Clinical and Experimental Pharmacology & Physiology (1 978) 5,655-663.
THE EFFECTS OF DIAZEPAM ON RAT ISOLATED HEART MUSCLE J. Sugimoto, M. Nagata and Y.Ikeda Department of Pharmacology, Kansai Medical School, Moriguchi City, Osaka, Japan
(Received 25 January 1977;revision received 28 November 1977)
SUMMARY
1. The effects of diazepam on spontaneously beating right atrium, left atrium and papillary muscle isolated from a rat heart were compared to those of chlorpromazine and phenytoin. 2. Arrhythmic contractions induced by threshold electrical stimulation in the rhythmic spontaneous contractions of the right atrium were prevented by diazepam (10 pglml). These arrhythmic contractions were not prevented by chlorpromazine (0.01-10 pg/ml), or by phenytoin (0.1-10 pg/ml). 3. The refractory periods of the heart muscle, estimated by use of Govier’s method, was increased by diazepam (10 pg/ml) by 90.5% in the left atrium and 54.4% in the papillary muscles. Chlorpromazine (1 .O pg/ml) and phenytoin (10 pgl ml) increased the refractory period of papillary muscle by 42.8 and 54.1%, respectively, but had little effect on the refractory period of the left atrium. 4. The maximum driving frequency of the left atrium was decreased by diazepam (10 pdrnl) by 25.1% and by chlorpromazine (1.0 pglml) by 33.3%, while that of papillary muscle was little influenced by the drugs at these concentrations. Phenytoin (0.1-10 pglml) had little effect on the maximum driving frequency of the left atrium or of papillary muscle. 5. The rate and force of spontaneous contractions of the right atrium were decreased by phenytoin (10 pg/ml) by 36.7% and 97.0%, respectively. Diazepam (0.1-10 pglml) had little effect on rate and produced a concentration-dependent increase in force up to 151.5%. Chlorpromazine (0.01-1.0 pg/ml) had little effect on either the rate or force of spontaneous contraction of the right atrium. 6. The contractile force of the left atrium and of papillary muscle driven by electrical stimulation was depressed by diazepam (20 pgcglml), chlorpromazine (2.0 pg/ml) and phenytoin (20 pglml). Diazepam (0.5-10 pg/ml) produced a concentrationdependent increase in force of up to 204.7% in the left atrium and 163.1% in papillary muscle. Chlorpromazine (0.01-1 .O pg/ml) had little effect on the force of the driven left atrium, but produced a concentrationdependent decrease in force of papillary muscle down to 40.1%. Phenytoin (10 pg/ml) reduced the force of contraction by 84.6 and 92.7%, respectively, in the left atrium and papillary muscle.
Key words: diazepam, chlorpromazine, phenytoin. Correspondence: Dr Jiro Sugimoto, Department of Pharmacology, Kansai Medical School, Moriguchi City, Osaka 570, Japan.
0305-1870/78/11004655$02.00 0 1978 Blackwell Scientific Publications
655
65 6
J. Sugimoto, M . Nagata and Y. Ikeda INTRODUCTION
The tranquilizing action of diazepam was reported by Randall et ul. (1961); since then, the drug has been widely used in clinical practice. Many clinicians regard it as the preferred tranquilizer in the management of patients with cardiac disease. Most investigations on its effects have been in relation t o its tranquilizing action rather than its cardiac actions. Prindle et ul. (1970) found that diazepam had n o significant effect on isometric tension in cat isolated papillary muscle. However, we observed that diazepam prolonged the refractory period of rat isolated cardiac muscle preparations and increased the force of contraction. This led to the present study on the effects of diazepam on spontaneously beating atria, as well as on the electrically driven atrial and papillary muscle and a comparison of its effects with those of chlorpromazine and phenytoin. METHODS Adult rats of both sexes weighing 350-450 g were killed by severing the common carotid arteries. Immediately thereafter, the heart was excised and a right atrium, left atrium and two or three papillary muscles were isolated and suspended in an organ bath containing Locke's solution saturated with pure oxygen at 30°C and pH 7.2-7.4. The Locke's solution used consisted of NaCl 9.0 g, KCl 0.42 g, CaClz 0.24 g, NaHC03 0.7 g and glucose 1.O g per litre of distilled water. Control values of the rate and force of spontaneous contractions of the right atrium were taken after equilibrium of the preparation for 30 t o 40 min in the organ bath, Then the drug to be tested was added t o the organ bath and its effects on the rate and force of contractions were taken 30 min later. Arrhythmic contractions in the rhythmic spontaneous contractions of the right atrium were induced by electrical stimulation at a threshold intensity with a pulse duration of 5.0 ms and a frequency of 2.0 Hz; this stimulation was given through a pair of electrodes (diameter 0.5 mm, length 5.0 mm) which were placed as close as possible to the preparation. The stimulation was given before and 30 min after addition of the drug being tested, and the change in the threshold intensity for producing arrhythmic contractions was determined. In experiments with the left atrium and papillary muscle, contractions were elicited by electrical stimulation, using electrodes as described above; stimulation was at twice the threshold intensity at frequencies of 1.0, 2.0 and 4.0 Hz and pulse durations of 5.0 ms for the left atrium and 10 ms for papillary muscle. The drug t o be tested was added to the organ bath 30 min after beginning stimulation, and its effect on the force of contractions was determined 30 min later. The refractory periods of the left atrium and of papillary muscle were determined by a modification of the method of Govier (1965). "he preparations were driven by electrical stimulation as described above, and additional test stimuli were given at 10 t o 500 ms after each driving stimulus. When the time interval between driving and test stimuli was shorter than the refractory period of the tissue, there was no contractile response t o the test stimulus. The time interval was gradually increased until the contractile responses to the test stimuli began to appear; this interval was taken as the mechanical refractory period of the preparation being tested. The preparation was driven regularly for 30 min before determining the control refractory period. Then the drug being tested was added t o the organ bath and its effect on the refractory period was determined 30 min later.
Cardiac effects o f diazepam
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The maximum driving frequencies of the left atrium and of papillary muscle were determined by a modification of the method of Tanz (1974). The preparation was driven by electrical stimulation at twice the threshold intensity with a pulse duration of 5.0 ms for the left atrium and 10 ms for papillary muscle. For the first 30 min, the frequency was 1.0 Hz; thereafter, the frequency was increased from 1.O to 13 Hz with increments at 15 s intervals. The maximum driving frequency was taken as the stimulation frequency at which the contractile responses just failed t o correspond to the driving stimuli. After determining the control value of the maximum driving frequency, the frequency was decreased t o 1.0 Hz and
200
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200
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Chlorpornazine ( p g / m l )
i L
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I
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Diozepom ( pg / ml )
Phenytoin ( p g / ml Fig. 1. Effects of chlorpromazine, diazepam and phenytoin on the rate and force of spontaneous contractions of isolated right rat atrium: the effects were determined 30 min after addition of the drugs. Each mean point and its standard error was obtained from values in six preparations.
J. Sugimoto, M . Nagata and Y. Ikeda
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the drug to be tested was added t o the organ bath; its effect on the maximum driving frequency was determined 30 min later. The electrical stimulators used were Nihon-Kohden MSE3R and JM (Tokyo). The contractions were measured with strain gauges and recorded on pen-writing oscillographs (Shinko-Tsushin UL-2 and AS3A, Tokyo). The drugs used were chlorpromazine hydrochloride (Takeda), diazepam (Takeda) and phenytoin (Dainihon). Diazepam was initially dissolved in ethanol and propylene glycol, and then diluted in Locke’s solution. The corresponding drug-free mixture was shown t o be devoid of effect on the preparations. Chlorpromazine and phenytoin were initially dissolved in distilled water and then diluted in Locke’s
Chlorprornozine ( p g / m l )
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10
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Phenytoin (pg/ml)
Fig 2. Effects of chlorpromazine, diazepam and phenytoin on the force of contraction of (a) the left atrium and of (b) papillary muscle driven by electrical stimulation at twice the threshold intensity with pulse durations of 5.0 ms for the left atrium and 10 ms for papillary muscle and frequencies of (0)1.0, (0)2.0 and (A) 4.0 Hz. The effects of drugs were determined 30 min after their addition to the organ bath. Each point is a mean of values from eight preparations, except for the points showing the effects of 1.0 pg/ml of chlorpromazine (ten preparations) and 10 pg/ml of diazepam (twelve preparations).
Cardiac effects of diazepam
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solution. The volume of the drug solution added t o the organ bath did not exceed 1% of the bath volume. Drug concentrations in the organ bath were expressed in terms of pg/ml. The effects of drugs were expressed as a percentage of change from the control values. RESULTS
Effects of chlorpromazine, diazepam and phenytoin on the rate and force of spontaneous contractions of the r&hratrium Chlorpromazine, diazepam and phenytoin in a concentration of 20 pg/ml arrested the 2cK
(b)
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Diazepam ( p g / m l )
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4 0.1
1.0
0.1 I0 Phenytoin (pg /mil
Fig. 3. Effects of chlorpromazine, diazepam and phenytoin on the refractory periods of (a) the left atrium and of (b) papillary muscle, estimated by the modified double-stimulus method of Govier (1965). Basal stimulation was givenat frequencies of (0)1.0, (0)2.0 and (a) 4.0 Hz, and the effects of drugs were determined 30 min after their addition t o the organ bath. Each point is the mean of values from eight preparations, except for the points showing the effects of 1.0 fig/ml of chlorpromazine (ten preparations) and 10 bg/ml of diazepam (twelve preparations).
660
J. Sugimoto, M. Nagata and Y. Ikeda
spontaneous contractions of the right atrium. Lower concentrations of chlorpromazine (0.0 1-1 .O pg/ml) had little effect on either the rate or force of contractions. Phenytoin in a concentration of 10 pg/ml decreased the rate by 36.7% (s.e.m. = 9.9, n = 6) and for force by 97.0% (s.e.m = 18.6, n = 6). However, diazepam produced a concentration-dependent increase in force up to 15 1.5% (s.e.m. = 5.6, n = 6) of the control over the range of 1.O to 10 pg/ml, but had little effect on the rate (Fig. 1).
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Fig. 4. Effects of chlorpromazine, diazepam and phenytoin on the maximum driving frequency of (a) the left atrium and of (b) papillary muscle, estimated by the modified method of Tanz (1974). The effects of drugs were determined 30 min after their addition to the organ bath. Each point and its standard error were calculated from values in eight preparations, except for the points showing the effects of 1.O pg/ml of chlorpromazine (ten preparations) and 5 .O and 10 p d m l of diazepam (twelve preparations).
Cardiac effects of diazepam
66 1
Effects of chlorpromazine, diazepam and phenytoin on the force of contraction of the left atrium and papillary muscle driven b y electrical stimulation Chlorpromazine (2.0 pdml), diazepam (20 pg/ml) and phenytoin (20 pg/ml) depressed the force of contractions of the left atrium and of papillary muscle driven by electrical stimulation. Lower concentrations of chlorpromazine (0.1 -1 .O pg/ml) caused a concentrationdependent decrease in the force of contraction of papillary muscle down to 40.1% (s.e.m. = 17.3, n = 10) of control. Phenytoin (10 pg/ml) decreased the force of contraction of the left atrium by 84.6% (s.e.m. = 18.5, n = 8) and that of papillary muscle by 92.7% (s.e.m. = 21.8, n = 8). However, diazepam caused a concentration-dependent increase in the force of contraction over a concentration range of 0.5 to 10 pg/ml up to 204.7% (s.em. = 25.1, n = 12) in the left atrium and 163.1% (s.e.m. = 13.9,n = 12) in papillary muscle. These values were obtained when the preparations were driven at a stimulation frequency of 4.0 Hz (Fig. 2).
Effects of the chlorpromazine, diazepam and phenytoin on the refractory periods of the left atrium and papillary muscle Diazepam (5.0 and 10 pg/ml, respectively) increased the refractory period of the left atrium by 54.6% (s.e.m. = 11.4, n = 8) and 90.5% (s.e.m. = 16.0, n = 12), and that of papillary muscle by 37.2% (s.e.m. = 11.3, n = 8) and 54.4% (s.e.m. = 5.7, n = 12) of the control value for each tissue. The effects of chlorpromazine and phenytoin on the refractory periods of both tissues were less than that of diazepam: chlorpromazine (1 .O pdrnl) and phenytoin (10 pg/ml) increased that of papillary muscle by 42.8% (s.e.m. = 10.1, n = 10) and 54.1% (s.e.m. = 37.6, n = 8) of the control, respectively; they had little effect on the refractory period of the left atrium. These values were obtained when the preparations were driven at a frequency of 4.0 Hz (Fig. 3). Effects of chlorpromazine, diazepam and phenytoin on the maximum driving frequency of the left atrium and of papillary muscle Diazepam (1 0 pglml) and chlorpromazine (1 .O pg/ml) reduced the maximum driving frequency of the left atrium by 21.5% (s.e.m. = 3.3, n = 12) and 33.3% (s.e.m. = 4.9,n = lo), respectively, but had little effect on that of papillary muscle (Fig. 4). Phenytoin (0.1-10 pg/ml) had little effect on the maximum driving frequency of either tissue.
Fig. 5. Spontaneous contractions of isolated right atrium and arrhythmic contractions induced by electrical stimulation at intensities of 2.0, 2.5 and 3.0 V with a pulse duration of 5.0 ms and a frequency of 2.0 Hz. The lines under the record show the periods of 15 s during which the stimulation was given. In the presence of diazepam (10 fig/ml), it was necessary to increase the stimulation intensity to induce arrhythmic contractions (and the amplitude of contractions was increased). Numerals in parentheses above the record indicate the rate of spontaneous contractions per min. At (W) the diazepam was washed out of the organ both, and at ( S ) the record was stopped for 30 min.
662
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Preventive effect of diazepam on arrhythmic contractions induced b y electrical stimulation given during spontaneous contractions of the ripht atrium Electrical stimulation at a threshold intensity induced arrhythmic contractions in the spontaneously beating right atrium. The addition of diazepam (1 0 pg/ml) increased the threshold intensity to induce the arrhythmic contractions (Fig. S), whereas neither chlorpromazine (0.01-10 pg/ml) nor phenytoin (0.1-10 pg/ml) affected the threshold intensity.
DISCUSSION In our previous papers (Sugimoto, Nagata & Morita, 1972, 1978), in which the effects of several kinds of antiarrhythmic drugs were compared in prolonging the refractory period of isolated heart muscle, ajmaline, cocaine, lignocaine, phenytoin, procainamide, propranolol, quinidine and trimetazidine were found to produce concentration-dependent depressions of the force of contraction of the heart muscle at the higher concentrations used. In the present study diazepam was also found t o depress the force of contraction of rat heart muscle in concentrations greater than 20 pg/ml. However, in concentrations of 0.5 t o 10 pg/ml, diazepam increased the force of contraction. Over this concentration range, diazepam had little effect on the rate of spontaneous contractions of the right atrium and prolonged the refractory period of the left atrium and of papillary muscle. Therefore, the cardiac actions of diazepam appear t o differ from those of quinidine and other antiarrhythmic drugs. Diazepam prolonged the refractory period of the left atrium by about 100% and that of papillary muscle by about SO%, at maximum, and decreased the maximum driving frequency of the left atrium but not that of papillary muscle. These observations suggest that the cardiac action of diazepam is more marked in atrial than in ventricular muscle. "he actions of diazepam partially resemble those of cocaine and trimetazidine, which prolong the refractory period of atrial muscle but scarcely affect that of papillary muscle, without having a suppressant effect on the force of contractions, although these drugs (unlike diazepam) never augmented the force of contractions at any of the concentrations used (Sugimoto et al., 1972,1978). Chlorpromazine also prolonged the refractory period of papillary muscle. However, the prolongation was less than 50% of control at most and its effect was less than that of diazepam. Although the effect of chlorpromazine on the maximum driving frequency of the left atrium was greater than that of diazepam, the concentration producing these effects caused a pronounced decrease in the force of contraction of papillary muscle. The effects of phenytoin on the refractory period and maximum driving frequency of the left atrium and papillary muscle were weak. The arrhythmic contractions induced by electrical stimulation during spontaneous contractions of the right atrium were prevented by diazepam, but not by chlorpromazine or phenytoin. Therefore, the cardiac actions of diazepam do not appear to be a common property of tranquilizer drugs. Regarding the experimental techniques, the effects of drugs on the force of contraction of electrically driven cardiac muscle and on the refractory period are dependent on the driving frequency. In subsequent studies it will be necessary to determine which frequency of stimulation is most appropriate for extrapolation of the effects of drugs t o those on the heart in situ. Tanz (1974) determined the effects of drugs on the refractory period of cardiac muscle using the maximum driving frequency of the muscle; however, the results of the present study suggest that the effects of drugs on the refractory period and the maxi-
Cardiac effects of diazepam
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mum driving frequency are not always the same. Essential differences between these two effects will be reported in a subsequent paper. REFERENCES Govier, W.C. (1965) The mechanism of the atrial refractory period change produced by ouabain. Journal o f Pharmacology and Experimental Therapeutics, 148,100-105. Prindle, K.H., Jr., Gold, H.K., Cardon, P.V. & Epstein, S.E. (1970) Effects of psychopharmacologic agents on myocardial contractility. Journal of Pharmacology and Experimental Therapeutics, 173, 133137. Randall, LO., Heise, G.A., Schalleck, W., Bagdon, RE, Baziger, R, Boris, A., Moe, RA. & A b m , W.B. (1961) Pharmacological and clinical studies on Valium (T.M.), a new psychotherapeutic agent of the benzodiazepine class. a r r e n t Therapeutic Research, 3,405-425. Sugimoto, J., Nagata, M. & Morita, M. (1972) Comparative studies on actions of anti-arrhythmic drugs in guinea-pig atria. Abstract of the Fifth International Congress on Pharmacology, San Francisco, p. 225, No. 1346. Sugimoto, J., Nagata, M.& Morita, M. (1978) Comparative studies on the pharmacological actions of antiarrhythmic drugs in isolated rat papillary muscle. Clinical and Experimental Pharmacology & Physiology, 5, 17-21. Tanz, R (1974) Pharmacology of aconitine-induced automaticity on in vitro cat myocardial preparations. 11. Effects of refractory period prolongation, reduced sodium and tetrodotoxin. Journal of Pharmacology and Experimental Therapeutics, 191,232-240.
THE EFFECTS OF DIAZEPAM ON RAT ISOLATED HEART MUSCLE J. Sugimoto, M. Nagata and Y.Ikeda Department of Pharmacology, Kansai Medical School, Moriguchi City, Osaka, Japan
(Received 25 January 1977;revision received 28 November 1977)
SUMMARY
1. The effects of diazepam on spontaneously beating right atrium, left atrium and papillary muscle isolated from a rat heart were compared to those of chlorpromazine and phenytoin. 2. Arrhythmic contractions induced by threshold electrical stimulation in the rhythmic spontaneous contractions of the right atrium were prevented by diazepam (10 pglml). These arrhythmic contractions were not prevented by chlorpromazine (0.01-10 pg/ml), or by phenytoin (0.1-10 pg/ml). 3. The refractory periods of the heart muscle, estimated by use of Govier’s method, was increased by diazepam (10 pg/ml) by 90.5% in the left atrium and 54.4% in the papillary muscles. Chlorpromazine (1 .O pg/ml) and phenytoin (10 pgl ml) increased the refractory period of papillary muscle by 42.8 and 54.1%, respectively, but had little effect on the refractory period of the left atrium. 4. The maximum driving frequency of the left atrium was decreased by diazepam (10 pdrnl) by 25.1% and by chlorpromazine (1.0 pglml) by 33.3%, while that of papillary muscle was little influenced by the drugs at these concentrations. Phenytoin (0.1-10 pglml) had little effect on the maximum driving frequency of the left atrium or of papillary muscle. 5. The rate and force of spontaneous contractions of the right atrium were decreased by phenytoin (10 pg/ml) by 36.7% and 97.0%, respectively. Diazepam (0.1-10 pglml) had little effect on rate and produced a concentration-dependent increase in force up to 151.5%. Chlorpromazine (0.01-1.0 pg/ml) had little effect on either the rate or force of spontaneous contraction of the right atrium. 6. The contractile force of the left atrium and of papillary muscle driven by electrical stimulation was depressed by diazepam (20 pgcglml), chlorpromazine (2.0 pg/ml) and phenytoin (20 pglml). Diazepam (0.5-10 pg/ml) produced a concentrationdependent increase in force of up to 204.7% in the left atrium and 163.1% in papillary muscle. Chlorpromazine (0.01-1 .O pg/ml) had little effect on the force of the driven left atrium, but produced a concentrationdependent decrease in force of papillary muscle down to 40.1%. Phenytoin (10 pg/ml) reduced the force of contraction by 84.6 and 92.7%, respectively, in the left atrium and papillary muscle.
Key words: diazepam, chlorpromazine, phenytoin. Correspondence: Dr Jiro Sugimoto, Department of Pharmacology, Kansai Medical School, Moriguchi City, Osaka 570, Japan.
0305-1870/78/11004655$02.00 0 1978 Blackwell Scientific Publications
655
65 6
J. Sugimoto, M . Nagata and Y. Ikeda INTRODUCTION
The tranquilizing action of diazepam was reported by Randall et ul. (1961); since then, the drug has been widely used in clinical practice. Many clinicians regard it as the preferred tranquilizer in the management of patients with cardiac disease. Most investigations on its effects have been in relation t o its tranquilizing action rather than its cardiac actions. Prindle et ul. (1970) found that diazepam had n o significant effect on isometric tension in cat isolated papillary muscle. However, we observed that diazepam prolonged the refractory period of rat isolated cardiac muscle preparations and increased the force of contraction. This led to the present study on the effects of diazepam on spontaneously beating atria, as well as on the electrically driven atrial and papillary muscle and a comparison of its effects with those of chlorpromazine and phenytoin. METHODS Adult rats of both sexes weighing 350-450 g were killed by severing the common carotid arteries. Immediately thereafter, the heart was excised and a right atrium, left atrium and two or three papillary muscles were isolated and suspended in an organ bath containing Locke's solution saturated with pure oxygen at 30°C and pH 7.2-7.4. The Locke's solution used consisted of NaCl 9.0 g, KCl 0.42 g, CaClz 0.24 g, NaHC03 0.7 g and glucose 1.O g per litre of distilled water. Control values of the rate and force of spontaneous contractions of the right atrium were taken after equilibrium of the preparation for 30 t o 40 min in the organ bath, Then the drug to be tested was added t o the organ bath and its effects on the rate and force of contractions were taken 30 min later. Arrhythmic contractions in the rhythmic spontaneous contractions of the right atrium were induced by electrical stimulation at a threshold intensity with a pulse duration of 5.0 ms and a frequency of 2.0 Hz; this stimulation was given through a pair of electrodes (diameter 0.5 mm, length 5.0 mm) which were placed as close as possible to the preparation. The stimulation was given before and 30 min after addition of the drug being tested, and the change in the threshold intensity for producing arrhythmic contractions was determined. In experiments with the left atrium and papillary muscle, contractions were elicited by electrical stimulation, using electrodes as described above; stimulation was at twice the threshold intensity at frequencies of 1.0, 2.0 and 4.0 Hz and pulse durations of 5.0 ms for the left atrium and 10 ms for papillary muscle. The drug t o be tested was added to the organ bath 30 min after beginning stimulation, and its effect on the force of contractions was determined 30 min later. The refractory periods of the left atrium and of papillary muscle were determined by a modification of the method of Govier (1965). "he preparations were driven by electrical stimulation as described above, and additional test stimuli were given at 10 t o 500 ms after each driving stimulus. When the time interval between driving and test stimuli was shorter than the refractory period of the tissue, there was no contractile response t o the test stimulus. The time interval was gradually increased until the contractile responses to the test stimuli began to appear; this interval was taken as the mechanical refractory period of the preparation being tested. The preparation was driven regularly for 30 min before determining the control refractory period. Then the drug being tested was added t o the organ bath and its effect on the refractory period was determined 30 min later.
Cardiac effects o f diazepam
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The maximum driving frequencies of the left atrium and of papillary muscle were determined by a modification of the method of Tanz (1974). The preparation was driven by electrical stimulation at twice the threshold intensity with a pulse duration of 5.0 ms for the left atrium and 10 ms for papillary muscle. For the first 30 min, the frequency was 1.0 Hz; thereafter, the frequency was increased from 1.O to 13 Hz with increments at 15 s intervals. The maximum driving frequency was taken as the stimulation frequency at which the contractile responses just failed t o correspond to the driving stimuli. After determining the control value of the maximum driving frequency, the frequency was decreased t o 1.0 Hz and
200
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200
-
Chlorpornazine ( p g / m l )
i L
r
*O0I
I
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Diozepom ( pg / ml )
Phenytoin ( p g / ml Fig. 1. Effects of chlorpromazine, diazepam and phenytoin on the rate and force of spontaneous contractions of isolated right rat atrium: the effects were determined 30 min after addition of the drugs. Each mean point and its standard error was obtained from values in six preparations.
J. Sugimoto, M . Nagata and Y. Ikeda
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the drug to be tested was added t o the organ bath; its effect on the maximum driving frequency was determined 30 min later. The electrical stimulators used were Nihon-Kohden MSE3R and JM (Tokyo). The contractions were measured with strain gauges and recorded on pen-writing oscillographs (Shinko-Tsushin UL-2 and AS3A, Tokyo). The drugs used were chlorpromazine hydrochloride (Takeda), diazepam (Takeda) and phenytoin (Dainihon). Diazepam was initially dissolved in ethanol and propylene glycol, and then diluted in Locke’s solution. The corresponding drug-free mixture was shown t o be devoid of effect on the preparations. Chlorpromazine and phenytoin were initially dissolved in distilled water and then diluted in Locke’s
Chlorprornozine ( p g / m l )
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Phenytoin (pg/ml)
Fig 2. Effects of chlorpromazine, diazepam and phenytoin on the force of contraction of (a) the left atrium and of (b) papillary muscle driven by electrical stimulation at twice the threshold intensity with pulse durations of 5.0 ms for the left atrium and 10 ms for papillary muscle and frequencies of (0)1.0, (0)2.0 and (A) 4.0 Hz. The effects of drugs were determined 30 min after their addition to the organ bath. Each point is a mean of values from eight preparations, except for the points showing the effects of 1.0 pg/ml of chlorpromazine (ten preparations) and 10 pg/ml of diazepam (twelve preparations).
Cardiac effects of diazepam
659
solution. The volume of the drug solution added t o the organ bath did not exceed 1% of the bath volume. Drug concentrations in the organ bath were expressed in terms of pg/ml. The effects of drugs were expressed as a percentage of change from the control values. RESULTS
Effects of chlorpromazine, diazepam and phenytoin on the rate and force of spontaneous contractions of the r&hratrium Chlorpromazine, diazepam and phenytoin in a concentration of 20 pg/ml arrested the 2cK
(b)
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Fig. 3. Effects of chlorpromazine, diazepam and phenytoin on the refractory periods of (a) the left atrium and of (b) papillary muscle, estimated by the modified double-stimulus method of Govier (1965). Basal stimulation was givenat frequencies of (0)1.0, (0)2.0 and (a) 4.0 Hz, and the effects of drugs were determined 30 min after their addition t o the organ bath. Each point is the mean of values from eight preparations, except for the points showing the effects of 1.0 fig/ml of chlorpromazine (ten preparations) and 10 bg/ml of diazepam (twelve preparations).
660
J. Sugimoto, M. Nagata and Y. Ikeda
spontaneous contractions of the right atrium. Lower concentrations of chlorpromazine (0.0 1-1 .O pg/ml) had little effect on either the rate or force of contractions. Phenytoin in a concentration of 10 pg/ml decreased the rate by 36.7% (s.e.m. = 9.9, n = 6) and for force by 97.0% (s.e.m = 18.6, n = 6). However, diazepam produced a concentration-dependent increase in force up to 15 1.5% (s.e.m. = 5.6, n = 6) of the control over the range of 1.O to 10 pg/ml, but had little effect on the rate (Fig. 1).
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Fig. 4. Effects of chlorpromazine, diazepam and phenytoin on the maximum driving frequency of (a) the left atrium and of (b) papillary muscle, estimated by the modified method of Tanz (1974). The effects of drugs were determined 30 min after their addition to the organ bath. Each point and its standard error were calculated from values in eight preparations, except for the points showing the effects of 1.O pg/ml of chlorpromazine (ten preparations) and 5 .O and 10 p d m l of diazepam (twelve preparations).
Cardiac effects of diazepam
66 1
Effects of chlorpromazine, diazepam and phenytoin on the force of contraction of the left atrium and papillary muscle driven b y electrical stimulation Chlorpromazine (2.0 pdml), diazepam (20 pg/ml) and phenytoin (20 pg/ml) depressed the force of contractions of the left atrium and of papillary muscle driven by electrical stimulation. Lower concentrations of chlorpromazine (0.1 -1 .O pg/ml) caused a concentrationdependent decrease in the force of contraction of papillary muscle down to 40.1% (s.e.m. = 17.3, n = 10) of control. Phenytoin (10 pg/ml) decreased the force of contraction of the left atrium by 84.6% (s.e.m. = 18.5, n = 8) and that of papillary muscle by 92.7% (s.e.m. = 21.8, n = 8). However, diazepam caused a concentration-dependent increase in the force of contraction over a concentration range of 0.5 to 10 pg/ml up to 204.7% (s.em. = 25.1, n = 12) in the left atrium and 163.1% (s.e.m. = 13.9,n = 12) in papillary muscle. These values were obtained when the preparations were driven at a stimulation frequency of 4.0 Hz (Fig. 2).
Effects of the chlorpromazine, diazepam and phenytoin on the refractory periods of the left atrium and papillary muscle Diazepam (5.0 and 10 pg/ml, respectively) increased the refractory period of the left atrium by 54.6% (s.e.m. = 11.4, n = 8) and 90.5% (s.e.m. = 16.0, n = 12), and that of papillary muscle by 37.2% (s.e.m. = 11.3, n = 8) and 54.4% (s.e.m. = 5.7, n = 12) of the control value for each tissue. The effects of chlorpromazine and phenytoin on the refractory periods of both tissues were less than that of diazepam: chlorpromazine (1 .O pdrnl) and phenytoin (10 pg/ml) increased that of papillary muscle by 42.8% (s.e.m. = 10.1, n = 10) and 54.1% (s.e.m. = 37.6, n = 8) of the control, respectively; they had little effect on the refractory period of the left atrium. These values were obtained when the preparations were driven at a frequency of 4.0 Hz (Fig. 3). Effects of chlorpromazine, diazepam and phenytoin on the maximum driving frequency of the left atrium and of papillary muscle Diazepam (1 0 pglml) and chlorpromazine (1 .O pg/ml) reduced the maximum driving frequency of the left atrium by 21.5% (s.e.m. = 3.3, n = 12) and 33.3% (s.e.m. = 4.9,n = lo), respectively, but had little effect on that of papillary muscle (Fig. 4). Phenytoin (0.1-10 pg/ml) had little effect on the maximum driving frequency of either tissue.
Fig. 5. Spontaneous contractions of isolated right atrium and arrhythmic contractions induced by electrical stimulation at intensities of 2.0, 2.5 and 3.0 V with a pulse duration of 5.0 ms and a frequency of 2.0 Hz. The lines under the record show the periods of 15 s during which the stimulation was given. In the presence of diazepam (10 fig/ml), it was necessary to increase the stimulation intensity to induce arrhythmic contractions (and the amplitude of contractions was increased). Numerals in parentheses above the record indicate the rate of spontaneous contractions per min. At (W) the diazepam was washed out of the organ both, and at ( S ) the record was stopped for 30 min.
662
J. Sugimoto, M. Nagata and Y. Ikeda
Preventive effect of diazepam on arrhythmic contractions induced b y electrical stimulation given during spontaneous contractions of the ripht atrium Electrical stimulation at a threshold intensity induced arrhythmic contractions in the spontaneously beating right atrium. The addition of diazepam (1 0 pg/ml) increased the threshold intensity to induce the arrhythmic contractions (Fig. S), whereas neither chlorpromazine (0.01-10 pg/ml) nor phenytoin (0.1-10 pg/ml) affected the threshold intensity.
DISCUSSION In our previous papers (Sugimoto, Nagata & Morita, 1972, 1978), in which the effects of several kinds of antiarrhythmic drugs were compared in prolonging the refractory period of isolated heart muscle, ajmaline, cocaine, lignocaine, phenytoin, procainamide, propranolol, quinidine and trimetazidine were found to produce concentration-dependent depressions of the force of contraction of the heart muscle at the higher concentrations used. In the present study diazepam was also found t o depress the force of contraction of rat heart muscle in concentrations greater than 20 pg/ml. However, in concentrations of 0.5 t o 10 pg/ml, diazepam increased the force of contraction. Over this concentration range, diazepam had little effect on the rate of spontaneous contractions of the right atrium and prolonged the refractory period of the left atrium and of papillary muscle. Therefore, the cardiac actions of diazepam appear t o differ from those of quinidine and other antiarrhythmic drugs. Diazepam prolonged the refractory period of the left atrium by about 100% and that of papillary muscle by about SO%, at maximum, and decreased the maximum driving frequency of the left atrium but not that of papillary muscle. These observations suggest that the cardiac action of diazepam is more marked in atrial than in ventricular muscle. "he actions of diazepam partially resemble those of cocaine and trimetazidine, which prolong the refractory period of atrial muscle but scarcely affect that of papillary muscle, without having a suppressant effect on the force of contractions, although these drugs (unlike diazepam) never augmented the force of contractions at any of the concentrations used (Sugimoto et al., 1972,1978). Chlorpromazine also prolonged the refractory period of papillary muscle. However, the prolongation was less than 50% of control at most and its effect was less than that of diazepam. Although the effect of chlorpromazine on the maximum driving frequency of the left atrium was greater than that of diazepam, the concentration producing these effects caused a pronounced decrease in the force of contraction of papillary muscle. The effects of phenytoin on the refractory period and maximum driving frequency of the left atrium and papillary muscle were weak. The arrhythmic contractions induced by electrical stimulation during spontaneous contractions of the right atrium were prevented by diazepam, but not by chlorpromazine or phenytoin. Therefore, the cardiac actions of diazepam do not appear to be a common property of tranquilizer drugs. Regarding the experimental techniques, the effects of drugs on the force of contraction of electrically driven cardiac muscle and on the refractory period are dependent on the driving frequency. In subsequent studies it will be necessary to determine which frequency of stimulation is most appropriate for extrapolation of the effects of drugs t o those on the heart in situ. Tanz (1974) determined the effects of drugs on the refractory period of cardiac muscle using the maximum driving frequency of the muscle; however, the results of the present study suggest that the effects of drugs on the refractory period and the maxi-
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mum driving frequency are not always the same. Essential differences between these two effects will be reported in a subsequent paper. REFERENCES Govier, W.C. (1965) The mechanism of the atrial refractory period change produced by ouabain. Journal o f Pharmacology and Experimental Therapeutics, 148,100-105. Prindle, K.H., Jr., Gold, H.K., Cardon, P.V. & Epstein, S.E. (1970) Effects of psychopharmacologic agents on myocardial contractility. Journal of Pharmacology and Experimental Therapeutics, 173, 133137. Randall, LO., Heise, G.A., Schalleck, W., Bagdon, RE, Baziger, R, Boris, A., Moe, RA. & A b m , W.B. (1961) Pharmacological and clinical studies on Valium (T.M.), a new psychotherapeutic agent of the benzodiazepine class. a r r e n t Therapeutic Research, 3,405-425. Sugimoto, J., Nagata, M. & Morita, M. (1972) Comparative studies on actions of anti-arrhythmic drugs in guinea-pig atria. Abstract of the Fifth International Congress on Pharmacology, San Francisco, p. 225, No. 1346. Sugimoto, J., Nagata, M.& Morita, M. (1978) Comparative studies on the pharmacological actions of antiarrhythmic drugs in isolated rat papillary muscle. Clinical and Experimental Pharmacology & Physiology, 5, 17-21. Tanz, R (1974) Pharmacology of aconitine-induced automaticity on in vitro cat myocardial preparations. 11. Effects of refractory period prolongation, reduced sodium and tetrodotoxin. Journal of Pharmacology and Experimental Therapeutics, 191,232-240.
