyclic pyridones as functionally selective human GABAA alpha 2/3 receptor-ion channel ligands

A series of tricyclic pyridones has been evaluated as benzodiazepine site ligands with functional selectivity for the alpha(3) over the alpha(1) containing subtype of the human GABA(A) receptor ion channel. This investigation led to the identification of a high affinity, functionally selective, orally bioavailable benzodiazepine site ligand that demonstrated activity in rodent anxiolysis models and reduced sedation relative to diazepam.     

CGS8216 noncompetitively antagonizes the discriminative effects of diazepam in rats

The benzodiazepine antagonist properties of CGS8216 were evaluated in rats trained to discriminate between saline and 1.0 mg/kg of diazepam in a two-choice, stimulus-shock termination procedure. CGS8216 (0.3 to 100 mg/kg) administered alone, either s.c., p.o. or i.p., occasioned only saline-appropriate responding. When administered concomitantly with a constant 1.0 mg/kg dose of diazepam, CGS8216 produced dose-related decreases in drug-appropriate responding. CGS8216 was most potent by the i.p. route, and approximately tenfold less potent by the oral route. CGS8216 was dermatotoxic after s.c. administration. CGS8216 i.p. had a long duration of action. A dose of 30 mg/kg completely antagonized the discriminative effects of the 1.0 mg/kg training dose of diazepam when the antagonist was administered 8 hr before the start of the test session. In order to determine the type of antagonism by CGS8216, the dose-effect curve for diazepam was redetermined in the presence of varying doses of CGS8216 (0.3 to 3.0 mg/kg, i.p.). CGS8216 produced a dose-related rightward shift in the diazepam dose-effect curve, but also decreased the slope and appeared to decrease the maximal effect. These results are consistent with the interpretation that CGS8216 antagonizes diazepam in a noncompetitive manner. It may do so because either it interacts with a subpopulation of benzodiazepine receptors, it functions as a pseudo-irreversible antagonist due to its high affinity, or because it is an antagonist with agonist properties.     

Feedforward sympathetic coronary vasodilation in exercising dogs.

The hypothesis that exercise-induced coronary vasodilation is a result of sympathetic activation of coronary smooth muscle beta-adrenoceptors was tested. Ten dogs were chronically instrumented with a flow transducer on the circumflex coronary artery and catheters in the aorta and coronary sinus. During treadmill exercise, coronary venous oxygen tension decreased with increasing myocardial oxygen consumption, indicating an imperfect match between myocardial blood flow and oxygen consumption. This match was improved after alpha-adrenoceptor blockade with phentolamine but was significantly worse than control after alpha + beta-adrenoceptor blockade with phentolamine plus propranolol. The response after alpha-adrenoceptor blockade included local metabolic vasodilation plus a beta-adrenoceptor vasodilator component, whereas the response after alpha + beta-adrenoceptor blockade contained only the local metabolic vasodilator component. The large difference in coronary venous oxygen tensions during exercise between alpha-adrenoceptor blockade and alpha + beta-adrenoceptor blockade indicates that there is significant feedforward beta-adrenoceptor coronary vasodilation in exercising dogs. Coronary venous and estimated myocardial interstitial adenosine concentrations did not increase during exercise before or after alpha + beta-adrenoceptor blockade, indicating that adenosine levels did not increase to compensate for the loss of feedforward beta-adrenoceptor-mediated coronary vasodilation. These results indicate a meaningful role for feedforward beta-receptor-mediated sympathetic coronary vasodilation during exercise.     

Comparative study of the effect of 3-carboxy-beta-carboline methylamide and diazepam on rat behavior

The effects of benzodiazepine receptor agonist, diazepam, and inverse agonist, FG 7142, were examined. Strong antagonism between FG 7142 (10 mg/kg) and diazepam (1 mg/kg) activity was revealed in the open field test. On the other hand, both FG 7142 and diazepam inhibited isolation-induced intraspecies aggressive behaviour of rats. FG 7142 also reduced interspecies aggression of mouse-killing rats. The findings suggest that both diazepam and FG 7142 have antiaggressive properties in the isolation-induced aggression model, which are mediated by benzodiazepine receptors of the central nervous system.