Cardiac effects of benzodiazepine receptor agonists and antagonists in the isolated rat heart: a comparative study.

Effects of PK 11195 and flumazenil on cardiac responses to diazepam, clonazepam and zolpidem were compared. Coronary flow rate was increased at relatively low doses of diazepam and decreased at higher doses. Clonazepam induced a dose-dependent increase, and zolpidem a decrease of coronary flow rate. PK 11195 reduced the diazepam-induced increase of coronary flow rate, and flumazenil was ineffective. Neither antagonist evoked substantial changes in the decrease of coronary flow rate. PK 11195, and less so flumazenil, antagonized the clonazepam-induced increase. PK 11195 and flumazenil only in their highest doses suppressed and respectively potentiated the zolpidem-induced decrease. Inotropy showed a biphasic response in the presence of diazepam, i.e. an initial transient decrease, followed by a dose-dependent increase in two steps. Clonazepam induced a similar response. Zolpidem increased the inotropy. The negative inotropic response induced by diazepam did not change significantly in the presence of PK 11195 or flumazenil. The positive inotropic response was suppressed by PK 11195, and less so by flumazenil. The negative response to clonazepam was antagonized by both PK 11195 and flumazenil; the positive response was not significantly changed. In the presence of lower doses of PK 11195, the zolpidem-induced response was potentiated, whereas higher doses produced reversal; flumazenil potentiated the response. In conclusion, the results support earlier suggestions, involving receptor mechanisms with cardiac effects of benzodiazepines. Both agonists and antagonists (inter)act in a different manner, suggesting that rather ambiguous receptor mechanisms are involved in benzodiazepine effects in the heart.

Modification of cardiac actions of RO 05-4864 by PK 11195 and flumazenil in the perfused rat heart.

The benzodiazepine analogue Ro 05-4864 [chlorodiazepam] (2.10[-5] to 4.10[-4] M) induced a concentration-dependent increase of coronary flow rate (Emax 82.4% [+/- 2.2 SEM]) and an increase of contraction force (Emax 68.3% [+/- 4.7 SEM]) of the retrograde perfused, isolated Langendorff rat heart. The influence of PK 11195, antagonist of the peripheral type benzodiazepine receptor, and flumazenil (Anexate), antagonist of the central type benzodiazepine receptor, on these responses to Ro 5-4864 was studied. In concentrations of 10(-7) to 5.10(-5) M, PK 11195 significantly reduced both the increase of coronary flow rate and of contraction force, without affecting these functions by itself; the positive inotropic response produced by Ro 05-4864 was even abolished in the presence of 5.10(-5) M PK 11195. The Emax values of Ro 05-4864 on both coronary flow and inotropy were reduced significantly by PK 11195. In the presence of flumazenil, 10(-7) to 10(-5) M, both the vasodilatory and the positive inotropic response induced by Ro 05-4864 were significantly counteracted as well. The Emax values of Ro 05-4864 were reduced significantly. In conclusion, the results support earlier suggestions that it is tempting to involve peripheral type benzodiazepine receptors in cardiac actions of benzodiazepines. The finding that the centrally acting benzodiazepine antagonist flumazenil reduced the cardiac actions of Ro 05-4864 is as yet difficult to explain. On the other hand concentrations of both agonist and antagonist employed are so-much high that interference of other receptors than benzodiazepine receptors must be considered as well.

Flunarizine but not theophylline modulates inotropic responses of the isolated rat heart to diazepam.

Diazepam (2 x 10(-5)-6 x 10(-4) M) induced a concentration-dependent positive inotropic effect on the perfused rat heart which was preceded by a transient concentration-dependent negative inotropic response. The influence of the Ca(2+)-entry blocking drug, flunarizine, and the adenosine receptor blocking drug, theophylline on these inotropic responses was studied. Flunarizine in concentrations of 10(-9)-10(-6) M antagonized the positive inotropic response to diazepam significantly; the negative inotropic response was reduced as well. At the lower concentrations of diazepam the negative inotropic response was completely abolished in the presence of flunarizine. The actions of the Ca(2+)-entry blocker were related to the concentrations used. Theophylline in concentrations up to 5 x 10(-5) M did not interfere with either inotropic response to diazepam. The results suggest that Ca2+ currents in the myocardium are involved with the response of the isolated heart to diazepam. It is concluded that the finding that the negative inotropic effect of diazepam was almost abolished by flunarizine suggests that the site of this response most be associated with Ca(2+)-current mechanisms.

PK 11195 antagonizes the positive inotropic response of the isolated rat heart to diazepam but not the negative inotropic response.

The influence of the ligand PK 11195 (1-(2-chlorophenyl)-N-methyl-N-(1- methylpropyl)-3-isoquinolinecarboxamide), antagonist of the peripheral-type benzodiazepine receptor, on the inotropic response of the perfused rat heart to diazepam (7-chloro-5-phenyl-methyl- 1,3-dihydro-2H-1,4-benzodiazepin-2-one) was studied. Diazepam induced a positive inotropic response which was preceded by a transient negative inotropic response. Concentrations of 10(-7) M PK 11195 were ineffective, whereas concentrations of 10(-6) and 10(-5) M PK 11195 reduced the positive inotropic response significantly. At 5 x 10(-5) M PK 11195 the response was completely abolished. The negative inotropic response was not changed by either concentration of PK 11195 used. It is concluded that the positive inotropic response of the isolated rat heart to diazepam may well be mediated through peripheral-type benzodiazepine receptors; the negative inotropic response must be related to other (more complex) mechanisms.

Actions of benzodiazepines on the inotropy of the perfused rat heart.

The inotropic responses of four benzodiazepines (diazepam, midazolam and the more recently developed adinazolam and alprazolam) have been studied in a Langendorff heart preparation of the rat. Added to the perfusate in increasing concentrations (2 x 10(-5) to 6 x 10(-4) M), diazepam induced a concentration-dependent biphasic effect on the contractile force (n = 12), and, in low concentrations (2 x 10(-5) to 1 x 10(-4) M), a significant enhancement which diminished after concentrations higher than 1 x 10(-4) M were applied. The increase in contractile force was preceded by a transient short-lasting concentration-dependent inhibition. Midazolam (2 x 10(-5) to 6 x 10(-4) M) produced a significant concentration-dependent increase in heart contractile force which diminished at the highest concentrations. The maximum increase was only half that caused by diazepam (60 and 140%, respectively). Adinazolam and alprazolam, in the range of 2 x 10(-8) to 2 x 10(-7) M and 6 x 10(-7) to 1 x 10(-5) M, respectively, produced a marked concentration-dependent and short-lasting increase in inotropy (maximum response = 290 and 180%, respectively). Propranolol (10(-7) M) antagonized the inotropic effects of both diazepam and midazolam, whereas the positive inotropic response to alprazolam remained unchanged. This study shows that benzodiazepines may elicit both positive and negative concentration-dependent inotropic responses in the isolated rat heart. Differences between the drugs tested are both qualitative and quantitative. The newer benzodiazepines adinazolam and alprazolam are more powerful in increasing contractile force, as judged from the maximum response, even at relatively low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes induced by diazepam in the synaptic vesicle size of rat phrenic nerve terminals.

The effect of diazepam (Valium), administered by i.p. injection on the cross-sectional area of synaptic vesicle profiles of the endplate-rich area of the rate diaphragm was studied by electron microscopy at 15-180 min after treatment. In the dose range of 0.25-10 mg/kg, diazepam induced a significant increase of the size of the synaptic vesicles. This increase was dose-related within a certain margin, dependent on the treatment time, while the effect of each dose increased significantly with the period after treatment. At 2.5-10 mg/kg a maximum seemed to be reached at 90 min.

pH modulates cisplatin toxicity.

Cisplatin in normal saline of pH 2.5 caused haemolysis of rat erythrocytes, whereas cisplatin in normal saline of pH 3.5 did not. Even a difference of 0.2 pH units appeared to be of significant importance: haemolysis of rat erythrocytes was observed with cisplatin in saline of pH 3.0 but not with cisplatin in saline of pH 3.2. The LD in mice was 15.4 mg/kg for cisplatin in saline of pH 2.5 versus 24.0 mg/kg for cisplatin in saline of pH 3.5. Experiments with cisplatin should include careful control of pH.

Alterations in high affinity choline accumulation rate in rat cerebral cortex during anaesthesia with ketamine and pentobarbital.

After i.p. injection of ketamine (75 mg/kg) or pentobarbital-Na (40 mg/kg) to rats, there was a rapid, then a steady decrease of the sodium-dependent high affinity choline (HAC) accumulation rate to a minimum. This minimum was followed by a rapid increase (ketamine) or a gradual rise (pentobarbital-Na). Immediately after the rats came out of anaesthesia, the accumulation rate had not yet completely recovered. We suggest the ketamine or pentobarbital-Na induce alterations in central cholinergic systems, i.e. changes in choline uptake and incorporation into acetylcholine. It is conceivable that interactions of cholinesterase inhibitors or corticosteroids with the anaesthetics are based upon some modification of these changes.

Interactions of cholinesterase inhibitors and glucocorticoids with ketamine and pentobarbitone-induced general anaesthesia in the rat: possible effects on central cholinergic activity.

Doses of 100, 150 and 200 micrograms kg-1 of the cholinesterase inhibitor neostigmine reverse the anaesthetic action of ketamine. The antagonistic effect is increased as the dose is increased. The duration of anaesthesia induced by pentobarbitone is reversed by the cholinesterase inhibitor in doses of 150, 200 and 250 micrograms kg-1. Choline, in a dose of 50 mg kg-1, significantly antagonizes the action of the two anaesthetics, whereas hemicholinium-3, an inhibitor of the uptake of choline and the synthesis of acetylcholine, markedly potentiates their action. Dexamethasone induces a significant reduction of the duration of anaesthesia produced by ketamine and pentobarbitone. The potentiation of the anaesthetic effect caused by hemicholinium-3 is also reversed by dexamethasone. The acetylcholine content in rat cerebral cortex is increased after treatment with ketamine and pentobarbitone. Measurements of the course of the plasma level of pentobarbitone do not reveal alterations in the pharmacokinetic profile by either neostigmine or dexamethasone. These results indicate that central cholinergic systems may somehow be involved in the anaesthesia induced by ketamine and pentobarbitone and that the interactions described in this paper may be the result of modification by neostigmine and dexamethasone of the alterations in cholinergic activity caused by the two anaesthetics.

Interactions of cholinesterase inhibitors and corticosteroids with the hypnotic effect of benzodiazepines in mice.

Interactions of cholinesterase inhibitors or dexamethasone with the hypnotic effect of benzodiazepines appear to be strongly dependent on the dose of the cholinesterase inhibitor used and to a lesser extent on the dose of the hypnotic. The duration of the loss of righting reflex caused by the benzodiazepines, diazepam or chlorodiazepoxide, is markedly prolonged by 500 micrograms/kg, significantly antagonized both diazepam and chlorodiazepoxide. Relatively higher doses of physostigmine have no significant effect on either diazepam or chlorodiazepoxide. Dexamethasone, in relatively low doses of 250 and 500 micrograms/kg or 500 micrograms/kg respectively, significantly reverses the effect of diazepam and chlorodiazepoxide, while in relatively high doses of 1000 micrograms/kg the hormone significantly potentiates the hypnosis produced by the benzodiazepines. It is suggested that the interactions described may be the result of a modification of possible changes in cholinergic activity caused by the benzodiazepines. Although as yet no unequivocal explanation can be offered for the different effects, the observations described in this paper may explain the highly contradictory findings of other authors in both animal and human studies.

Glucocorticoid-induced changes in synaptic vesicles of rat phrenic nerve terminals.

The mean cross sectional area of synaptic vesicle profiles of the myoneural junction of rat diaphragm is increased significantly when rats are injected i.p. with single doses of 200 micrograms/kg dexamethasone 1/2 hr to 8 hr previously to a control injection of 0.9% NaCl. A maximum effect is observed at 8 hr after pretreatment with dexamethasone. The treatment of rats with hemicholinium-3 (300 micrograms/kg) induces a significant reduction of the vesicle size. Complete prevention of this decrease in vesicle size is already observed when the animals are pretreated with a single dose of 200 micrograms/kg dexamethasone 1/2 hr before treatment with 300 micrograms/kg hemicholinium-3. Dexamethasone in the same dose causes an optimum increase of the mean cross sectional area when it is given 1 hr before treatment of the animals with hemicholinium-3. It is concluded that glucocorticoids have direct actions in both "normal" motor nerve terminals and in motor nerve terminals with a deficient choline transport system, which may contribute to their proposed beneficial effects in certain cases of muscle weakness.

Efficacy of theophylline and its N-7-substituted derivatives in experimentally induced bronchial asthma in the guinea-pig.

A study has been made of the bronchospasmolytic actions of theophylline and some ot its N-7-substituted derivatives administered by i.v infusion in anaesthetized guinea pigs, in which experimental bronchial asthma was induced by i.v. administration of histamine, 5-hydroxytryptamine and bradykinin. Bronchoconstriction was measured as changes in tidal volume, airflow rate, intrapleural pressure fluctuations and respiratory frequency. Dynamic lung compliance and pulmonary resistance was computed and recorded simultaneously. In addition blood pressure and heart rate were recorded. Theophylline, proxyphylline, diprophylline and etophylline when given alone had hardly an effect on lung function; relatively high doses of the drugs caused a fall in blood pressure and an increase in heart rate. Acephylline infusion in relatively high doses produced a decrease of dynamic lung compliance and an increase of pulmonary resistance. Some animals died. Theophylline, proxyphylline, diprophylline and etophylline were effective in reducing the mediator-induced bronchoconstriction. Protective effects correlated considerably (R = 0.75-0.82) with the plasma concentrations. The magnitude of these protective effects increased with the plasma concentration. Effective doses of proxyphylline, diprophylline and etophylline were much higher than those of theophylline. Acephylline was completely inactive in reversing the mediator-induced bronchoconstriction. At relatively high doses it increased the bronchoconstrictive effects of the mediators. It is concluded that diprophylline, proxyphylline and etophylline, like theophylline, are highly effective bronchospasmolytics, but does of the individual drugs must be adjusted according to the derivative used. Theophylline is by far the most effective of the four compounds. On the basis of this study and recent pharmacokinetic data in man the therapeutic value of acephylline in asthmatics seems doubtful.

Effects of corticosteroids on neuromuscular blocking actions of d-tubocurarine.

Dexamethasone (50 microgram/kg) significantly increased the LD50 of d-tubocurarine (d-TC) when administered i.p. simultaneously with d-TC. Choline (50 and 100 mg/kg) gave some protection against the lethal effects of d-TC and the cholinesterase inhibitors neostigmine (250 microgram/kg) and physostigmine (1000 microgram/kg) provided full protection against doses of d-TC twice the LD50. The blocking effect of d-TC (75 microgram/kg) on the sciatic nerve-tibialis anterior muscle preparation was antagonized by dexamethasone. Prednisolone delayed the occurrence of a complete neuromuscular block caused by d-TC in the phrenic nerve-diaphragm preparation, and antagonized the effect of d-TC on short tetanic contractions. d-TC (5 mumol/l) inhibited the [14C]choline uptake in the endplate-rich region of the rat diaphragm during stimulation. This inhibition was antagonized by dexamethasone as well as by physostigmine. The incorporation of radioactive choline into acetylcholine was inhibited in the presence of d-TC (15 mumol/l), and both dexamethasone and physostigmine counteracted this inhibition. It is concluded from these experiments that d-TC very probably has an effect on the choline carrier system. These experimental results support the hypothesis that glucocorticoids may improve reduced muscle performance by direct presynaptic effects at the neuromuscular junction.

Effect of corticosteroids on sciatic nerve-tibialis anterior muscle of rats treated with hemicholinium-3. An experimental approach to a possible mechanism of action of corticosteroids in myasthenia gravis.

We studied the effect of intraperitoneally administered corticosteroids on the neuromuscular transmission in the sciatic nerve-tibialis anterior muscle preparation of the anesthetized rat stimulated at a rate of 10 Hz. Administered simultaneously with hemicholinium-3 (HC-3), 80 mug per kilogram (that is, half the lethal dose for 50 percent survival), prednisolone and dexamethasone cause a marked reversal of the block of the neuromuscular transmission caused by HC-3. The effect of aldosterone is very small. The blocking action of d-tubocurarine is not antagonized by either prednisolone or dexamethasone. Choline provides total protection against the HC-3 blockade, whereas physostigmine, in a just sublethal dose, is ineffective. We tentatively conclude that in myasthenia gravis the carrier-mediated transport of choline into the nerve endings may be deficient and that the beneficial effect of corticosteroids in this condition is based on their ability to ameliorate the deficient choline transport.

Effect of corticosteroids on the phrenic nerve-diaphragm of preparation treated with hemicholinium. A possible model of myasthenia gravis.

A marked protective action of the corticosteroids prednisolone, hydrocortisone, and dexamethasone has been shown on a hypothetical model of myasthenia gravis, using the rat phrenic nerve-diaphragm preparation treated with hemicholinium-3. Dexamethasone is much more effective than prednisolone, and hydrocortisone is the least effective. Prednisolone has no effect on a neuromuscular block caused by choline, decamethonium, physostigmine, d-tubocurarine, and a high or low calcium ion concentration. The possible implications of the present study for myasthenic disease are discussed. It is tentatively concluded that the site of action of corticosteroids in myasthenia gravis is located presynaptically.

The effect of corticosteroids and hemicholinium-3 on choline uptake and incorporation into acetylcholine in rat diaphragm.

The glucocorticoids prednisolone and dexamethasone antagonize the inhibition by hemicholinium-3 of both the rate of choline uptake and the incorporation of choline into acetylcholine in the rat diaphragm. Aldosterone has no such effects. It is concluded that the beneficial effect of glucocorticoids in the treatment of myasthenia gravis may be due not only to immunosuppression, but also to some direct effect on presynaptic events perhaps via a choline carrier or an enzyme of choline metabolism.

An analysis of an apparent sex difference in the desoxyribonucleic acid content of rat liver.

Using the extraction procedure of Schmidt & Thannhauser (1945) and the indole reaction for DNA according to Ceriotti (1952), the DNA content of female rat liver was about one and a half times that of male liver. Castration of male rats, with or without administration of testosterone propionate, had no effect on the liver DNA content. Spaying of female rats (5-6 weeks of age) caused a decrease of the liver DNA content. Substitution with oestradiol benzoate restored the amount of DNA. No significant sex difference was observed in the DNA content of either rat brain, kidney, spleen and thymus, or mouse liver. Dische's diphenylamine reaction showed no significant sex difference in the rat liver DNA content. It was concluded that rat liver may contain a substance which is controlled by oestrogens and which interferes with the indole reaction. The interfering factor is present in the protein fraction of the liver extract. The possible nature of this interfering substance is discussed.

The effects of three benzodiazepines and of meprobamate on the action of smooth muscle stimulants on the guinea-pig ileum.

The benzodiazepines chlorodiazepoxide, diazepam and flurazepam and meprobamate depress the response of the guinea-pig ileum to acetylcholine, histamine and 5-hydroxytryptamine. As compared to the benzodiazepines the action of meprobamate is very weak. Chlorodiazepoxide has a weaker anti-acetylcholine activity than diazepam and flurazepam. On the other hand chlorodiazepoxide possesses a realtively strong anti-histamine activity. The three benzodiazeptines tested are about equally effective in reducing the contraction of the guinea-pig ileum caused by 5-hydroxytryptamine. The potency of diazepam and flurazepam in blocking the effect of the three smooth muscle stimulants appears to be rather similar.