Role of benzodiazepine receptors in realizing the anxiolytic effect of compounds on intact rats and on animals with a physical dependence ethanol

Anxiolytic activity of DSIP, sodium hydroxybutyrate, nicotinoyl-GABA, mebicar, some derivatives of aminoandrostane and beta-carboline was not, like in the case of diazepam and beta C-3CEE, related to benzodiazepine receptors. The degree of the decrease in anxiolytic activity of these compounds did not correspond to increasing Ki binding of 3H diazepam in alcoholic rats.     

Characteristics of the psychotropic spectrum of action of mebicar

Under group interaction in cats, a new Soviet tranquilizer mebicar eliminates fear-alarm induced by stimulatin of the emotiogenic zone of the hypothalamus. This action is not associated with myorelaxant or hypnotic action. Mebicar decreases the brain noradrenaline level, exerts no effect on the dopaminergic systems, increases the brain serotonin level, and does not elicit cholinolytic action.     

Involvement of GABA-A receptor chloride channel complex in isolation stress-induced free choice ethanol consumption in rats

The present study revealed the effect of diazepam, a benzodiazepine, and progesterone, a pregnane precursor of neurosteroids, which act via modulating GABA-A chloride channel complex on the isolation stress-induced free choice ethanol consumption in adult rats. Isolation stress for 24 hr over a period of 6 days produced a significant increase in ethanol consumption, which persisted during the 6-day recovery period. Pretreating the animals with diazepam (5 mg/kg, i.p.), or progesterone (5 mg/kg, i.p.), blocked the isolation stress-induced increase in ethanol consumption. Bicuculline (2 mg/kg, i.p.), a GABA-A receptor antagonist significantly attenuated the effect of both diazepam and progesterone on stress-induced modulation of ethanol consumption. Isolation stress also caused an increase in total fluid consumption, which was antagonised by both diazepam and progesterone. Like ethanol consumption, this effect of diazepam and progesterone on isolation stress-induced increase in total fluid consumption was attenuated by bicuculline. Neither diazepam nor progesterone produced an increase in ethanol consumption in non-stressed rats. However, unlike diazepam, progesterone administration to non-stressed rats caused a significant increase in total fluid consumption. Results of the present study thus show that GABAergic mechanisms may be playing an important role in isolation stress-induced increase in ethanol consumption.     

An endogenous inhibitor of GABA receptor binding

An endogenous inhibitor of GABA receptor binding was prepared from synaptic membrane of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA for GABA binding sites. The inhibition of GABA receptor binding by the endogenous inhibitor was blocked by the allosteric effect of diazepam. In the presence of diazepam, specific [³H]GABA binding was greater in a medium containing the endogenous inhibitor than in one containing an equal inhibitory potency of GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself.     

In Vivo Administration of Ethanol Enhances the Function of the γ-Aminobutyric Acid-Dependent Chloride Channel in the Rat Cerebral Cortex

The effect of in vivo administration of ethanol on the gamma-aminobutyric acidA (GABAA) receptor-coupled chloride channel was studied by measuring ex vivo t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding in the rat cerebral cortex. Intragastric administration of ethanol (0.5-1 g/kg) elicited in 40 min a significant decrease of [35S]TBPS binding to unwashed cortical membrane preparations, an effect mimicked by diazepam (0.5-1 mg/kg, i.p.). However, Scatchard plot analysis indicated that, unlike the case with diazepam, the decrease was entirely due to a reduction in the apparent affinity of [35S]TBPS receptors with no change in the total number of binding sites. Moreover, ethanol, like diazepam, reduced the increase of [35S]TBPS binding elicited by isoniazid (350 mg/kg, s.c.), an inhibitor of the GABAergic transmission. Finally, ethanol markedly potentiated the inhibitory action of diazepam on [35S]TBPS binding. The results suggest that ethanol, like benzodiazepines, enhances the function of the GABAA-coupled chloride channel.     

Marked variation in diazepam sensitivity in Swiss albino mice

Using the righting reflex as the critical level, sleep was measured in Swiss albino mice at a dose of 35 mg/kg diazepam, i.p. Sleep times varied markedly from zero to 120 min with a mean +/- s.d. of 44 +/- 37 (N = 202). The distribution is skewed to the left with a coefficient of skewness of 0.33 +/- 0.17. The sleep times of the two sexes, when analyzed separately, showed similar range, mean and s.d., except that the distribution tended to be more clearly bimodal in males than in females. These animals also exhibited marked variations in their response to either ethanol (4 g/kg) or pentobarbital (45 mg/kg). The diazepam sleep time failed to correlate with the ethanol sleep time. Significant correlation, however, was obtained between diazepam and pentobarbital sleep times. On further analysis with least-squares fit to a straight line, the data yielded a line with a slope of 0.16; thus despite the correlation reaching a significant level, there is no significant difference in the pentobarbital sleep times between mice that have the longest or the shortest diazepam sleep times. By monitoring the plasma and brain levels of diazepam and N-desmethyldiazepam in mice at awakening, it was found that the variations in sleep time cannot be explained by individual differences in drug disposition. The phenomenon is discussed in terms of individual variations in diazepam sensitivity and the possibility of development of tolerance to diazepam almost immediately after diazepam administration.     

Cannabinoids and Their Interactions with Diazepam on Modulation of Serum Corticosterone Concentration in Male Mice

Experimental results indicate a mutual interaction between cannabinoidergic and GABAergic systems; however, the interaction between these systems on corticosterone release has not been fully investigated. In this study, we treated male mice with either cannabinoid compounds alone or in combination with diazepam. Blood samples were collected at 60 min post-injection. The serum corticosterone (CORT) level was measured using ELISA technique. Acute treatment of mice by cannabinoid receptor agonist WIN55212-2 (2.5 mg/kg; i.p.) resulted in a significant reduction of CORT, while treatment with either endocannabinoid reuptake inhibitor AM404 or endocannabinoid degradation enzyme inhibitor URB597 increased CORT compared to control group. Co-administration of AM404 or URB597 with cannabinoid CB1 receptor antagonist AM251 blocked the effect of these compounds on CORT. Treatment of mice with different doses of diazepam alone did not alter CORT compared to control group. However, co-administration of diazepam and either AM404 or WIN55212-2 significantly reduced CORT compared to the respective group treated with cannabinoid compound alone. Co-administration of ineffective dose of URB597 and ineffective dose of diazepam increased CORT level compared to groups treated with each compound alone. In conclusion, our findings suggest that the endogenous cannabinoid system is active as a modulator of CORT in mice and diazepam can alter the effect of cannabinoid system in the modulation of neuroendocrine functions.     

Positive modulation of diazepam activity by cortexolone in alcoholic rats

It has been shown that glucocorticoid receptor antagonist-cortexolone--increased anxiolytic action of diazepam in alcoholic rats. Neither diazepam (2 mg/kg), nor cortexolone (20 mg/kg) alone influenced voluntary ethanol consumption in alcoholic rats during 14 days of administration, however, combined administration of diazepam and cortexolone diminished ethanol consumption. Receptor and permissive mechanism of gluco- and antiglucocorticoid effect on the action of diazepam are being discussed.     

Effect of ethanol on the arachidonic acid metabolism in mouse peritoneal macrophages

Macrophages play an important role in the development of chronic inflammatory states. Ethanol has been shown to impair a number of membrane-linked phenomena. The synthesis and secretion of oxygenated metabolites of arachidonic acid is triggered at the cytoplasma membrane level. The present study was carried out in order to investigate the effect of ethanol on the arachidonic acid metabolism in mouse peritoneal macrophages. Two types of experiments were performed: with endogenous radiolabeled arachidonic acid and with exogenously added radiolabeled arachidonic acid. Our data show that ethanol in vitro activates the release of arachidonic acid from intracellular pools, while the proportion of endogenous substrate metabolized in the presence of ethanol is similar to that in controls. From the exogenous it seems clear that ethanol induces different effects depending whether the arachidonic acid is endogenous or added exogenously.     

Ethanol and diazepam withdrawal convulsions are extensively codetermined in WSP and WSR mice

Selective breeding was used to produce lines of mice which differ markedly in their genetically-mediated vulnerability to handling-induced convulsions (HIC) associated with the ethanol withdrawal syndrome. These are known as the ethanol withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) selection lines. As a result of 5 generations of selective breeding with ethanol, a 3.4-fold difference between WSP and WSR mice was seen in HIC associated with ethanol withdrawal. When diazepam was used as the dependence-producing drug, a 2.4-fold difference emerged. After 6 more generations of selective breeding with ethanol, an approximate 10-fold difference was seen with ethanol, while with diazepam, this difference in HIC scores was also about 10-fold. This close parallel between ethanol and diazepam indicates that physical dependence on both drugs, as indexed by handling-induced convulsions, is extensively codetermined by the same genes, and thus by the same mechanisms, in these selectively-bred mice.     

A Comparative Study of UV-Spectrophotometry and First-Order Derivative UV-Spectrophotometry Methods for the Estimation of Diazepam in Presence of Tween-20 and Propylene Glycol

Nonionic surfactants like polysorbates (Tweens) and co-surfactant like propylene glycol are used in pharmaceutical dosage forms, like microemulsion of diazepam. These additives interfere significantly with the estimation of diazepam by UV spectrophotomery method. The aim of this work was to develop a first-order derivative UV-spectrophotometry method that can estimate diazepam in presence of Tween-20 and propylene glycol. The experimental results clearly suggested that, in comparison with the UV-spectrophotometry method, the first-order derivative UV-spectrophotometry is a simple method to estimate diazepam with sufficient accuracy, specificity, and precision even in the presence of 282-times Tween-20 and 2,072-times propylene glycol.     

Neurophysiological analysis of the correction by nootropic substances of disorders in the bioelectric activity of the brain in animals during chronic administration of ethanol

The influence of some drugs (piracetam and 3-oxypyridine derivative) having a nootropic effect on ethanol-induced changes of bioelectrical activity was studied in experiments on freely moving rats. Discontinuation of ethanol administration (1, 2 g/kg, i.p. for 40 days) has been found to provoke destructuring of Fourier's spectral power of sensorimotor cortex and dorsal hippocamp on the EEG. Long-term administration of piracetam or 3-oxypyridine derivative (300 and 50 mg/kg, respectively, i.p. for 40 days) with ethanol has a protective effect and normalizes EEG at the cortical level. The authors discuss possible neurophysiological mechanisms of nootropic drug action in ethanol-induced pathology.     

Use of the overturn end point in goldfish to measure the interaction of diazepam and ethanol and the effects of the binding of diazepam to bovine serum albumin

Over the ranges 2.8 X 10(-5) to 8.78 X 10(-5) M diazepam and 4.85 X 10(-2) to 1.22 X 10(-1) M ethanol, addition of the effects of these agents on the overturn end point in goldfish was observed. The addition of bovine serum albumin (1.56 X 10(-5) M) to aqueous solutions of diazepam modifies the diazepam effect by reducing the "free" drug concentrations.     

Interaction of piracetam with 3H-imipramine binding sites and the GAMA-benzodiazepine receptor complex of brain membranes

Piracetam at a concentration of 10(-6) M was shown to behave as a noncompetitive inhibitor of 3H-imipramine specific binding to rat brain membranes. At the same time piracetam failed to influence specific binding of 3H-mianserin to membranes of guinea-pig cerebellum, which is indicative of its inability to suppress histamine H1 receptors, a component of 3H-imipramine specific binding sites. At a concentration of 10(-4) M piracetam does not change specific binding of 3H-flunitrazepam to rat hippocampal membranes in the absence of GABA, but in the presence of 5 X 10(-5) M GABA, like atypical tranquilizer mebicar, acts as a competitor of 3H-flunitrasepam binding. Though Ro-15 1788 did not suppress anxyolytic piracetam (and mebicar) effect, our results give evidence of a possible involvement of GABA-benzodiazepine supramolecular complex in the anxiolytic activity of piracetam.     

Acetaldehyde Is a Causal Agent Responsible for Ethanol-Induced Ripening Inhibition in Tomato Fruit

Inhibition of tomato (Lycopersicon esculentum Mill.) fruit ripening by exogenously applied ethanol was shown to be caused by elevated endogenous levels of acetaldehyde (AA). Exposure of excised pericarp discs of mature-green tomato fruit to ethanol or AA vapors produced elevated levels of both compounds in the tissue, but only the levels of AA were associated with ripening inhibition. Ripening inhibition was dependent on both the applied concentration and the duration of exposure. Discs treated with inhibitory levels of AA had levels of ethanol that were elevated but below that associated with inhibition of ripening. The in vivo activity of alcohol dehydrogenase was inhibited 40 to 60% by 4-methylpyrazole (4-MP), a competitive inhibitor of this enzyme. The inhibitory effect of ethanol on ripening was reduced by the simultaneous application of 4-MP. Tissue treated with 4-MP plus AA vapors had higher endogenous levels of AA and ripening was inhibited longer than in tissue without 4-MP. The tissue AA level resulting from ethanol or AA application appears to be the critical determinant of ripening inhibition.     

Anxiolytic effects of Equisetum arvense Linn. extracts in mice

The petroleum ether (PE), chloroform (CH), ethanol (ETH) and water extracts of E. arvense stems were evaluated for anti-anxiety activity in mice using elevated plus maze model. Ketamine induced hypnosis and actophotometer was used to evaluate sedative effect with various extracts in mice. The results were compared with standard drug diazepam. The ethanolic extract of E. arvense (50 and 100 mg/kg) significantly increased the time-spent and the percentage of the open arm entries in the elevated plus-maze model which was comparable to diazepam. Ethanolic extract (100 mg/kg) prolonged the ketamine-induced total sleeping time and decreased the locomotor activity in mice. The results suggest that the ethanolic extract of E. arvense seems to possess anxiolytic effect with lower sedative activity than that of diazepam. The results could be attributed to the flavonoid content of the ethanolic extract.     

Antagonism of diazepam-induced feeding in rats by antisera to opioid peptides

Diazepam-induced feeding in rats is antagonized not only by the opiate antagonist naloxone but also intraventricular administration of specific antisera to the endogenous opioid peptides met-enkephalin or beta-endorphin. Pituitary beta-endorphin is probably not implicated in the diazepam effect since blockade with the glucocorticoid dexamethasone of the release of beta-endorphin from the anterior pituitary does not modify the diazepam-induced feeding, which is however prevented by TRH, a suggested physiological antagonist of some of the effects of opioid peptides. The possible central participation of both beta-endorphin and met-enkephalin in the ingestive behavior induced by diazepam gives further support to the postulated physiological role of endogenous opioids in appetite regulation.     

Possible involvement of GABA mechanisms in central cardiovascular and respiratory control. Studies on the interaction between diazepam, picrotoxin and clonidine

Various doses (0.1-1 mg/kg) of diazepam were given to chloralose anesthetized rats, with both systemic (i.p.) and central injections being tested. Arterial pressure, heart rate, respiration depth and frequency were recorded. Diazepam caused a dose-dependent decrease in the arterial pressure after systemic administration and also decreased it after central administration. However, only intraventricular but not intracisternal injections of diazepam were effective. The hypotensive effect of systemic diazepam was competitively counteracted by pretreatment with picrotoxin, a putative gamma-aminobutyric acid receptor blocking agent. The hypotensive effect of the centrally acting alpha-adrenoreceptor agonist clonidine was not influenced by picrotoxin pretreatment. The effect of diazepam on heart rate was inconsistent. Diazepam caused a reduction of respiratory frequency, which was not counteracted by picrotoxin pretreatment. It is concluded that central gabergic mechanisms are to some extent involved in the hypotensive effect of diazepam, probably at a supramedullary level. The hypotensive effect of a threshold dose of diazepam was blocked by a small dose of clonidine. Likewise, diazepam pretreatment could counteract the hypotension, bradycardia and respiratory frequency reduction caused by a threshold dose of clonidine. These results suggest that gabergic and/or other benzodiazepine-sensitive receptors may interact with alpha-adrenoreceptors in the control of central cardiovascular and respiratory mechanisms.     

Effect on Root Growth of Endogenous and Applied IAA and ABA: A Critical Reexamination

Applications of indole-3yl-acetic acid (IAA) and abscisic acid (ABA) were done on two-day-old intact maize (cv LG 11) roots. The effect of the treatment on the root growth depends on their initial elongation rate. The slow growing roots were all inhibited by exogenous IAA and ABA at any concentrations used whereas for the fast growing roots their elongation was promoted by these two hormones at low concentrations. Quantitative analyses of endogenous IAA and ABA were performed using the gas chromatography-mass spectrometry technique. Detection and quantification of endogenous IAA and ABA were done on the zone of the root implicated in elongation. These techniques were achieved by electron impact on the IAA-Me-heptafluorobutyryl derivative and by negative ion chemical ionization with NH₃ on the ABA-Me ester derivative. A negative correlation between the growth and the endogenous content of these two hormones was obtained. ABA presented a larger range of endogenous level than IAA on the whole population of roots tested. When using applied IAA and ABA at different concentrations the same differentiating effect on the growth was observed. This allowed us to conclude that for identical concentrations, IAA has a more powerful effect on root elongation than ABA. Present results are discussed in relation to previous data related to the role of IAA and ABA in the growth and gravireaction of maize roots.     

Tofizopam selectively increases the action of anticonvulsants

The effect of tofizopam, a 3,4-benzodiazepine (BZ) derivative, in modulating the anticonvulsive action of various drugs was investigated in mice. Electric shock and intravenous infusion of bicuculline were used as convulsive agents. Tofizopam increased the action of clonazepam, diazepam and flunitrazepam against bicuculline. The anticonvulsive effect of diazepam against electroshocks was augmented only slightly. Tofizopam failed to alter the actions of carbamazepine, phenobarbital, phenytoin, or sodium valproate against either of the convulsive stimuli. Both in vitro and in vivo, tofizopam has been shown to stimulate the binding of 1,4-BZs (e.g., flunitrazepam) to BZ receptors. Similarly, tofizopam enhances the binding of muscimol to GABA receptors. Although several anticonvulsants act on the GABA-BZ receptor complex, tofizopam seems to modify selectively the anticonvulsive action of 1,4-BZs, and this effect is seen better in bicuculline-induced seizures than in electroshocks.