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Effect of dantrolene sodium on the transmembrane potentials and contractility of guinea pig atrial myocardium
European Journal of Pharmacology, 74 (1981 ) 181- 188 Elsevier/North-Holland Biomedical Press
181
E F F E C T OF D A N T R O L E N E S O D I U M O N T H E T R A N S M E M B R A N E CONTRACTILITY OF GUINEA PIG ATRIAL MYOCARDIUM *
POTENTIALS AND
JANOS MlkSZAROS 1, VALI~RIA KECSKEMI~TI 2,** and JOZSEF SZEGI i' I Department of Pharmacology, Medical University of Debrecen, H-4012 Debrecen, and e Department of Pharmacology, Semmelweis Universi O' of Medicine, Nagyvarad tbr 4, H-1445 Budapest, Hunga~.
Received 6 May 1981, accepted 11 June 1981
J. MESZAROS, V. KECSKEMIb.TI and J. SZEGI, Effect of dantrolene sodium on the transmembrane potentials and contractility of guinea pig atrial myocardum, European J. Pharmacol. 74 (1981) 181-188. Dantrolene sodium (3 × 10 4 M) exerted a biphasic effect on the mechanical and electrical activity of the isolated guinea pig left atrium. In the first phase, the drug increased the contractile force by about 200%, and prolonged the action potential duration. This transient positive inotropic effect was antagonized by D-600, a slow Caz+ channel inhibitor. In the later phase, the drug gradually decreased the amplitude of contractions, shortened the action potential duration and induced contracture. Dantrolene sodium increased the rate of depolarization and overshoot of CaZ+-mediated slow action potentials. The results suggest that dantrolene sodium increases the slow inward C a 2+ current, causing a positive inotropic effect in the atrial myocardium. The late negative inotropic effect of the drug could be due to a secondary inhibition of the slow inward Ca2+ current due to an increased intracellular free Ca2+ concentration as a consequence of other actions of the drug. Guinea pig atrium
Dantrolene sodium
Action potential
1. Introduction D a n t r o l e n e s o d i u m is a muscle r e l a x a n t used clinically in the t r e a t m e n t of spasticity ( S n y d e r et al., 1967; C h i a t t e et al., 1971; C h i p m a n et al., 1974); it depresses e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g b y i n h i b i t i n g calcium release from s a r c o p l a s m i c r e t i c u l u m (Ellis a n d Bryant, 1972; Ellis a n d C a r p e n t e r , 1972; P u t n e y a n d Bianchi, 1974; N o t t a n d B o w m a n , 1974; H a i n a u t a n d D e s m e d t , 1974; Brocklehurst, 1975; D e s m e d t a n d H a i n a u t , 1977; M o r g a n a n d Bryant, 1977). D a n t r o l e n e s o d i u m has no effect on n e u r o m u s c u l a r t r a n s m i s s i o n (Ellis a n d Carpenter, 1972; Z o r y c h t a et al., 1971; Lowndes, 1975; K u r i h a r a and Brooks, 1975) or on the electrical p r o p e r t i e s of the skeletal muscle m e m b r a n e (Ellis a n d Bryant, 1972; P u t n e y a n d Bianchi, 1974; H a t a e et al., 1980). The d a t a so far * A preliminary report of this work was presented at the XXVIII International Congress of Physiological Sciences, Budapest, 1980 (Mrszbxos et al., 1980). ** To whom all correspondence should be addressed. 0014-2999/81/0000-0000/$02.50
Contractility
Slow responses
p u b l i s h e d are inconsistent r e g a r d i n g the effect of d a n t r o l e n e s o d i u m on c a r d i a c muscle. It has been shown that d a n t r o l e n e s o d i u m has o n l y negligible effects on the c a r d i o v a s c u l a r system in dogs a n d sheep in vivo (Ellis et al., 1973, 1975, 1976) a n d on the isolated rat a n d guinea pig heart (Ellis et al., 1976; G o l l a n a n d M c D e r m o t t , 1979). O n the other h a n d , M e y l e r et al. (1976) r e p o r t e d that d a n t r o l e n e s o d i u m depressed the contractility of the isolated rat heart, b u t the heart frequency r e m a i n e d unchanged. D a n t r o l e n e s o d i u m caused depression in s p o n t a n e o u s l y b e a t i n g or electrically p a c e d atrial p r e p a r a t i o n s of guinea pig ( B o w m a n a n d K h a n , 1977). H a t a e et al. (1980) showed that d a n t r o l e n e s o d i u m increased the slow i n w a r d C a 2+ current which c o n s e q u e n t l y increased the twitch tension in the isolated left a t r i u m of guinea pig a n d the isolated frog atrium. T h e p u r p o s e of the present work was to investigate the effect of a relatively high conc e n t r a t i o n of d a n t r o l e n e s o d i u m (3 × 1 0 - 4 M ) on the t r a n s m e m b r a n e p o t e n t i a l s a n d c o n t r a c t i l e force in the isolated left a t r i u m of guinea pig.
© 1981 Elsevier/North-Holland Biomedical Press
182
2. Materials and methods Guinea pigs of either sex weighing 250-450 g were killed by a blow on the head. The heart was removed quickly and the atrium was dissected and mounted in an organ chamber. The preparations were pre-equilibrated for 30 min in Krebs solution of the following composition (mM): NaC1 1t8; KC1 4.7; CaC1 2.5; NaHzPO 4 1.0; MgCI 2 1.2; NaHCO 3 24.9; glucose 11.5 The solution was aerated with a gas mixture of 95% 02 and 5% CO2; the pH was 7.4 and the temperature was maintained at 37°C.
2.1. Contractions Isometric contractions of isolated left atrial preparations of guinea pigs were measured by means of an isometric force transducer. The contractions were monitored on a DISA oscilloscope and recorded on film and on a polygraph. The preparations were paced at a constant frequency of 2 Hz by isolated rectangular pulses of 1 msec duration and 3 times the threshold voltage, via bipolar platinum electrodes placed on the tissue. The resting tension was determined at which the contractile responses reached the maximal value. Doseresponse curves for the inotropic effect of different concentrations (10 -5 -10 -3 M) of dantrolene sodium were made with and without D-600 (1/~M). In order to prove the positive inotropic effect of dantrolene sodium, whether mediated by pharmacological receptors or direct stimulation of slow Ca2+ channels, the isolated atria were pretreated for 10 min with the following blockers: a /3adrenoceptor blocker pindolol (LB-46, 4 × 10-6 M), an a-adrenoceptor blocker phentolamine (10 5 M), a histamine H~-receptor blocker mepyramine (10 -5 M), a histamine H2-receptor blocker cimetidine (10 6 M) and a slow Ca 2+ channel blocker D-600 (1/~M).
2.2. Action potentials Resting and action potentials were recorded by means of conventional glass microelectrodes filled with 3 M KC1 having resistances 5-15 megohms, connected to a cathode follower and a DC ampli-
tier (MIKI-1623). They were displayed on a DISA oscilloscope and were photographed (Kelemen et al., 1968). The depolarization phase of the action potential was differentiated with an analog differentiator and the maximal rate of depolarization (Vma~) was recorded as described previously by Kecskem6ti et al. (1973). The parameters of electrical stimulation were same as described above. In some experiments, dantrolene sodium was tested for its effects on the fast Na + channel in atria in which the slow Ca 2+ channel was blocked by D-600 (5/~M). In other experiments, dantrolene sodium was tested for its effects on the slow responses induced by caffeine (2 mM) in K depolarized atria in which the fast Na + channel was inactivated (Pappano, 1970; Schneider and Sperelakis, 1975). In our experiments the fast Na + channel was inactivated by means of elevated K + (26 mM)-Krebs solution (isosmolar substitution of K + for Na+). Details of the method have been described previously (Kecskem6ti, 1978). Under such conditions the myocardium was inexcitable despite intense electrical stimulation. The K +depolarized preparations were paced at a rate of 0.5 Hz. The stimulation voltage was elevated up to 10 times the normal threshold. D-600 was applied to eliminate the slow responses.
2.3. Drugs used The drugs used were D-600 (Knoll AG.), pindolol (LB-46; Visken) (EGYT), phentolamine (regitine, CIBA), mepyramine maleate (May and Baker), cimetidine (Smith Kline and French Lab. Ltd.), dantrolene sodium (Norwich Pharmacal Comp.). Dantrolene sodium was dissolved in 20% propylene glycol, to prepare a 10 2 M stock solution. The substance remained dissolved only in a warm solution. The final 3× 10-4M concentration of the drug was achieved by quickly diluting 150 ~tl of warm solution with a Gilson micropipette in 5 ml of Krebs solution thermostated at 37°C in the organ chamber. All values presented are means+S.E.M.. The statistical significance of differences from the control was estimated using Student's t-test. A P value of less than 0.05 was considered significant.
183 3. Results
3.1. Effect of dantrolene sodium on contractions The action of dantrolene sodium on the isometric contractions was studied in relation to time and concentration in the isolated left atria of guinea pigs. The drug was added to the bathing solution after a pre-incubation period of 30 min. The records of tension development of guinea pig a t r i u m in r e s p o n s e to d a n t r o l e n e s o d i u m ( 3 X I 0 - 4 M ) are shown in fig. 1BC. As can be seen in the figure, dantrolene sodium exerted a time-dependent dual effect on the contractile force of the atria. In the initial period of the treatment, the drug had a strong positive inotropic effect on the m y o c a r d i u m ; the increasing contraction
80mY
amplitude reached the m a x i m u m value ( + 2 1 0 -+- 15.3%) by 5 - 10 min, and then successively decreased and returned to the control level in about 40 min. The drug did not affect the time to peak tension (fig. 1B). In the later period of exposure, the amplitude of twitch contractions decreased further and in most cases contractile activity was abolished by about the 120th min of the experiment. In one of the control series contractile force remained practically unchanged for 120 min. In the later period of dantrolene action, contracture also appeared. The positive inotropic effect of the drug was concentration-dependent. The enhancement of contractions occurred at 10 5 M and above. Dose-response relationships for the positive inotropic effect of dantrolene sodium on left atrium are shown in fig. 2. Each concentration of dantrolene was tested on different preparations before and after application of D-600 (1/~M). Dantrolene sodium was washed out for 30 min after the maxim u m value of its positive inotropic effect had been reached, the atrium was pretreated with D-600
!Jl ¢1!
lOOV/sec LOmsec
B
200
40 msec
C
3
b
I
5
..........
6OO
w LL LL W
_~ lOO
/ /
-F,L .1.2.0.m.i.n
lOmin
Fig. 1. (A) Effect of dantrolene sodium (3 X 10 4 M) on transmembrane action potentials of the guinea pig left atrium. Tracings, from top to bottom: zero potential level; action potentials before (I) and after (2-5) application of dantrolene; rate of depolarization of action potential. Bottom tracings were shifted to the right for easier analysis of ~'m~,. (B) Twitch contractions of left atrium before (1) and after (2-5) addition of dantrolene. (C) Time course of the biphasic effect of dantrolene sodium (DS) on the contractility of left atrium, recorded at a slow sweep speed. The numbers show the time of recording of the action potentials and twitch contractions shown in A and B.
.j/,/' 5
4 - LOG [ DS] M
3
Fig. 2. Dose-response relationships for the positive inotropic effect of dantrolene sodium, with and without D-600, in guinea pig left atrium. Filled circles (C): effect of dantrolene sodium alone. Open circles (D-600): effect of dantrolene sodium after 10 min pretreatment with D-600 (1 ffM). Values are means + S.E.M. of 5 preparations.
184
(1/~M) for 10 rain and dantrolene was then added again at the same dose. D-600, a slow Ca z+ channel blocker, decreased the positive inotropic effect of dantrolene; the dose-response curve was shifted to the right. D-600 had a slight negative inotropic effect at this concentration. The propylene glycol in which the dantrolene sodium was dissolved had no effect by itself on the contractions. At the highest concentration of dantrolene used (10 -3 M), the propylene glycol concentration in the incubation solution was 2%. This concentration of solvent had a negligible effect on the contractions of the atria. The inital positive inotropic effect of dantrolene sodium (3 × 10-4 M) was investigated in the presence of different receptor antagonists, in order to show whether the dantrolene action is mediated by pharmacological receptors or has a direct effect specifically on the slow Ca 2+ channel. It was found that neither the a-adrenoceptor blocker phentolamine (10 -5 M) nor the fl-adrenoceptor blocker pindolol (LB-46, 4 × 10 - 6 M) was able to prevent the positive inotropic effect of dantrolene sodium after a preincubation period of 10 min (fig. 3A). The histamine Hi-receptor blocker mepyramine (10-5 M) and histamine Hz-receptor blocker cimetidine (10-6 M) did not influence the positive effect of dantrolene sodium (fig. 3B). The effects of phentolamine and cimetidine are not shown.
C D-6OO +DS 0.5g 10rain
Fig. 3. Effect of dantrolene sodium (DS) (3 x 10 -4 M) on the contractility of guinea pig left atrium pretreated with (A) fl-adrenoceptor blocker pindolol (LB-46, 4X l0 6 M), (B) the histamine Hi-receptor antagonist mepyramine (Mep, l0-5 M) and (C) the slow Ca 2+ channel blocker D-600 (1/~M).
A Control
B .DS
3x10-4M
C *Ca"
/.mM
4Omsec
1
02g
1rain
Fig. 4. Calcium reversal of dantrolene sodium-abolished plateau phase and contractility in guinea pig left atrium. (A) Control action potential and contractions. (B) Effect of dantrolene sodium (3 × 10-4 M) on the action potential and contractility during long-lasting treatment (120 rain). (C) Addition of 4 mM Ca 2+ to the dantrolene-treated atrium restored the plateau and the contractions.
The positive inotropic effect of dantrolene was completely inhibited by pretreatment with I # M D-600 (fig. 3C). The later negative inotropic effect of dantrolene sodium was antagonized when the external Ca 2+ concentration was increased to 6.5 mM (fig. 4).
3.2. Effect of dantrolene sodium on normal action potentials The control action potentials obtained in 5 isolated left atrial preparations of guinea pigs had the following parameters: overshoot, + 20.1 ± 1.6 mV; maximal rate of depolarization, 185.3 ___2.7 V/sec; duration of the action potential at 50% repolarization, 36.5 ± 1.1 msec. The resting potential was about - 8 0 mV. Dantrolene sodium was added at a concentration of 3 × 10-4 M after a preincubation period of 30 min. In this concentration the drug had a time-dependent dual effect on the action potentials. In the first stage of action, the duration of the action potential was prolonged homogeneously, the rate of depolarization was slightly reduced and the overshoot was increased (table 1 and fig. 1A). At this time a strong increase in 'contractile force was observed. In the later period of dantrolene action when the drug produced a negative ifiotropic effect, the action potential duration was shortened, the overshoot de-
185 TABLE l Effect of dantrolene sodium (3 × 10 4 M) on the parameters of action potential and relative contractile force in isolated left atrial preparations of guinea pigs. Mean ± S.E.M. of 5 experiments. Treatment
Control Dantrolene 5-10 mix 30-50 min 90-130 mix
Resting potential (mV)
Overshoot (mV)
Vm~, (V/sec)
Action potential duration at repolarization (msec)
Relative contractile force (%)
20%
50%
90%
- 8 2 . 9 -+- 1.8
+20.1 ± 1.6
185.3±2.7
20.4±0.6
36.5± 1.1
70.1 ± 1.9
100
-81.8±2.9 -80.5±2.1 -75.7__+1.7 *
+22.3±2.2 +18.3±1.2 +15.0___0.9"
172.6±3.9" 168.3±1.4" 141.6__+1.8 *
28.3±1.6" 19.5±1.4 6.7±0.5*
50.1±1.0" 36.5±1.2 13.5+1.1 *
85.0±2.7* 71.3±1.8 41.6±1.3"
310 95 0
* Statistically significant difference from the control (P<0.05).
creased, the rate of depolarization was decreased further, and the membrane was slightly depolarized. Elevation of external Ca2+ concentration to 6.5 mM transiently restored (for 3 - 6 mix) the plateau phase without a marked increase in the duration of action potential (fig. 4). Dantrolene sodium was also tested for its effects on the fast Na ÷ channel in atria in which the slow Ca2+ channel had first been blocked by 5 ~tM D-600. Fig. 5B shows that the plateau phase of the action potential was abolished, but the late repolarization time was prolonged by D-600. Dantrolene sodium (3 × 10 -4 M) shortened the late repolarization time of the action potential, but did not affect the early repolarization phase (fig. 5).
partially or even completely, according to the value of the depolarization whereas the slow Ca 2+ channel remains fully available. Under this condition a Ca2+-mediated slow action potential was induced by catecholamines (Pappano, 1970) by histamine (Inui and Imamura, 1976; Kecskemrti, 1978) or by methylxanthines (Schneider and Sperelakis, 1975). In the presence of elevated K + (26 mM)-Krebs solution our preparation became inexcitable i.e., action potential and tension development were gradually depressed and finally abolished, and only a resting potential could be recorded (fig. 6B). The A Control
B *K" (26 mM)
C *Caffeine
2 rnM
w:\ 185~
3.3. Effect of dantrolene sodium on slow action potentials When a myocardial fiber undergoes partial depolarization, the fast Na ÷ channel is inactivated A Control
B +D-600 5kaM
C +DS
D 3xl0-/'M
D *DS
3x10"~M
E ~-D-600 2)aM
V/ 12
40mV f
/,0mse¢ Fig. 5. Effect of dantrolene sodium on action potential mediated by the fast Na + channel in isolated left atrium. (A) Control action potential. (B) D-600 (5 ~M)-induced shortening of action potential duration. (C) 15 rain, (D) 120 rain after addition of dantrolene sodium (3?< 10 4 M).
~
/'0 mse~
Fig. 6. Effect of dantrolene sodium on the slow action potential induced by caffeine in K+-depolarized left atrium of guinea pig. (A) Control action potential. (B) Application of K + rich (26 raM) Krebs solution depolarized the membrane to about - 4 0 mV and abolished the excitability. (C) Caffeine (2 mM)induced slow action potential. (D) Addition of dantrolene sodium (3 × 10 -4 M) increased the maximal rate of depolarization, overshoot and duration of slow action potential within l0 mix. (E) D-600 (2/tM) completely abolished the slow responses within 3 mix.
186
K +-depolarized atria were stimulated at a rate of 0.5 Hz and stimulation intensities of 10 times the control threshold. Caffeine at a concentration of 2 mM rapidly (within 3 min) induced slow action potentials (fig. 6C). Ca2÷-mediated action potentials were characterized by a slow rate of depolarization (10.5__+0.9 V/sec). The overshoot was + 20.1 ± 0.5 mV, and the duration at 50% repolarization was 45.2___ 1.6 msec. After addition of dantrolene sodium (3× 10 -4 M) the rate of depolarization of slow responses was significantly increased up to 12.5 ± 0 . 3 V/sec. Furthermore, the overshoot of the Ca2÷-mediated action potential was elevated to + 2 3 . 3 ± 0 . 8 mV. Moreover, in all experiments (in seven atria) dantrolene sodium significantly prolonged the duration of slow action potentials to 51.5___ 1.3 msec (fig. 6D). These slow responses were completely abolished by D-600, 2 #M (fig. 6E). In 5 of 9 experiments dantrolene sodium was able to induce slow responses by itself at a concentration of 3 × 10-4 M.
4. Discussion We have shown that dantrolene sodium produced a dose- and time-dependent action on the contractility of isolated guinea pig left atrium. Our observations on the long-lasting negative inotropic effect of dantrolene sodium were consistent with reports of others (Meyler et al., 1976; Bowman and Khan, 1977). On the other hand, in the first period of the experiment the contractile force was increased by dantrolene sodium (up to 10 -5 M). This dose-dependent positive inotropic effect of dantrolene sodium was accompanied by the lengthening of action potential duration. The contractile force was transiently potentiated over 10 min then decreased gradually. The transient positive effect of dantrolene was independent of adrenergic fl-receptors and of histamine receptors but was prevented by D-600, a Ca 2+ channel blocker. Dantrolene sodium enhanced the amplitude and the rate of depolarization of the slow responses induced by caffeine in guinea pig atria partially depolarized by elevated K + and in some experiments dantrolene itself was able to induce slow responses. As the slow responses were
mainly Ca 2+ dependent and the increase in the plateau phase was probably due to the increase of the slow inward Ca 2+ current, the potentiation of the contractile force by dantrolene sodium may have been due to an augmentation of the slow inward Ca 2+ current. Our findings are in good agreement with the data of others (Oba and Hotta, 1978; Hatae et al., 1980) showing a positive inotropic action of dantrolene sodium in frog skeletal muscle and in guinea pig atrial muscle. However, other explanations for the observed positive inotropic effect of the drug cannot be excluded. Since the outward current is not negligible during the slow inward Ca 2÷ current, drugs added or alterations of the intracellular Ca 2+ concentration can indirectly influence the slow inward Ca 2÷ current by modifying the outward current (Pappano, 1976; Isenberg, 1975, 1977). The depressive effect on the outward current, the decrease of the calcium efflux or an effect on Ca 2÷ sequestration into the intracellular reservoir might also explain the positive inotropic effect of dantrolene sodium. In the present study dantrolene sodium exerted a negative inotropic effect after 40 min, accompanied by the shortening of the action potential duration. Later the contractions were abolished and a slow contracture developed. It is not probable that this late negative effect of the drug can be explained by the inhibition of activator calcium release from the sarcoplasmic reticulum (Ellis and Bryant, 1972; Putney and Bianchi, 1974; Van Winkle, 1976). The observed late negative inotropic effect of dantrolene sodium could be conceived as resulting from a direct inhibitory effect on the slow Ca 2÷ channel. This possibility is excluded since Desmedt and Hainaut (1977) observed that 45Ca efflux from barnacle muscle fiber was decreased by dantrolene sodium, whereas 45Ca influx was not significantly changed. The most plausible theory is that dantrolene s o d i u m - - b y facilitating the slow inward Ca 2+ current and additionally inhibiting the permeability of the sarcoplasmic reticulum membrane to calcium ions thus decreasing Ca 2+ efflux--causes lessening of the transmembrane Ca 2+ concentration gradient due to an intracellular accumulation of free Ca 2÷. This hypothesis is supported by the finding of the
187 s h o r t a c t i o n p o t e n t i a l d u r a t i o n a n d o f the c o n t r a c ture. K o h l h a r d t et al. (1974) g a v e a s i m i l a r exp l a n a t i o n for t h e b i p h a s i c e f f e c t of c a f f e i n e o n t h e t r a n s m e m b r a n e C a 2÷ c u r r e n t in the right v e n t r i c u lar p a p i l l a r y m u s c l e o f the cat. H o w e v e r , o t h e r e x p l a n a t i o n s e.g., the e f f e c t o n C a 2+ e x c h a n g e at the m i t o c h o n d r i a l m e m b r a n e c a n also b e o f f e r e d .
Acknowledgements We are indebted to Norwich Pharmacal Comp. for the supply of dantrolene sodium, to Knoll AG. for D-600 and to Smith Kline and French Lab. LTD for cimetidine.
References Bowman, W.C. and H.H. Khan, 1977, Effects of dantrolene sodium on isolated skeletal, smooth and cardiac muscle of the guinea-pig, J. Pharm. Pharmacol. 29, 628. Brocklehurst, L., 1975, Dantrolene sodium and 'skinned; muscle fibers, Nature 254, 364. Chipman, M., S. Kaul and M. Lambi, 1974, Efficacy of dantrolene sodium in the treatment of spasticity, Dis. Nerv. Syst. 35,427. Chiatte, S.B., J.H. Birdsong and B.A. Bergman, 1971, The effects of dantrolene sodium on spasticity and motor performance in hemiplegia, South. Med. J. 64, 180. Desmedt, J.E. and K. Hainaut, 1977, Inhibition of the intracellular release of calcium by dantrolene in barnacle giant muscle fibres, J. Physiol. 265, 565. Ellis, K.O. and S.H. Bryant, 1972, Excitation-contraction uncoupling in skeletal muscle by dantrolene sodium, NaunynSchmiedeb. Arch. Pharmacol. 274, 107. Ellis, K.O., J.L. Butterfield, F.L. Wessels and J.F. Carpenter, 1976, A comparison of skeletal, cardiac and smooth muscle actons of dantrolene sodium--a skeletal muscle relaxant, Arch. Int. Pharmacodyn. 244, 118. Ellis, K.O. and J.F. Carpenter, 1972, Studies on the mechanism of action of dantrolene sodium a skeletal muscle relaxant, Naunyn-Schmiedeb. Arch. Pharmacol. 275, 83. Ellis, K.O., A.W. Castellion, L.J. Honkomp, F.L. Wessels, J.F. Carpenter and R.P. Halliday, 1973, Dantrolene, a direct acting skeletal muscle relaxant, J. Pharm. Sci. 62, 948. Ellis, R.H., P. Simpson, P. Tatham, M. Leighton and J. Williams, 1975, The cardiovascular effects of dantrolene sodium in dogs, Anaesthesia 30, 318. Gollan, F. and J. McDermott, 1979, Effect of the skeletal muscle relaxant dantrolene sodium on the isolated, perfused heart, Proc. Soc. Exp. Biol. Med. 160, 42. Hainaut, K. and J.E. Desmedt, 1974, Effect of dantrolene sodium on calcium movements in single muscle fibers, Nature 252, 728.
Hatae, J., M. Ohba and H. Kawata, 1980, Effects of dantrolene sodium on the excitation-contraction coupling of the mammalian and amphibian cardiac muscle, J. Mol. Cell. Cardiol. 12, 857. Inui, I. and H. Imamura, 1976, Restoration by histamine of the calcium-dependent electrical and mechanical response in the guinea pig papillary muscle partially depolarized by potassium, Naunyn-Schmiedeb. Arch. Pharmacol. 294, 261. Isenberg, G., 1975, Is potassium conductance of cardiac Purkinje fibers controlled by [Call?, Nature 253, 273. Isenberg, G., 1977, Cardiac Purkinje fibers. The slow inward current component under the influence of modified [Ca]i , Pfliigers Arch. 371, 61. Kecskem~ti, V., 1978, Restorative effect of histamine on the transmembrane action potentials in potassium-depolarized guinea-pig auricle, Pol. J. Pharmacol. Pharm. 30, 377. Kecskemeti, V., K. Kelemen and J. Knoll, 1973, Effect of prostaglandin E L on the cardiac transmembrane potentials, European J. Pharmacol. 24, 289. Kelemen, K., V. Kecskem~ti, L. Scult~ty and T. Friedmann, 1968, Activity index of frog heart ventricle cells. Analysis of the correlation between resting potential and activity of the sodium-carrier system in the frog heart by means of celluline and adrenaline, Acta Physiol. Acad. Sci. Hung. 33, 269. Kohlhardt, M., M. Ktibler and E. Hansi, 1974, Ambiguous effect of caffeine upon the transmembrane Ca current in mammalian ventricular myocardium, Experientia 30, 254. Kurihara, T. and J.E. Brooks, 1975, The effect of hyperosmolar glycerol solution and dantrolene sodium on mammalian muscle in vitro, Arch. Neurol. 32, 92. Lowndes, H.E., 1975, Dantrolene effects on neuromuscular function in cat soleus muscle, European J. Pharmacol. 32, 267. Mtszhros, J., V. Kecskem~ti and J. Szegi, 1980, Effect of dantrolene sodium on cardiac transmembrane potentials and contractility, Abstracts book of the XXVIIIth International Congress of Physiological Sciences, p. 579, Budapest. Meyler, W.J., H. Wesseling and S. Agoston, 1976, The effects of dantrolene sodium on cardiac and skeletal muscle in rats, European J. Pharmacol. 39, 127. Morgan, K.G. and S.H. Bryant, 1977, The mechanism of action of dantrolene sodium, J. Pharmacol. Exp. Ther. 201, 138. N0tt, M.W. and W.C. Bowman, 1974, Actions of dantrolene sodium on contractions of the tibialis anterior and soleus muscles of cats under chloralose anaesthesia, Cln. Exp. Pharmacol. Physiol. 1, 113. Oba, T. and K. Hotta, 1978, Transient effect of intracellular dantrolene on E-C coupling in skeletal muscle, European J. Pharmacol. 51, 8 I. Pappano, A.J., 1970, Calcium dependent action potentials produced by catecholamines in guinea pig atrial fibers depolarized by potassium, Circulation Res. 27, 379. Pappano, A.J., 1976, Action potentials in chick atria. Ontogenetic changes in the dependence of tetrodotoxin-resistance action potentials on calcium, strontium, and barium, Circulation Res. 39, 99. Putney, J.W. and C.P. Bianchi, 1974, Site of action of dantro-
188 lene in frog sartorius muscle, J. Pharmacol. Exp. Ther. 189, 202. Schneider, J.A. and N. Sperelakis, 1975, Slow Ca 2+ an Na ~ responses induced by isopreterenol and methylxanthines in isolated perfused guinea pig hearts exposed to elevated K ÷, J. Mol. Cell, Cardiol. 7, 249. Snyder, H.R., C.S. Davis, R.K. Bickerton and R.P. Halliday, 1967, l-[(5-Arylfurfurylidene)-amino] hydantoins, A new class of muscle relaxants, J. med. Chem. 10, 807.
Van Winkle, W.B., 1976, Calcium release from skeletal muscle sarcoplasmic reticulum: Site of action of dantrolene sodium?, Science 193, 1130. Zorychta, E., D.W. Esplin, R. Capek and A. Lastowecka, 1971, The actions of dantrolene on extrafusal and intrafusal striated muscle, Federation Proc. 30, 669.
181
E F F E C T OF D A N T R O L E N E S O D I U M O N T H E T R A N S M E M B R A N E CONTRACTILITY OF GUINEA PIG ATRIAL MYOCARDIUM *
POTENTIALS AND
JANOS MlkSZAROS 1, VALI~RIA KECSKEMI~TI 2,** and JOZSEF SZEGI i' I Department of Pharmacology, Medical University of Debrecen, H-4012 Debrecen, and e Department of Pharmacology, Semmelweis Universi O' of Medicine, Nagyvarad tbr 4, H-1445 Budapest, Hunga~.
Received 6 May 1981, accepted 11 June 1981
J. MESZAROS, V. KECSKEMIb.TI and J. SZEGI, Effect of dantrolene sodium on the transmembrane potentials and contractility of guinea pig atrial myocardum, European J. Pharmacol. 74 (1981) 181-188. Dantrolene sodium (3 × 10 4 M) exerted a biphasic effect on the mechanical and electrical activity of the isolated guinea pig left atrium. In the first phase, the drug increased the contractile force by about 200%, and prolonged the action potential duration. This transient positive inotropic effect was antagonized by D-600, a slow Caz+ channel inhibitor. In the later phase, the drug gradually decreased the amplitude of contractions, shortened the action potential duration and induced contracture. Dantrolene sodium increased the rate of depolarization and overshoot of CaZ+-mediated slow action potentials. The results suggest that dantrolene sodium increases the slow inward C a 2+ current, causing a positive inotropic effect in the atrial myocardium. The late negative inotropic effect of the drug could be due to a secondary inhibition of the slow inward Ca2+ current due to an increased intracellular free Ca2+ concentration as a consequence of other actions of the drug. Guinea pig atrium
Dantrolene sodium
Action potential
1. Introduction D a n t r o l e n e s o d i u m is a muscle r e l a x a n t used clinically in the t r e a t m e n t of spasticity ( S n y d e r et al., 1967; C h i a t t e et al., 1971; C h i p m a n et al., 1974); it depresses e x c i t a t i o n - c o n t r a c t i o n c o u p l i n g b y i n h i b i t i n g calcium release from s a r c o p l a s m i c r e t i c u l u m (Ellis a n d Bryant, 1972; Ellis a n d C a r p e n t e r , 1972; P u t n e y a n d Bianchi, 1974; N o t t a n d B o w m a n , 1974; H a i n a u t a n d D e s m e d t , 1974; Brocklehurst, 1975; D e s m e d t a n d H a i n a u t , 1977; M o r g a n a n d Bryant, 1977). D a n t r o l e n e s o d i u m has no effect on n e u r o m u s c u l a r t r a n s m i s s i o n (Ellis a n d Carpenter, 1972; Z o r y c h t a et al., 1971; Lowndes, 1975; K u r i h a r a and Brooks, 1975) or on the electrical p r o p e r t i e s of the skeletal muscle m e m b r a n e (Ellis a n d Bryant, 1972; P u t n e y a n d Bianchi, 1974; H a t a e et al., 1980). The d a t a so far * A preliminary report of this work was presented at the XXVIII International Congress of Physiological Sciences, Budapest, 1980 (Mrszbxos et al., 1980). ** To whom all correspondence should be addressed. 0014-2999/81/0000-0000/$02.50
Contractility
Slow responses
p u b l i s h e d are inconsistent r e g a r d i n g the effect of d a n t r o l e n e s o d i u m on c a r d i a c muscle. It has been shown that d a n t r o l e n e s o d i u m has o n l y negligible effects on the c a r d i o v a s c u l a r system in dogs a n d sheep in vivo (Ellis et al., 1973, 1975, 1976) a n d on the isolated rat a n d guinea pig heart (Ellis et al., 1976; G o l l a n a n d M c D e r m o t t , 1979). O n the other h a n d , M e y l e r et al. (1976) r e p o r t e d that d a n t r o l e n e s o d i u m depressed the contractility of the isolated rat heart, b u t the heart frequency r e m a i n e d unchanged. D a n t r o l e n e s o d i u m caused depression in s p o n t a n e o u s l y b e a t i n g or electrically p a c e d atrial p r e p a r a t i o n s of guinea pig ( B o w m a n a n d K h a n , 1977). H a t a e et al. (1980) showed that d a n t r o l e n e s o d i u m increased the slow i n w a r d C a 2+ current which c o n s e q u e n t l y increased the twitch tension in the isolated left a t r i u m of guinea pig a n d the isolated frog atrium. T h e p u r p o s e of the present work was to investigate the effect of a relatively high conc e n t r a t i o n of d a n t r o l e n e s o d i u m (3 × 1 0 - 4 M ) on the t r a n s m e m b r a n e p o t e n t i a l s a n d c o n t r a c t i l e force in the isolated left a t r i u m of guinea pig.
© 1981 Elsevier/North-Holland Biomedical Press
182
2. Materials and methods Guinea pigs of either sex weighing 250-450 g were killed by a blow on the head. The heart was removed quickly and the atrium was dissected and mounted in an organ chamber. The preparations were pre-equilibrated for 30 min in Krebs solution of the following composition (mM): NaC1 1t8; KC1 4.7; CaC1 2.5; NaHzPO 4 1.0; MgCI 2 1.2; NaHCO 3 24.9; glucose 11.5 The solution was aerated with a gas mixture of 95% 02 and 5% CO2; the pH was 7.4 and the temperature was maintained at 37°C.
2.1. Contractions Isometric contractions of isolated left atrial preparations of guinea pigs were measured by means of an isometric force transducer. The contractions were monitored on a DISA oscilloscope and recorded on film and on a polygraph. The preparations were paced at a constant frequency of 2 Hz by isolated rectangular pulses of 1 msec duration and 3 times the threshold voltage, via bipolar platinum electrodes placed on the tissue. The resting tension was determined at which the contractile responses reached the maximal value. Doseresponse curves for the inotropic effect of different concentrations (10 -5 -10 -3 M) of dantrolene sodium were made with and without D-600 (1/~M). In order to prove the positive inotropic effect of dantrolene sodium, whether mediated by pharmacological receptors or direct stimulation of slow Ca2+ channels, the isolated atria were pretreated for 10 min with the following blockers: a /3adrenoceptor blocker pindolol (LB-46, 4 × 10-6 M), an a-adrenoceptor blocker phentolamine (10 5 M), a histamine H~-receptor blocker mepyramine (10 -5 M), a histamine H2-receptor blocker cimetidine (10 6 M) and a slow Ca 2+ channel blocker D-600 (1/~M).
2.2. Action potentials Resting and action potentials were recorded by means of conventional glass microelectrodes filled with 3 M KC1 having resistances 5-15 megohms, connected to a cathode follower and a DC ampli-
tier (MIKI-1623). They were displayed on a DISA oscilloscope and were photographed (Kelemen et al., 1968). The depolarization phase of the action potential was differentiated with an analog differentiator and the maximal rate of depolarization (Vma~) was recorded as described previously by Kecskem6ti et al. (1973). The parameters of electrical stimulation were same as described above. In some experiments, dantrolene sodium was tested for its effects on the fast Na + channel in atria in which the slow Ca 2+ channel was blocked by D-600 (5/~M). In other experiments, dantrolene sodium was tested for its effects on the slow responses induced by caffeine (2 mM) in K depolarized atria in which the fast Na + channel was inactivated (Pappano, 1970; Schneider and Sperelakis, 1975). In our experiments the fast Na + channel was inactivated by means of elevated K + (26 mM)-Krebs solution (isosmolar substitution of K + for Na+). Details of the method have been described previously (Kecskem6ti, 1978). Under such conditions the myocardium was inexcitable despite intense electrical stimulation. The K +depolarized preparations were paced at a rate of 0.5 Hz. The stimulation voltage was elevated up to 10 times the normal threshold. D-600 was applied to eliminate the slow responses.
2.3. Drugs used The drugs used were D-600 (Knoll AG.), pindolol (LB-46; Visken) (EGYT), phentolamine (regitine, CIBA), mepyramine maleate (May and Baker), cimetidine (Smith Kline and French Lab. Ltd.), dantrolene sodium (Norwich Pharmacal Comp.). Dantrolene sodium was dissolved in 20% propylene glycol, to prepare a 10 2 M stock solution. The substance remained dissolved only in a warm solution. The final 3× 10-4M concentration of the drug was achieved by quickly diluting 150 ~tl of warm solution with a Gilson micropipette in 5 ml of Krebs solution thermostated at 37°C in the organ chamber. All values presented are means+S.E.M.. The statistical significance of differences from the control was estimated using Student's t-test. A P value of less than 0.05 was considered significant.
183 3. Results
3.1. Effect of dantrolene sodium on contractions The action of dantrolene sodium on the isometric contractions was studied in relation to time and concentration in the isolated left atria of guinea pigs. The drug was added to the bathing solution after a pre-incubation period of 30 min. The records of tension development of guinea pig a t r i u m in r e s p o n s e to d a n t r o l e n e s o d i u m ( 3 X I 0 - 4 M ) are shown in fig. 1BC. As can be seen in the figure, dantrolene sodium exerted a time-dependent dual effect on the contractile force of the atria. In the initial period of the treatment, the drug had a strong positive inotropic effect on the m y o c a r d i u m ; the increasing contraction
80mY
amplitude reached the m a x i m u m value ( + 2 1 0 -+- 15.3%) by 5 - 10 min, and then successively decreased and returned to the control level in about 40 min. The drug did not affect the time to peak tension (fig. 1B). In the later period of exposure, the amplitude of twitch contractions decreased further and in most cases contractile activity was abolished by about the 120th min of the experiment. In one of the control series contractile force remained practically unchanged for 120 min. In the later period of dantrolene action, contracture also appeared. The positive inotropic effect of the drug was concentration-dependent. The enhancement of contractions occurred at 10 5 M and above. Dose-response relationships for the positive inotropic effect of dantrolene sodium on left atrium are shown in fig. 2. Each concentration of dantrolene was tested on different preparations before and after application of D-600 (1/~M). Dantrolene sodium was washed out for 30 min after the maxim u m value of its positive inotropic effect had been reached, the atrium was pretreated with D-600
!Jl ¢1!
lOOV/sec LOmsec
B
200
40 msec
C
3
b
I
5
..........
6OO
w LL LL W
_~ lOO
/ /
-F,L .1.2.0.m.i.n
lOmin
Fig. 1. (A) Effect of dantrolene sodium (3 X 10 4 M) on transmembrane action potentials of the guinea pig left atrium. Tracings, from top to bottom: zero potential level; action potentials before (I) and after (2-5) application of dantrolene; rate of depolarization of action potential. Bottom tracings were shifted to the right for easier analysis of ~'m~,. (B) Twitch contractions of left atrium before (1) and after (2-5) addition of dantrolene. (C) Time course of the biphasic effect of dantrolene sodium (DS) on the contractility of left atrium, recorded at a slow sweep speed. The numbers show the time of recording of the action potentials and twitch contractions shown in A and B.
.j/,/' 5
4 - LOG [ DS] M
3
Fig. 2. Dose-response relationships for the positive inotropic effect of dantrolene sodium, with and without D-600, in guinea pig left atrium. Filled circles (C): effect of dantrolene sodium alone. Open circles (D-600): effect of dantrolene sodium after 10 min pretreatment with D-600 (1 ffM). Values are means + S.E.M. of 5 preparations.
184
(1/~M) for 10 rain and dantrolene was then added again at the same dose. D-600, a slow Ca z+ channel blocker, decreased the positive inotropic effect of dantrolene; the dose-response curve was shifted to the right. D-600 had a slight negative inotropic effect at this concentration. The propylene glycol in which the dantrolene sodium was dissolved had no effect by itself on the contractions. At the highest concentration of dantrolene used (10 -3 M), the propylene glycol concentration in the incubation solution was 2%. This concentration of solvent had a negligible effect on the contractions of the atria. The inital positive inotropic effect of dantrolene sodium (3 × 10-4 M) was investigated in the presence of different receptor antagonists, in order to show whether the dantrolene action is mediated by pharmacological receptors or has a direct effect specifically on the slow Ca 2+ channel. It was found that neither the a-adrenoceptor blocker phentolamine (10 -5 M) nor the fl-adrenoceptor blocker pindolol (LB-46, 4 × 10 - 6 M) was able to prevent the positive inotropic effect of dantrolene sodium after a preincubation period of 10 min (fig. 3A). The histamine Hi-receptor blocker mepyramine (10-5 M) and histamine Hz-receptor blocker cimetidine (10-6 M) did not influence the positive effect of dantrolene sodium (fig. 3B). The effects of phentolamine and cimetidine are not shown.
C D-6OO +DS 0.5g 10rain
Fig. 3. Effect of dantrolene sodium (DS) (3 x 10 -4 M) on the contractility of guinea pig left atrium pretreated with (A) fl-adrenoceptor blocker pindolol (LB-46, 4X l0 6 M), (B) the histamine Hi-receptor antagonist mepyramine (Mep, l0-5 M) and (C) the slow Ca 2+ channel blocker D-600 (1/~M).
A Control
B .DS
3x10-4M
C *Ca"
/.mM
4Omsec
1
02g
1rain
Fig. 4. Calcium reversal of dantrolene sodium-abolished plateau phase and contractility in guinea pig left atrium. (A) Control action potential and contractions. (B) Effect of dantrolene sodium (3 × 10-4 M) on the action potential and contractility during long-lasting treatment (120 rain). (C) Addition of 4 mM Ca 2+ to the dantrolene-treated atrium restored the plateau and the contractions.
The positive inotropic effect of dantrolene was completely inhibited by pretreatment with I # M D-600 (fig. 3C). The later negative inotropic effect of dantrolene sodium was antagonized when the external Ca 2+ concentration was increased to 6.5 mM (fig. 4).
3.2. Effect of dantrolene sodium on normal action potentials The control action potentials obtained in 5 isolated left atrial preparations of guinea pigs had the following parameters: overshoot, + 20.1 ± 1.6 mV; maximal rate of depolarization, 185.3 ___2.7 V/sec; duration of the action potential at 50% repolarization, 36.5 ± 1.1 msec. The resting potential was about - 8 0 mV. Dantrolene sodium was added at a concentration of 3 × 10-4 M after a preincubation period of 30 min. In this concentration the drug had a time-dependent dual effect on the action potentials. In the first stage of action, the duration of the action potential was prolonged homogeneously, the rate of depolarization was slightly reduced and the overshoot was increased (table 1 and fig. 1A). At this time a strong increase in 'contractile force was observed. In the later period of dantrolene action when the drug produced a negative ifiotropic effect, the action potential duration was shortened, the overshoot de-
185 TABLE l Effect of dantrolene sodium (3 × 10 4 M) on the parameters of action potential and relative contractile force in isolated left atrial preparations of guinea pigs. Mean ± S.E.M. of 5 experiments. Treatment
Control Dantrolene 5-10 mix 30-50 min 90-130 mix
Resting potential (mV)
Overshoot (mV)
Vm~, (V/sec)
Action potential duration at repolarization (msec)
Relative contractile force (%)
20%
50%
90%
- 8 2 . 9 -+- 1.8
+20.1 ± 1.6
185.3±2.7
20.4±0.6
36.5± 1.1
70.1 ± 1.9
100
-81.8±2.9 -80.5±2.1 -75.7__+1.7 *
+22.3±2.2 +18.3±1.2 +15.0___0.9"
172.6±3.9" 168.3±1.4" 141.6__+1.8 *
28.3±1.6" 19.5±1.4 6.7±0.5*
50.1±1.0" 36.5±1.2 13.5+1.1 *
85.0±2.7* 71.3±1.8 41.6±1.3"
310 95 0
* Statistically significant difference from the control (P<0.05).
creased, the rate of depolarization was decreased further, and the membrane was slightly depolarized. Elevation of external Ca2+ concentration to 6.5 mM transiently restored (for 3 - 6 mix) the plateau phase without a marked increase in the duration of action potential (fig. 4). Dantrolene sodium was also tested for its effects on the fast Na ÷ channel in atria in which the slow Ca2+ channel had first been blocked by 5 ~tM D-600. Fig. 5B shows that the plateau phase of the action potential was abolished, but the late repolarization time was prolonged by D-600. Dantrolene sodium (3 × 10 -4 M) shortened the late repolarization time of the action potential, but did not affect the early repolarization phase (fig. 5).
partially or even completely, according to the value of the depolarization whereas the slow Ca 2+ channel remains fully available. Under this condition a Ca2+-mediated slow action potential was induced by catecholamines (Pappano, 1970) by histamine (Inui and Imamura, 1976; Kecskemrti, 1978) or by methylxanthines (Schneider and Sperelakis, 1975). In the presence of elevated K + (26 mM)-Krebs solution our preparation became inexcitable i.e., action potential and tension development were gradually depressed and finally abolished, and only a resting potential could be recorded (fig. 6B). The A Control
B *K" (26 mM)
C *Caffeine
2 rnM
w:\ 185~
3.3. Effect of dantrolene sodium on slow action potentials When a myocardial fiber undergoes partial depolarization, the fast Na ÷ channel is inactivated A Control
B +D-600 5kaM
C +DS
D 3xl0-/'M
D *DS
3x10"~M
E ~-D-600 2)aM
V/ 12
40mV f
/,0mse¢ Fig. 5. Effect of dantrolene sodium on action potential mediated by the fast Na + channel in isolated left atrium. (A) Control action potential. (B) D-600 (5 ~M)-induced shortening of action potential duration. (C) 15 rain, (D) 120 rain after addition of dantrolene sodium (3?< 10 4 M).
~
/'0 mse~
Fig. 6. Effect of dantrolene sodium on the slow action potential induced by caffeine in K+-depolarized left atrium of guinea pig. (A) Control action potential. (B) Application of K + rich (26 raM) Krebs solution depolarized the membrane to about - 4 0 mV and abolished the excitability. (C) Caffeine (2 mM)induced slow action potential. (D) Addition of dantrolene sodium (3 × 10 -4 M) increased the maximal rate of depolarization, overshoot and duration of slow action potential within l0 mix. (E) D-600 (2/tM) completely abolished the slow responses within 3 mix.
186
K +-depolarized atria were stimulated at a rate of 0.5 Hz and stimulation intensities of 10 times the control threshold. Caffeine at a concentration of 2 mM rapidly (within 3 min) induced slow action potentials (fig. 6C). Ca2÷-mediated action potentials were characterized by a slow rate of depolarization (10.5__+0.9 V/sec). The overshoot was + 20.1 ± 0.5 mV, and the duration at 50% repolarization was 45.2___ 1.6 msec. After addition of dantrolene sodium (3× 10 -4 M) the rate of depolarization of slow responses was significantly increased up to 12.5 ± 0 . 3 V/sec. Furthermore, the overshoot of the Ca2÷-mediated action potential was elevated to + 2 3 . 3 ± 0 . 8 mV. Moreover, in all experiments (in seven atria) dantrolene sodium significantly prolonged the duration of slow action potentials to 51.5___ 1.3 msec (fig. 6D). These slow responses were completely abolished by D-600, 2 #M (fig. 6E). In 5 of 9 experiments dantrolene sodium was able to induce slow responses by itself at a concentration of 3 × 10-4 M.
4. Discussion We have shown that dantrolene sodium produced a dose- and time-dependent action on the contractility of isolated guinea pig left atrium. Our observations on the long-lasting negative inotropic effect of dantrolene sodium were consistent with reports of others (Meyler et al., 1976; Bowman and Khan, 1977). On the other hand, in the first period of the experiment the contractile force was increased by dantrolene sodium (up to 10 -5 M). This dose-dependent positive inotropic effect of dantrolene sodium was accompanied by the lengthening of action potential duration. The contractile force was transiently potentiated over 10 min then decreased gradually. The transient positive effect of dantrolene was independent of adrenergic fl-receptors and of histamine receptors but was prevented by D-600, a Ca 2+ channel blocker. Dantrolene sodium enhanced the amplitude and the rate of depolarization of the slow responses induced by caffeine in guinea pig atria partially depolarized by elevated K + and in some experiments dantrolene itself was able to induce slow responses. As the slow responses were
mainly Ca 2+ dependent and the increase in the plateau phase was probably due to the increase of the slow inward Ca 2+ current, the potentiation of the contractile force by dantrolene sodium may have been due to an augmentation of the slow inward Ca 2+ current. Our findings are in good agreement with the data of others (Oba and Hotta, 1978; Hatae et al., 1980) showing a positive inotropic action of dantrolene sodium in frog skeletal muscle and in guinea pig atrial muscle. However, other explanations for the observed positive inotropic effect of the drug cannot be excluded. Since the outward current is not negligible during the slow inward Ca 2÷ current, drugs added or alterations of the intracellular Ca 2+ concentration can indirectly influence the slow inward Ca 2÷ current by modifying the outward current (Pappano, 1976; Isenberg, 1975, 1977). The depressive effect on the outward current, the decrease of the calcium efflux or an effect on Ca 2÷ sequestration into the intracellular reservoir might also explain the positive inotropic effect of dantrolene sodium. In the present study dantrolene sodium exerted a negative inotropic effect after 40 min, accompanied by the shortening of the action potential duration. Later the contractions were abolished and a slow contracture developed. It is not probable that this late negative effect of the drug can be explained by the inhibition of activator calcium release from the sarcoplasmic reticulum (Ellis and Bryant, 1972; Putney and Bianchi, 1974; Van Winkle, 1976). The observed late negative inotropic effect of dantrolene sodium could be conceived as resulting from a direct inhibitory effect on the slow Ca 2÷ channel. This possibility is excluded since Desmedt and Hainaut (1977) observed that 45Ca efflux from barnacle muscle fiber was decreased by dantrolene sodium, whereas 45Ca influx was not significantly changed. The most plausible theory is that dantrolene s o d i u m - - b y facilitating the slow inward Ca 2+ current and additionally inhibiting the permeability of the sarcoplasmic reticulum membrane to calcium ions thus decreasing Ca 2+ efflux--causes lessening of the transmembrane Ca 2+ concentration gradient due to an intracellular accumulation of free Ca 2÷. This hypothesis is supported by the finding of the
187 s h o r t a c t i o n p o t e n t i a l d u r a t i o n a n d o f the c o n t r a c ture. K o h l h a r d t et al. (1974) g a v e a s i m i l a r exp l a n a t i o n for t h e b i p h a s i c e f f e c t of c a f f e i n e o n t h e t r a n s m e m b r a n e C a 2÷ c u r r e n t in the right v e n t r i c u lar p a p i l l a r y m u s c l e o f the cat. H o w e v e r , o t h e r e x p l a n a t i o n s e.g., the e f f e c t o n C a 2+ e x c h a n g e at the m i t o c h o n d r i a l m e m b r a n e c a n also b e o f f e r e d .
Acknowledgements We are indebted to Norwich Pharmacal Comp. for the supply of dantrolene sodium, to Knoll AG. for D-600 and to Smith Kline and French Lab. LTD for cimetidine.
References Bowman, W.C. and H.H. Khan, 1977, Effects of dantrolene sodium on isolated skeletal, smooth and cardiac muscle of the guinea-pig, J. Pharm. Pharmacol. 29, 628. Brocklehurst, L., 1975, Dantrolene sodium and 'skinned; muscle fibers, Nature 254, 364. Chipman, M., S. Kaul and M. Lambi, 1974, Efficacy of dantrolene sodium in the treatment of spasticity, Dis. Nerv. Syst. 35,427. Chiatte, S.B., J.H. Birdsong and B.A. Bergman, 1971, The effects of dantrolene sodium on spasticity and motor performance in hemiplegia, South. Med. J. 64, 180. Desmedt, J.E. and K. Hainaut, 1977, Inhibition of the intracellular release of calcium by dantrolene in barnacle giant muscle fibres, J. Physiol. 265, 565. Ellis, K.O. and S.H. Bryant, 1972, Excitation-contraction uncoupling in skeletal muscle by dantrolene sodium, NaunynSchmiedeb. Arch. Pharmacol. 274, 107. Ellis, K.O., J.L. Butterfield, F.L. Wessels and J.F. Carpenter, 1976, A comparison of skeletal, cardiac and smooth muscle actons of dantrolene sodium--a skeletal muscle relaxant, Arch. Int. Pharmacodyn. 244, 118. Ellis, K.O. and J.F. Carpenter, 1972, Studies on the mechanism of action of dantrolene sodium a skeletal muscle relaxant, Naunyn-Schmiedeb. Arch. Pharmacol. 275, 83. Ellis, K.O., A.W. Castellion, L.J. Honkomp, F.L. Wessels, J.F. Carpenter and R.P. Halliday, 1973, Dantrolene, a direct acting skeletal muscle relaxant, J. Pharm. Sci. 62, 948. Ellis, R.H., P. Simpson, P. Tatham, M. Leighton and J. Williams, 1975, The cardiovascular effects of dantrolene sodium in dogs, Anaesthesia 30, 318. Gollan, F. and J. McDermott, 1979, Effect of the skeletal muscle relaxant dantrolene sodium on the isolated, perfused heart, Proc. Soc. Exp. Biol. Med. 160, 42. Hainaut, K. and J.E. Desmedt, 1974, Effect of dantrolene sodium on calcium movements in single muscle fibers, Nature 252, 728.
Hatae, J., M. Ohba and H. Kawata, 1980, Effects of dantrolene sodium on the excitation-contraction coupling of the mammalian and amphibian cardiac muscle, J. Mol. Cell. Cardiol. 12, 857. Inui, I. and H. Imamura, 1976, Restoration by histamine of the calcium-dependent electrical and mechanical response in the guinea pig papillary muscle partially depolarized by potassium, Naunyn-Schmiedeb. Arch. Pharmacol. 294, 261. Isenberg, G., 1975, Is potassium conductance of cardiac Purkinje fibers controlled by [Call?, Nature 253, 273. Isenberg, G., 1977, Cardiac Purkinje fibers. The slow inward current component under the influence of modified [Ca]i , Pfliigers Arch. 371, 61. Kecskem~ti, V., 1978, Restorative effect of histamine on the transmembrane action potentials in potassium-depolarized guinea-pig auricle, Pol. J. Pharmacol. Pharm. 30, 377. Kecskemeti, V., K. Kelemen and J. Knoll, 1973, Effect of prostaglandin E L on the cardiac transmembrane potentials, European J. Pharmacol. 24, 289. Kelemen, K., V. Kecskem~ti, L. Scult~ty and T. Friedmann, 1968, Activity index of frog heart ventricle cells. Analysis of the correlation between resting potential and activity of the sodium-carrier system in the frog heart by means of celluline and adrenaline, Acta Physiol. Acad. Sci. Hung. 33, 269. Kohlhardt, M., M. Ktibler and E. Hansi, 1974, Ambiguous effect of caffeine upon the transmembrane Ca current in mammalian ventricular myocardium, Experientia 30, 254. Kurihara, T. and J.E. Brooks, 1975, The effect of hyperosmolar glycerol solution and dantrolene sodium on mammalian muscle in vitro, Arch. Neurol. 32, 92. Lowndes, H.E., 1975, Dantrolene effects on neuromuscular function in cat soleus muscle, European J. Pharmacol. 32, 267. Mtszhros, J., V. Kecskem~ti and J. Szegi, 1980, Effect of dantrolene sodium on cardiac transmembrane potentials and contractility, Abstracts book of the XXVIIIth International Congress of Physiological Sciences, p. 579, Budapest. Meyler, W.J., H. Wesseling and S. Agoston, 1976, The effects of dantrolene sodium on cardiac and skeletal muscle in rats, European J. Pharmacol. 39, 127. Morgan, K.G. and S.H. Bryant, 1977, The mechanism of action of dantrolene sodium, J. Pharmacol. Exp. Ther. 201, 138. N0tt, M.W. and W.C. Bowman, 1974, Actions of dantrolene sodium on contractions of the tibialis anterior and soleus muscles of cats under chloralose anaesthesia, Cln. Exp. Pharmacol. Physiol. 1, 113. Oba, T. and K. Hotta, 1978, Transient effect of intracellular dantrolene on E-C coupling in skeletal muscle, European J. Pharmacol. 51, 8 I. Pappano, A.J., 1970, Calcium dependent action potentials produced by catecholamines in guinea pig atrial fibers depolarized by potassium, Circulation Res. 27, 379. Pappano, A.J., 1976, Action potentials in chick atria. Ontogenetic changes in the dependence of tetrodotoxin-resistance action potentials on calcium, strontium, and barium, Circulation Res. 39, 99. Putney, J.W. and C.P. Bianchi, 1974, Site of action of dantro-
188 lene in frog sartorius muscle, J. Pharmacol. Exp. Ther. 189, 202. Schneider, J.A. and N. Sperelakis, 1975, Slow Ca 2+ an Na ~ responses induced by isopreterenol and methylxanthines in isolated perfused guinea pig hearts exposed to elevated K ÷, J. Mol. Cell, Cardiol. 7, 249. Snyder, H.R., C.S. Davis, R.K. Bickerton and R.P. Halliday, 1967, l-[(5-Arylfurfurylidene)-amino] hydantoins, A new class of muscle relaxants, J. med. Chem. 10, 807.
Van Winkle, W.B., 1976, Calcium release from skeletal muscle sarcoplasmic reticulum: Site of action of dantrolene sodium?, Science 193, 1130. Zorychta, E., D.W. Esplin, R. Capek and A. Lastowecka, 1971, The actions of dantrolene on extrafusal and intrafusal striated muscle, Federation Proc. 30, 669.