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The influence of ouabain on twitch contractions in the presence of veratridine
Gen. Pharmac. Vol. 20, No. 1, pp. 23-30, 1989 Printed in Great Britain. All rights reserved
0306-3623/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press pie
THE I N F L U E N C E OF OUABAIN ON TWITCH CONTRACTIONS IN THE PRESENCE OF V E R A T R I D I N E MASAKAZU NISHIMURA, HIROYA OHTANI a n d OSAMU YAGASAKI Department of Veterinary Pharmacology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan [Tel. 0722-52-1161]
(Received 18 April 1988) Abstraet--l. Direct stimulation evoked twitches in mouse diaphragm muscles in presence of 10/~M D-tubocurarine in vitro. Effects of ouabain and their dependence on K ÷ were examined on the twitch responses and action potentials in the presence and absence of twitch potentiators. 2. Ouabain inhibited twitch contractions only in the presence of veratridine, aconitine and monensin while it had no inhibitory effect on control twitches. The interactions between ouabain and these twitch potentiators depended on the presence of external K +, except in the case of monensin. 3. Removal of Ca 2÷ from a bathing solution accelerated the potentiating effect of veratridine and the antagonizing effect of ouabain. 4. Caffeine further potentiated the twitches which had been attenuated by ouabain combined with veratridine. 5. Ouabain combined with veratridine consistently decreased resting membrane potentials, action potentials and overshoot potentials and prolonged time to peak of and duration of the muscle action potentials. 6. Tetraethylammonium, 4-aminopyridine, and caffeine produced twitch potentiation which was insensitive to ouabain or the removal of K ÷. 7. These results suggest that twitch contractions in the presence of activators of sodium channels link with activation of Na+-K+-ATPase. Accumulation of Na + inside the muscle fibres may uncouple the excitation-contraction system. 8. This uncoupling may not include the caffeine-sensitive process that controls the release of Ca 2+ from the sarcoplasmic reticulum. Na ÷ accumulation may decrease transmembraneous gradient of this ions, thereby causing a reduction in excitation coupled with twitch contraction.
INTRODUCTION
4-aminopyridine, a n d caffeine. A c t i o n potentials were m e a s u r e d intracellularly in the presence o f veratridine c o m b i n e d with or w i t h o u t ouabain.
Veratridine increases m e m b r a n e permeability to N a ÷ by acting o n voltage-sensitive s o d i u m c h a n n e l s (Meves, 1966; Ulbricht, 1969; O h t a et al., 1973; Catterall, 1975; Stallcup, 1977; Krueger a n d Blaustein, 1980; M c K i n n e y , 1984) a n d can depolarize excitable m e m b r a n e s ( M c K i n n e y , 1984). In fact, veratridine e n h a n c e s the influx o f N a ÷, m e a s u r e d as influx o f radiolabeled N a ÷, a n d causes a c c u m u l a t i o n o f N a ÷ inside the muscle fibres in frog skeletal muscle ( M c K i n n e y a n d Ratzlaff, 1987). A c c u m u l a t i o n o f N a ÷ inside the muscle cells reduces muscle contraction (Bose, 1974, 1975; K i s h i m o t o et al., 1980), b u t veratridine potentiates twitch c o n t r a c t i o n s ( M e l d r u m et al., 1982). Thus, a possible i n h i b i t o r y effect o f N a ÷ inside the muscle fibre m a y be m a s k e d by o t h e r mechanisms. It has been suggested t h a t the action o f the N a + - K + p u m p is p r o b a b l y stimulated by intracellular increases in levels o f N a ÷ (Creese a n d Mitchell, 1981). It is, thus, predictable t h a t the twitch p o t e n t i a t i o n m a y be linked with a m e c h a n i s m for the extrusion o f N a ÷. If this hypothesis is correct, the effect o f veratridine should be a n t a g o n i z e d by inhibitors o f this p u m p system. To test this possibility, o u a b a i n a n d external levels o f K ÷ were e x a m i n e d for their effects o n twitch p o t e n t i a t i o n induced by veratridine in m o u s e diap h r a g m muscles. The results were c o m p a r e d with results o b t a i n e d from twitch c o n t r a c t i o n s in the presence of aconitine, m o n e n s i n , t e t r a e t h y l a m m o n i u m ,
MATERIALS AND METHODS
Media and preparation of tissue Left hemidiaphragrn muscles were isolated from male mice of the ddY strain with a body weight of 28-33 g (8-12 weeks old). The preparation was soaked in Krebs-Ringer solution of the following composition (mM): NaC1, 136; KCI, 5; CaCI 2, 2: CaCI 2, 2: MgCI 2, 1; NaHCO 3, 15; glucose, 1 I. A K+-free bathing solution was prepared by replacing 5 mM KC1 with equimolar NaC1. A solution containing no added CaC12 was also prepared. All the solutions included 10/zM D-tubocurarine to block neuromuscular transmission. The solution was bubbled with a mixture of 95% 02 and 5*/o CO2 and maintained at pH 7.3 and 36°C. The muscle preparations were equilibrated in these solutions for at least 30 min before addition of any agents. The temperature of the fluid in the bath was monitored by a thermistor (Shibaura Electric Co., Tokyo, Model MGA-II) and held constant by means of an external water jacket and a thermoregulatory device (Taiyo, Tokyo, Thermominder Mini 80). Tension recordings One end of the site of insertion of the rib cage in the hemidiaphragm was fixed and the other central tendon was attached to a force~lisplacement transducer (Nihon Kohden, Tokyo, TB-612T). Muscle layers were stimulated directly through bipolar electrodes connected to an electronic square pulse generator (Nihon Kohden, SEN-3201) with a constant 23
24
MASAKAZU NISHIMURA e l al.
was effective at concentrations of 100 nM or higher and produced maximum effect at 300 nM (471 _ 33% of control, m e a n + S E M , N = 14, at a plateau state). The effect progressively increased and reached a maximum level at least 15 min after the addition of 300 nM veratridine (Fig. 1). Most of the potentiating effect of veratridine was blocked in the presence of 5/1M ouabain which, itself, had no inhibitory effect on the twitch responses (Fig. 1). 2,4-Dinitrophenol (10 # M ) reduced the muscle twitches in the presence of 300 nM veratridine while it produced only a slight decrease in the amplitude of control twitch contractions. Tetrodotoxin (5 nM) also effectively reduced the twitch responses, while it had almost no inhibitory effect on control twitches. Omission of KCl from a standard bathing solution abolished most of the potentiating effect of veratridine (Fig. 2). Reinclusion of KC1, at a final concentration of 2.5 mM, immediately restored the potentiating effect of veratridine while it had no potentiating effect on the twitch responses in the absence of veratridine. The KCl-dependent effect of veratridine was completely abolished by 5 # M ouabain. The inhibitory effect of ouabain required a latent period during which ouabain slightly potentiated the twitch contractions for several minutes. Veratridine altered not only the amplitude but also the duration of the twitch contractions (Fig. 3). In a standard bathing solution, the time course of twitching gave a phasic profile with durations from 40 to 100msec. Veratridine (300nM) increased both the amplitude and duration of the twitch contractions without changing the rate of rise of the twitches. It prolonged the duration of twitches by 5-fold or more of the control value. Ouabain ( 5 # M ) significantly reduced the amplitude and rate of rise of the twitches in the presence of veratridine, while it partially shortened the duration of the twitches in the presence of veratridine. In a K+-free medium, veratridine scarcely affected the amplitude, duration, or rate of rise of the twitch contractions (Fig. 3, lower panel). Reinclusion of KCI at a final concentration of
voltage output. All stimuli were supramaximal and of 0.7msec duration at 0.1 Hz. Resulting isometric muscle twitches were recorded on a pen-writing recorder (Nihon Kohden, WI-641G) or on an analyzing recorder (Yokogawa Hokushin Electric Co., Tokyo). Electrical parameters
Resting membrane potentials, action potentials, overshoot potentials, time to peak of the action potentials, and 10% duration of the action potentials were recorded from 5 fibres per each muscles in the standard bathing solution containing 10 p M o-tubocurarine. The 10% duration of the action potential means the duration of the action potential which is larger than 10% of the whole amplitude. The action potentials were evoked by application of an outward current through an intracellular electrode which was also used for measurement of the electrical activities. Input resistance of the electrode varied from 3 to 5 mf~. The outward current was 0.1 msec square wave pulses and varied from 150 to 250 nA. The outward current was given to fibres through a micro-electrode amplifier (Nihon Kohden, MEZ-8201) from an electronic square pulse generator (Nihon Kohden, SEN3201). Protocol for the measurement was as follows: control values were measured from 20 to 35 min of first incubating period in the standard bathing solution; values in the presence of veratridine or ouabain alone were from 20 to 35 min of first incubating period with either agent; values in the presence of both veratridine and ouabain were from 20 to 35 min after addition of ouabain which was done 20 min after the addition of veratridine. Drugs used were: o-tubocurarine chloride, veratridine, aconitine, monensin, (Sigma, St Louis), ouabain (Merck, Darmstadt), tetraethylammonium, 4-aminopyridine, caffeine (Wako Pure Chemicals, Osaka), 2,4-dinitrophenol (Maruwaka Chemicals, Osaka) and tetrodotoxin (Sankyo, Tokyo). RESULTS In the presence of 1 0 # M D-tubocurarine, diaphragm muscles of approx. 4 m m in width developed twitch responses with a constant amplitude of 1.44.4 g in response to direct activation of the muscle layer by electrical pulses, for at least 3 hr or longer. Veratridine persistently potentiated the twitch contractions in a concentration-dependent manner. It Standard
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Fig. 1. Potentiation by 300 nM veratridine of directly-evoked twitches in mouse diaphragm muscles bathed in a solution that contained I0/~M D-tubocurarine, and antagonism by 5 #M ouabain. Typical results, from 1 of 14 sets of similar observations, are presented. VRT: veratridine. OUA: ouabain.
Ouabain and Na channel activators
25
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Fig. 2. Typical results of restoration, by reinclusion of 2.5 mM KC1, of the effects of 300 nM veratridine and 5/~M ouabain on directly-evoked twitches in mouse diaphragm muscles in a K+-free bathing solution that contained 10/zM D-tubocurarine. VRT: veratridine. OUA: ouabain.
2.5 mM increased the amplitude and prolonged the duration of the muscle twitches. Ca 2÷ outside the cell is known to stabilize excitable membranes. Removal of Ca 2÷ from the bathing solution was examined for its effect on twitch potentiation by veratridine, since muscle twitches could be elicited in the Ca2*-free bathing solution (Fig. 4). The effect of 300 nM veratridine appeared, without a latent period, immediately after its addition, and reached a maximum level much more quickly in this Ca2÷-free solution than in the standard solution. The inhibitory effect of 5/~M ouabain also occurred immediately after its addition to this solution. Caffeine has been shown to act on the sarcoplasmic reticulum (Weber, 1966) to stimulate the Ca 2÷releasing mechanism of this structure (Endo et al., 1970). At 2 mM, it increased the amplitude of the twitch contractions by approx. 170% without effecting any changes in the base line of the muscle tone (Fig. 5). A similar potentiating effect of caffeine was observed in the presence of 5/~M ouabain which, itself, slightly but significantly augmented the twitch amplitude. Further, caffeine caused a two-fold or greater increase in the amplitude of the twitch contractions, in spite of the fact that the contractions had been attenuated by 5/~M ouabain in combination with 300 nM veratridine. Figure 6 summarizes the interaction between veratridine and ouabain on electrical parameters, as resting membrane potential, action potential, overshoot potential, time to peak of the action potential, and 10% duration of the action potential, of muscle fibres. Both veratridine and ouabain slightly but significantly increased the resting membrane potentials. The combination of veratridine and ouabain, however, significantly decreased the resting membrane potentials. Ouabain largely reduced the action potentials in the presence of veratridine which, itself, had no inhibitory effect on the action potentials. Ouabain alone caused only a little decrease of the action potentials. Both veratridine and ouabain decreased the overshoot potentials but their combination produced more decrease of them. Ouabain significantly lengthened both the time to peak and 10% duration of the action potentials only in the presence of veratridine. Thus, ouabain negatively
altered all the parameters tested only when combined with veratridine. Aconitine has been shown to activate sodium channels on the excitable membrane in a manner similar to that of veratridine (Peper and Trautwein, 1967). At concentrations lower than 1 #M, aconitine produced a slight but sustained increase in the amplitude of the twitch contractions (Fig. 7A). It had no prolonging effect on the duration of the twitch contractions (Fig. 8). A final concentration of 0.1% ethanol had no such a potentiating effect on the twitches, and the stock solution of aconitine was prepared in ethanol. Ouabain (5 # M) was then added to Standard bathing solution
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Fig. 3. Modification by 5 ,uM ouabain (upper panel), and by reinclusion of 2.5 mM KC1 (lower panel), of the effect of 300 nM veratridine on contractile curves of directly-evoked twitches in the mouse diaphragm muscles in solutions that contained 10 ~uM D-tubocurarine. Curve (a) in both panels: control. Curves (b) and (c) in upper panel: 15 rain after addition of 3011nM veratridine and 10 rain after further addition of 5 ,uM ouabain, respectively.Curves (b) and (c) in lower panel: 15 rain after addition of 300 nM veratridine to the K+-free bathing solution and 10 rain after reinclusion of 2.5 mM KC1 in addition to 300 nM veratridine, respectively. Each curve represents the average of results of 8 trials. Typical results, from 1 of 5 sets of similar observations are presented. OUA: ouabain. VRT: veratridine.
MASAKAZU NISHIMURA el al.
26
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Fig. 4. Representative results of facilitation, by removal of Ca 2÷, of the effects of 300 nM veratridine and 5 # M ouabain on directly-evoked twitches in the mouse diaphragm muscles in a solution that contained 10 g M o-tubocurarine. VRT: veratridine. OUA: Ouabain. m o s t o f the twitches evoked in the presence of m o n e n s i n (Fig. 9A). In a K+-free b a t h i n g solution, 30/a M m o n e n s i n was still able to increase the amplitude o f the twitches, b u t with a relatively longer latent period (Fig. 9B).
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Fig. 5. Effect of 2 mM caffeine on twitch responses driven electrically in mouse diaphragm muscles treated with 10 # M D-tubocurarine in bathing solutions that contained 5 # M ouabain alone or in combination with 300 nM veratridine. Twitch amplitude was measured 10 min after addition of caffeine or ouabain and 15 min after addition of veratridine. All values are expressed as percentages of the amplitude of control twitches in a solution without any agent except n-tubocurarine. Vertical bars show SEM of results from 5 or 6 experiments. Different preparations were used for the experiments to examine the effects of ouabain alone or in combination with caffeine and of veratridine alone or combined with ouabain and caffeine. Caffeine, ouabain, and veratridine, by themselves, significantly potentiated the directly-evoked twitches. *P < 0.05. CAF: caffeine. OUA: ouabain. VRT: veratridine.
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the b a t h i n g solution t h a t c o n t a i n e d 1/aM aconitine (Fig. 7B). The amplitude o f the twitch c o n t r a c t i o n s was transiently increased by o u a b a i n a n d this increase was followed by a sharp decline, until it was abolished. In a K+-free b a t h i n g solution, 1/aM aconitine caused only a n inhibitory effect o n the twitches (Fig. 7C). Higher c o n c e n t r a t i o n s o f aconitine, 2.5 or 5 / a M , a t t e n u a t e d the twitch c o n t r a c t i o n s with a transient p o t e n t i a t i o n o f the twitches (data n o t shown). M o n e n s i n increased the permeability o f m e m b r a n e to cations such as N a ÷ (Pressman, 1976). M o n e n s i n persistently p o t e n t i a t e d the twitch contractions, with a l m o s t n o latent period, at a c o n c e n t r a t i o n o f 3 0 / a M (Fig. 9A). It h a d n o p r o l o n g i n g effect o n the d u r a t i o n o f the twitches (Fig. 8). O u a b a i n ( 5 / a M ) abolished
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Fig. 6. Effects of 300nM veratridine, 5 #M ouabain and combination of veratridine and ouabain on electrical parameters of mouse diaphragm muscles in the standard bathing solution. (A) Resting membrane potentials (left side scale), (B) action potentials (right side scale), (C) overshoot potentials (right side scale), (D) time to peak, (E) 10% duration of action potentials. See text for their determinations in detail. "All parameters were derived from 30 fibres of 6 muscles (5 fibres in each preparation). Vertical bars show SEM of 30 determinations. *P < 0.05.
Ouabain and Na channel activators
27
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Fig. 7. Effects of I/aM aconitine on directly-evoked twitches in mouse diaphragm muscles in standard and K+-free bathing solutions that contained I0/aM D-tubocurarine, and the changes in these effects caused by 5/aM ouabain. Typical results, from 1 of 5 sets of similar observations are presented. ACN: aconitine. OUA: ouabain. Tetraethylammonium (Stanfieid, 1 9 8 3 ) and 4-aminopyridine (Yeh et al., 1976) are blockers of potassium channels on excitable membranes. Both tetraethylammonium (10 mM) and 4-aminopyridine (200pM) persistently increased the level of twitch amplitude (Figs 10A and 11A). They had almost no prolonging effect on the duration of the twitch contractions (Fig. 8). 5 p M ouabain had no inhibitory effect on the twitches when applied after treatment with either tetraethylammonium (Fig. 10A) or 4-
aminopyridine (Fig. 11A). These potentiating effects of tetraethylammonium and of 4-aminopyridine also occurred in a K+-free bathing solution (Figs 10B and lIB). DISCUSSION Veratridine, aconitine, monensin, tetraethylammonium, and 4-aminopyridine potentiated muscle contractions stimulated directly in the diaphragm
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Fig. 8. Effects of I/aM aconitine, 30/aM monensin, 10mM teraethylammonium, and 200/aM 4-aminopyridine on contractile curves of directly-evoked twitches in mouse diaphragm muscles in a standard bathing solution that contained 10/aM D-tubocurarine and the changes in these effects caused by 5/a M ouabain. (a) Control, (b) 15 min after addition of each twitch potentiator, (c) 10 min after further addition of ouabain, in addition to the twitch potentiators. Each trace represents the average of results from 8 trials. ACN: aconitine. MNS: monensin. TEA: tetraethylammonium. 4-AP: 4-aminopyridine. OUA: ouabain.
28
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Fig. 9. Twitch potentiation by 30gM monensin in mouse diaphragm muscles stimulated directly in standard (A) and K÷-free (B) bathing solutions that contained 10 # M D-tubocurarine, and the inhibition by 5 #M ouabain of the effect of monensin in the standard solution. Typical results, from 1 to 5 sets of similar observations, are presented. MNS: monensin. OUA: ouabain. muscles pretreated with 10/~ M D-tubocurarine. Such a potentiating effect has been previously observed in studies of directly stimulated contractions of phrenic nerve-diaphragm preparations of rats in the presence of veratridine (Meldrum et al., 1982). 10 gM D-tubocurarine completely abolished indirectly evoked twitches in those tissues. These results suggest that the potentiating effect of these agents on directly evoked twitch contractions occurs via mechanisms that are independent of the cholinergic system. Veratridine shifts the voltage dependence of activation of sodium channels and blocks inactivation of sodium channels to cause persistent activation of .H,
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Fig. 10. The absence of any effect of 5/~M ouabain or removal of K + from the bathing solution on twitch potentiation by l0 mM tetraethylammonium in mouse diaphragm muscles stimulated directly in solutions that contained l0/~M o-tubocurarine. (A) In a standard bathing solution. (B) In a K+-free bathing solution. Typical results, from I of 5 sets of similar observations, are presented. OUA: ouabain. TEA: tetraethylammonium.
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Fig. 11. Potentiating effects of 200#M 4-aminopyridine, which are resistant to 5 #M ouabain and to removal of K + from the bathing solution, on directly-evoked twitches in mouse diaphragm muscles in solutions that contained I0 # M D-tubocurarine. Typical results, from 1 of 5 sets of similar observations, are presented. 4-AP: 4-aminopyridine. OUA: ouabain. (Clausen, 1986). This restoration may be related to the observed hyperpolarization (Rang and Ritchie, 1968). The present experiments indicate that veratridine, aconitine, and monensin increase the amplitude of twitch contractions in response to direct shocks. Thus, it is suggested that the twitch contractions, in the presence of these agents, must be coupled with an increased influx of Na t . Further more, the twitch contractions are most probably maintained by the activation of the Na+-K+-ATPase. The latter hypothesis is supported by the observation that treatment with ouabain and removal of K + from a bathing solution reduced the amplitude of the twitch contractions in the presence of these agents. Both ouabain and K÷-free solutions have been shown to inhibit the activity of Na+-K+-ATPase (Clausen, 1986). When the sodium pump system is blocked, Na + accumulates in the muscle fibres. In such a case, the twitch contractions were decreased in amplitude in spite of the presence of twitch potentiators such as veratridine, aconitine, and monensin. Thus, it is suggested that the excess and persistent accumulation of Na t inside the muscle fibres uncouples the excitation-contraction process. This hypothesis is supported by observations that the accumulation of Na ÷ inside the cell is associated with an inhibition of contractile responses of preparations of smooth muscle to different stimulants (Bose, 1974, 1975; Kishimoto et aL, 1980). Present experiments indicate that, only when ouabain was added to a solution containing veratridine, it significantly decreased all electrical parameters tested as the resting membrane potentials, action potentials, overshoot potentials, time to peak of the action potentials, and 10% duration of the action potentials. Thus, the whole reduction of the electrical activity could be related to the inhibition of twitch contraction. Such an alteration may be derived from the possible accumulation of internal Na t .
Caffeine (2mM) potentiated electrically driven twiches without producing any contraction on the base line. This effect was not reduced by ouabain or by ouabain in combination with veratridine which would be expected to cause accumulation of more Na + inside the muscle fibres. The site of action of caffeine is on the sarcoplasmic reticulum and caffeine requires Ca 2÷ for its action (Weber, 1966). Caffeine stimulates the Ca2+-releasing mechanism of this structure, but does not reduce its capacity to accumulate Ca 2+ (Endo et al., 1970). Thus, it is conceivable that the mechanism whereby Na t inhibits twitch contractions is independent of the process activated by caffeine on the sarcoplasmic reticulum. An Na--Ca exchange system exists in a variety of tissues (Blaustein, 1974). In vascular smooth muscle, this exchange system has been proposed to cause the development of tension when the concentration of intracellular Na t is elevated (Ozaki and Urakawa, 1979). The twitch potentiation by activators of sodium channels, demonstrated in the present experiments, may be related to such an exchange of internal Na t with external Ca 2÷. However, this possibility is unlikely, because the potentiating effects of such sodium channel activators occurred rather rapidly in a Ca2÷-free bathing solution and were not stimulated by the presence of ouabain, which should have increased the internal concentration of Na t . Nonetheless, Ca 2+ can compete with Na t at as yet unidentified sites that carry an anionic charge on the surface of the cell membrane, causing an inhibition of the influx of Na t (Luttgau and Niedergerke, 1958). If the same holds true in the diaphragm muscle, it is possible that removal of Ca 2+ from the bathing solution facilitates the effects of the activators of the sodium channels and also the proposed inhibitory effect of the accumulated internal Na t . Twitch potentiation was evident in the diaphragm muscles stimulated directly in the presence of tetraethylammonium and 4-aminopyridine. This type of
30
MASAKAZUNISHIMURAet al.
twitch potentiation was resistant to ouabain or removal o f K +. Tetraethylammonium (Stanfield, 1983) and 4-aminopyridine (Yeh et al., 1976) can inhibit permeability of membrane to K ÷, causing prolongation of the action potential. Thus, the antagonizing effect of ouabain and omission of K + from a bathing solution is specific for the activators of sodium channels. These results support the hypothesis that twitch contractions in the presence o f activators of sodium channels are maintained by the synchronous activation of N a ÷ - K ÷-ATPase. In conclusion, twitch contractions in the presence of blockers of sodium channels are probably maintained by the synchronous activation of N a + - K ÷ ATPase. Na ÷ accumulated in the muscle cells may reduce the amplitude o f twitch contractions by a mechanism other than that which is stimulated by caffeine and involves the release of Ca 2+ from the sarcoplasmic reticulum.
REFERENCES
Blaustein M. P. (1974) The interrelationship between sodium and calcium fluxes across cell membranes. Rev. Physiol. Biochem. Pharmac. 70, 33-82. Bose D. (1974) Mechanism of inhibition of smooth muscle tension in guinea-pig taenia coli by ouabain. Can. J. Physiol. Pharmac. 52, 898-901. Bose D. (1975) Mechanism of mechanical inhibition of smooth muscle by ouabain. Br. 3. Pharmac. 55, 111-116. Catterall W. A. (1975) Activation of the action potential Na ÷ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin. J. biol. Chem. 250, 4053~,059. Clausen T. (1986) Regulation of active Na+-K ÷ transport in skeletal muscle. Physiol. Rev. 66, 542-580. Creese R., Head S. D. and Jenkinson D. F. (1987) The role of the sodium pump during prolonged end-plate currents in guinea-pig diaphragm. J. Physiol. 384, 377-403. Creese R. and Mitchell L. D. (1981) Spontaneous recovery from depolarizing drugs in rat diaphragm. J. Physiol. 313, 173-186. Endo M., Tanaka M. and Ogawa Y. (1970) Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature, Lond. 228, 34-36. Kishimoto T., Ozaki H. and Urakawa N. (1980) Quantitative relationship between cellular Na accumulation and relaxation produced by ouabain in depolarized smooth
muscle of guinea-pig taenia coli. Naunyn-Schmiedebergs Arch. Pharmac. 312, 199-207. Krueger B. K. and Blaustein M. P. (1980) Sodium channels in presynaptic nerve terminals: regulation by neurotoxins. J. gen. Physiol. 76, 287-313. Luttgau H. C. and Niedergerke R. (1958) The antagonism between Ca and Na ions on the frog's heart. J. Physiol. 143, 486-505. McKinney L. C. (1984) Effect of veratridine on membrane potential of sartorius muscle from Rana pipiens. Am. J. Physiol. 247, C309-C313. McKinney L. C. and Ratzlaff R. W. (1987) Sodium permeability of frog skeletal muscle in absence and presence of veratridine. Am. J. Physiol. 252, C190-C196. Meldrum M. J., Snider R. M. and Gerald M. D. (1982) Cholinergic-independent effects of amphetamine on mammalian skeletal muscle contractions. Neuropharmacology 21, 439-443. Meves H. (1966) The effect of veratridine on internally perfused giant axons. Pflfigers Arch. ges. Physiol. 290, 211-217. Narahashi T. (1974) Chemicals as tools in the study in excitable membranes. Physiol. Rev. 54, 813-889. Ohta M., Narahashi T. and Keeler R. F. (1973) Effects of veratrum alkaloids on membrane potential and conductance of squid and crayfish giant axons. J. Pharmac. exp. Ther. 184, 143-154. Ozaki H. and Urakawa N. (1979) Na42a exchange and tension development in guinea-pig aorta. NaunynSchmiedebergs Arch. Pharmac. 309, 171-178. Peper K. and Trautwein W. (1967) The effect of aconitine on the membrane current in cardiac muscle. Pflagers Arch. ges. Physiol. 296, 328-336. Pressman B. C. (1976) Biological applications of ionophores. A. Rev. Biochem. 45, 501-530. Rang H. P. and Ritchie J. M. (1968) On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations. J. Physiol. 196, 183-221. Stallcup W. B. (1977) Comparative pharmacology of voltagedependent sodium channels. Brain Res. 135, 37-53. Stanfield P. R. (1983) Tetraethylammonium ions and the potassium permeability of excitable ceils. Rev. Physiol. Biochem. Pharmac. 97, 1-67. Ulbricht W. (1969) The effect of veratridine on excitable membranes of nerve and muscle. Ergebn. Physiol. Biol. Chem. exp. Pharmak. 61, 18-71. Weber A. (1966) Energized calcium transport and relaxing factors. Curr. Topics Bioengng 1, 203-254. Yeh J. Z., Oxford G. S., Wu C. H. and Narahashi T. (1976) Dynamics of aminopyridine block of potassium channels in squid axon membrane. 3. gen. Physiol. 68, 519-535.
0306-3623/89 $3.00 + 0.00 Copyright © 1989 Pergamon Press pie
THE I N F L U E N C E OF OUABAIN ON TWITCH CONTRACTIONS IN THE PRESENCE OF V E R A T R I D I N E MASAKAZU NISHIMURA, HIROYA OHTANI a n d OSAMU YAGASAKI Department of Veterinary Pharmacology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 591, Japan [Tel. 0722-52-1161]
(Received 18 April 1988) Abstraet--l. Direct stimulation evoked twitches in mouse diaphragm muscles in presence of 10/~M D-tubocurarine in vitro. Effects of ouabain and their dependence on K ÷ were examined on the twitch responses and action potentials in the presence and absence of twitch potentiators. 2. Ouabain inhibited twitch contractions only in the presence of veratridine, aconitine and monensin while it had no inhibitory effect on control twitches. The interactions between ouabain and these twitch potentiators depended on the presence of external K +, except in the case of monensin. 3. Removal of Ca 2÷ from a bathing solution accelerated the potentiating effect of veratridine and the antagonizing effect of ouabain. 4. Caffeine further potentiated the twitches which had been attenuated by ouabain combined with veratridine. 5. Ouabain combined with veratridine consistently decreased resting membrane potentials, action potentials and overshoot potentials and prolonged time to peak of and duration of the muscle action potentials. 6. Tetraethylammonium, 4-aminopyridine, and caffeine produced twitch potentiation which was insensitive to ouabain or the removal of K ÷. 7. These results suggest that twitch contractions in the presence of activators of sodium channels link with activation of Na+-K+-ATPase. Accumulation of Na + inside the muscle fibres may uncouple the excitation-contraction system. 8. This uncoupling may not include the caffeine-sensitive process that controls the release of Ca 2+ from the sarcoplasmic reticulum. Na ÷ accumulation may decrease transmembraneous gradient of this ions, thereby causing a reduction in excitation coupled with twitch contraction.
INTRODUCTION
4-aminopyridine, a n d caffeine. A c t i o n potentials were m e a s u r e d intracellularly in the presence o f veratridine c o m b i n e d with or w i t h o u t ouabain.
Veratridine increases m e m b r a n e permeability to N a ÷ by acting o n voltage-sensitive s o d i u m c h a n n e l s (Meves, 1966; Ulbricht, 1969; O h t a et al., 1973; Catterall, 1975; Stallcup, 1977; Krueger a n d Blaustein, 1980; M c K i n n e y , 1984) a n d can depolarize excitable m e m b r a n e s ( M c K i n n e y , 1984). In fact, veratridine e n h a n c e s the influx o f N a ÷, m e a s u r e d as influx o f radiolabeled N a ÷, a n d causes a c c u m u l a t i o n o f N a ÷ inside the muscle fibres in frog skeletal muscle ( M c K i n n e y a n d Ratzlaff, 1987). A c c u m u l a t i o n o f N a ÷ inside the muscle cells reduces muscle contraction (Bose, 1974, 1975; K i s h i m o t o et al., 1980), b u t veratridine potentiates twitch c o n t r a c t i o n s ( M e l d r u m et al., 1982). Thus, a possible i n h i b i t o r y effect o f N a ÷ inside the muscle fibre m a y be m a s k e d by o t h e r mechanisms. It has been suggested t h a t the action o f the N a + - K + p u m p is p r o b a b l y stimulated by intracellular increases in levels o f N a ÷ (Creese a n d Mitchell, 1981). It is, thus, predictable t h a t the twitch p o t e n t i a t i o n m a y be linked with a m e c h a n i s m for the extrusion o f N a ÷. If this hypothesis is correct, the effect o f veratridine should be a n t a g o n i z e d by inhibitors o f this p u m p system. To test this possibility, o u a b a i n a n d external levels o f K ÷ were e x a m i n e d for their effects o n twitch p o t e n t i a t i o n induced by veratridine in m o u s e diap h r a g m muscles. The results were c o m p a r e d with results o b t a i n e d from twitch c o n t r a c t i o n s in the presence of aconitine, m o n e n s i n , t e t r a e t h y l a m m o n i u m ,
MATERIALS AND METHODS
Media and preparation of tissue Left hemidiaphragrn muscles were isolated from male mice of the ddY strain with a body weight of 28-33 g (8-12 weeks old). The preparation was soaked in Krebs-Ringer solution of the following composition (mM): NaC1, 136; KCI, 5; CaCI 2, 2: CaCI 2, 2: MgCI 2, 1; NaHCO 3, 15; glucose, 1 I. A K+-free bathing solution was prepared by replacing 5 mM KC1 with equimolar NaC1. A solution containing no added CaC12 was also prepared. All the solutions included 10/zM D-tubocurarine to block neuromuscular transmission. The solution was bubbled with a mixture of 95% 02 and 5*/o CO2 and maintained at pH 7.3 and 36°C. The muscle preparations were equilibrated in these solutions for at least 30 min before addition of any agents. The temperature of the fluid in the bath was monitored by a thermistor (Shibaura Electric Co., Tokyo, Model MGA-II) and held constant by means of an external water jacket and a thermoregulatory device (Taiyo, Tokyo, Thermominder Mini 80). Tension recordings One end of the site of insertion of the rib cage in the hemidiaphragm was fixed and the other central tendon was attached to a force~lisplacement transducer (Nihon Kohden, Tokyo, TB-612T). Muscle layers were stimulated directly through bipolar electrodes connected to an electronic square pulse generator (Nihon Kohden, SEN-3201) with a constant 23
24
MASAKAZU NISHIMURA e l al.
was effective at concentrations of 100 nM or higher and produced maximum effect at 300 nM (471 _ 33% of control, m e a n + S E M , N = 14, at a plateau state). The effect progressively increased and reached a maximum level at least 15 min after the addition of 300 nM veratridine (Fig. 1). Most of the potentiating effect of veratridine was blocked in the presence of 5/1M ouabain which, itself, had no inhibitory effect on the twitch responses (Fig. 1). 2,4-Dinitrophenol (10 # M ) reduced the muscle twitches in the presence of 300 nM veratridine while it produced only a slight decrease in the amplitude of control twitch contractions. Tetrodotoxin (5 nM) also effectively reduced the twitch responses, while it had almost no inhibitory effect on control twitches. Omission of KCl from a standard bathing solution abolished most of the potentiating effect of veratridine (Fig. 2). Reinclusion of KC1, at a final concentration of 2.5 mM, immediately restored the potentiating effect of veratridine while it had no potentiating effect on the twitch responses in the absence of veratridine. The KCl-dependent effect of veratridine was completely abolished by 5 # M ouabain. The inhibitory effect of ouabain required a latent period during which ouabain slightly potentiated the twitch contractions for several minutes. Veratridine altered not only the amplitude but also the duration of the twitch contractions (Fig. 3). In a standard bathing solution, the time course of twitching gave a phasic profile with durations from 40 to 100msec. Veratridine (300nM) increased both the amplitude and duration of the twitch contractions without changing the rate of rise of the twitches. It prolonged the duration of twitches by 5-fold or more of the control value. Ouabain ( 5 # M ) significantly reduced the amplitude and rate of rise of the twitches in the presence of veratridine, while it partially shortened the duration of the twitches in the presence of veratridine. In a K+-free medium, veratridine scarcely affected the amplitude, duration, or rate of rise of the twitch contractions (Fig. 3, lower panel). Reinclusion of KCI at a final concentration of
voltage output. All stimuli were supramaximal and of 0.7msec duration at 0.1 Hz. Resulting isometric muscle twitches were recorded on a pen-writing recorder (Nihon Kohden, WI-641G) or on an analyzing recorder (Yokogawa Hokushin Electric Co., Tokyo). Electrical parameters
Resting membrane potentials, action potentials, overshoot potentials, time to peak of the action potentials, and 10% duration of the action potentials were recorded from 5 fibres per each muscles in the standard bathing solution containing 10 p M o-tubocurarine. The 10% duration of the action potential means the duration of the action potential which is larger than 10% of the whole amplitude. The action potentials were evoked by application of an outward current through an intracellular electrode which was also used for measurement of the electrical activities. Input resistance of the electrode varied from 3 to 5 mf~. The outward current was 0.1 msec square wave pulses and varied from 150 to 250 nA. The outward current was given to fibres through a micro-electrode amplifier (Nihon Kohden, MEZ-8201) from an electronic square pulse generator (Nihon Kohden, SEN3201). Protocol for the measurement was as follows: control values were measured from 20 to 35 min of first incubating period in the standard bathing solution; values in the presence of veratridine or ouabain alone were from 20 to 35 min of first incubating period with either agent; values in the presence of both veratridine and ouabain were from 20 to 35 min after addition of ouabain which was done 20 min after the addition of veratridine. Drugs used were: o-tubocurarine chloride, veratridine, aconitine, monensin, (Sigma, St Louis), ouabain (Merck, Darmstadt), tetraethylammonium, 4-aminopyridine, caffeine (Wako Pure Chemicals, Osaka), 2,4-dinitrophenol (Maruwaka Chemicals, Osaka) and tetrodotoxin (Sankyo, Tokyo). RESULTS In the presence of 1 0 # M D-tubocurarine, diaphragm muscles of approx. 4 m m in width developed twitch responses with a constant amplitude of 1.44.4 g in response to direct activation of the muscle layer by electrical pulses, for at least 3 hr or longer. Veratridine persistently potentiated the twitch contractions in a concentration-dependent manner. It Standard
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Fig. 1. Potentiation by 300 nM veratridine of directly-evoked twitches in mouse diaphragm muscles bathed in a solution that contained I0/~M D-tubocurarine, and antagonism by 5 #M ouabain. Typical results, from 1 of 14 sets of similar observations, are presented. VRT: veratridine. OUA: ouabain.
Ouabain and Na channel activators
25
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Fig. 2. Typical results of restoration, by reinclusion of 2.5 mM KC1, of the effects of 300 nM veratridine and 5/~M ouabain on directly-evoked twitches in mouse diaphragm muscles in a K+-free bathing solution that contained 10/zM D-tubocurarine. VRT: veratridine. OUA: ouabain.
2.5 mM increased the amplitude and prolonged the duration of the muscle twitches. Ca 2÷ outside the cell is known to stabilize excitable membranes. Removal of Ca 2÷ from the bathing solution was examined for its effect on twitch potentiation by veratridine, since muscle twitches could be elicited in the Ca2*-free bathing solution (Fig. 4). The effect of 300 nM veratridine appeared, without a latent period, immediately after its addition, and reached a maximum level much more quickly in this Ca2÷-free solution than in the standard solution. The inhibitory effect of 5/~M ouabain also occurred immediately after its addition to this solution. Caffeine has been shown to act on the sarcoplasmic reticulum (Weber, 1966) to stimulate the Ca 2÷releasing mechanism of this structure (Endo et al., 1970). At 2 mM, it increased the amplitude of the twitch contractions by approx. 170% without effecting any changes in the base line of the muscle tone (Fig. 5). A similar potentiating effect of caffeine was observed in the presence of 5/~M ouabain which, itself, slightly but significantly augmented the twitch amplitude. Further, caffeine caused a two-fold or greater increase in the amplitude of the twitch contractions, in spite of the fact that the contractions had been attenuated by 5/~M ouabain in combination with 300 nM veratridine. Figure 6 summarizes the interaction between veratridine and ouabain on electrical parameters, as resting membrane potential, action potential, overshoot potential, time to peak of the action potential, and 10% duration of the action potential, of muscle fibres. Both veratridine and ouabain slightly but significantly increased the resting membrane potentials. The combination of veratridine and ouabain, however, significantly decreased the resting membrane potentials. Ouabain largely reduced the action potentials in the presence of veratridine which, itself, had no inhibitory effect on the action potentials. Ouabain alone caused only a little decrease of the action potentials. Both veratridine and ouabain decreased the overshoot potentials but their combination produced more decrease of them. Ouabain significantly lengthened both the time to peak and 10% duration of the action potentials only in the presence of veratridine. Thus, ouabain negatively
altered all the parameters tested only when combined with veratridine. Aconitine has been shown to activate sodium channels on the excitable membrane in a manner similar to that of veratridine (Peper and Trautwein, 1967). At concentrations lower than 1 #M, aconitine produced a slight but sustained increase in the amplitude of the twitch contractions (Fig. 7A). It had no prolonging effect on the duration of the twitch contractions (Fig. 8). A final concentration of 0.1% ethanol had no such a potentiating effect on the twitches, and the stock solution of aconitine was prepared in ethanol. Ouabain (5 # M) was then added to Standard bathing solution
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Fig. 3. Modification by 5 ,uM ouabain (upper panel), and by reinclusion of 2.5 mM KC1 (lower panel), of the effect of 300 nM veratridine on contractile curves of directly-evoked twitches in the mouse diaphragm muscles in solutions that contained 10 ~uM D-tubocurarine. Curve (a) in both panels: control. Curves (b) and (c) in upper panel: 15 rain after addition of 3011nM veratridine and 10 rain after further addition of 5 ,uM ouabain, respectively.Curves (b) and (c) in lower panel: 15 rain after addition of 300 nM veratridine to the K+-free bathing solution and 10 rain after reinclusion of 2.5 mM KC1 in addition to 300 nM veratridine, respectively. Each curve represents the average of results of 8 trials. Typical results, from 1 of 5 sets of similar observations are presented. OUA: ouabain. VRT: veratridine.
MASAKAZU NISHIMURA el al.
26
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Fig. 4. Representative results of facilitation, by removal of Ca 2÷, of the effects of 300 nM veratridine and 5 # M ouabain on directly-evoked twitches in the mouse diaphragm muscles in a solution that contained 10 g M o-tubocurarine. VRT: veratridine. OUA: Ouabain. m o s t o f the twitches evoked in the presence of m o n e n s i n (Fig. 9A). In a K+-free b a t h i n g solution, 30/a M m o n e n s i n was still able to increase the amplitude o f the twitches, b u t with a relatively longer latent period (Fig. 9B).
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Fig. 5. Effect of 2 mM caffeine on twitch responses driven electrically in mouse diaphragm muscles treated with 10 # M D-tubocurarine in bathing solutions that contained 5 # M ouabain alone or in combination with 300 nM veratridine. Twitch amplitude was measured 10 min after addition of caffeine or ouabain and 15 min after addition of veratridine. All values are expressed as percentages of the amplitude of control twitches in a solution without any agent except n-tubocurarine. Vertical bars show SEM of results from 5 or 6 experiments. Different preparations were used for the experiments to examine the effects of ouabain alone or in combination with caffeine and of veratridine alone or combined with ouabain and caffeine. Caffeine, ouabain, and veratridine, by themselves, significantly potentiated the directly-evoked twitches. *P < 0.05. CAF: caffeine. OUA: ouabain. VRT: veratridine.
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the b a t h i n g solution t h a t c o n t a i n e d 1/aM aconitine (Fig. 7B). The amplitude o f the twitch c o n t r a c t i o n s was transiently increased by o u a b a i n a n d this increase was followed by a sharp decline, until it was abolished. In a K+-free b a t h i n g solution, 1/aM aconitine caused only a n inhibitory effect o n the twitches (Fig. 7C). Higher c o n c e n t r a t i o n s o f aconitine, 2.5 or 5 / a M , a t t e n u a t e d the twitch c o n t r a c t i o n s with a transient p o t e n t i a t i o n o f the twitches (data n o t shown). M o n e n s i n increased the permeability o f m e m b r a n e to cations such as N a ÷ (Pressman, 1976). M o n e n s i n persistently p o t e n t i a t e d the twitch contractions, with a l m o s t n o latent period, at a c o n c e n t r a t i o n o f 3 0 / a M (Fig. 9A). It h a d n o p r o l o n g i n g effect o n the d u r a t i o n o f the twitches (Fig. 8). O u a b a i n ( 5 / a M ) abolished
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Fig. 6. Effects of 300nM veratridine, 5 #M ouabain and combination of veratridine and ouabain on electrical parameters of mouse diaphragm muscles in the standard bathing solution. (A) Resting membrane potentials (left side scale), (B) action potentials (right side scale), (C) overshoot potentials (right side scale), (D) time to peak, (E) 10% duration of action potentials. See text for their determinations in detail. "All parameters were derived from 30 fibres of 6 muscles (5 fibres in each preparation). Vertical bars show SEM of 30 determinations. *P < 0.05.
Ouabain and Na channel activators
27
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mmmmummmm=mm,mmm, mmmmmmmmmmumm,,+ nmnmliUWmH~mmmlmmmmmmm,m_+~++++ : -+'~-3;I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A C N 1/~M
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O ~m~i~m~M~HH~H~H~B~c~-~-~: IIIIItlIIIIHI~IIII~III~II~ItI~IHI~IIIIIIIIIItlHIIItlIII~III|IIt|IIII~II~7
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Htllll~llll~llalllllfllllllllltlllHIIIIWII | I I I I ~ E ~ - - ~ - - ~ - - = - - - - - - - - - = = - = - - ~ _~.. t|lllllMIWIlilltllllalll~llllflllmllllliq~l~lllllMIlliffllll~mEZ7 ---- - - z ~ ~ ]lllillltlliilllltll=llilllllllllllllllllg gIIIllglllllilgtlfllHIIIlilillllll~illlllllllil=]llllil~llilll~ ~1~~ 1 ~ ~ l l i ( ~ A C N 1/~M
Fig. 7. Effects of I/aM aconitine on directly-evoked twitches in mouse diaphragm muscles in standard and K+-free bathing solutions that contained I0/aM D-tubocurarine, and the changes in these effects caused by 5/aM ouabain. Typical results, from 1 of 5 sets of similar observations are presented. ACN: aconitine. OUA: ouabain. Tetraethylammonium (Stanfieid, 1 9 8 3 ) and 4-aminopyridine (Yeh et al., 1976) are blockers of potassium channels on excitable membranes. Both tetraethylammonium (10 mM) and 4-aminopyridine (200pM) persistently increased the level of twitch amplitude (Figs 10A and 11A). They had almost no prolonging effect on the duration of the twitch contractions (Fig. 8). 5 p M ouabain had no inhibitory effect on the twitches when applied after treatment with either tetraethylammonium (Fig. 10A) or 4-
aminopyridine (Fig. 11A). These potentiating effects of tetraethylammonium and of 4-aminopyridine also occurred in a K+-free bathing solution (Figs 10B and lIB). DISCUSSION Veratridine, aconitine, monensin, tetraethylammonium, and 4-aminopyridine potentiated muscle contractions stimulated directly in the diaphragm
b=.
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MNS 3 0 # M
c I
1
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+ E A l o mM
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i
i
i
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100
msec
Fig. 8. Effects of I/aM aconitine, 30/aM monensin, 10mM teraethylammonium, and 200/aM 4-aminopyridine on contractile curves of directly-evoked twitches in mouse diaphragm muscles in a standard bathing solution that contained 10/aM D-tubocurarine and the changes in these effects caused by 5/a M ouabain. (a) Control, (b) 15 min after addition of each twitch potentiator, (c) 10 min after further addition of ouabain, in addition to the twitch potentiators. Each trace represents the average of results from 8 trials. ACN: aconitine. MNS: monensin. TEA: tetraethylammonium. 4-AP: 4-aminopyridine. OUA: ouabain.
28
MASAKAZUNISHIMURAet al. ::z:
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~-
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~ ~~.~,~,,,lltlltllltlttlllltlllltllJl~ttll~tttlttllttttlllttllt~ttlltlllll~ ~lll!illl~lltllll~lttt~llltttll~lll~llt~tl~tttltttltltttllltttlttlttttttttlllt~ UtttlttllIIIttttttHUtlItlttttttttlI~III~t~IlItltlIItlIIHIItllIIItltlUltthi,
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Fig. 9. Twitch potentiation by 30gM monensin in mouse diaphragm muscles stimulated directly in standard (A) and K÷-free (B) bathing solutions that contained 10 # M D-tubocurarine, and the inhibition by 5 #M ouabain of the effect of monensin in the standard solution. Typical results, from 1 to 5 sets of similar observations, are presented. MNS: monensin. OUA: ouabain. muscles pretreated with 10/~ M D-tubocurarine. Such a potentiating effect has been previously observed in studies of directly stimulated contractions of phrenic nerve-diaphragm preparations of rats in the presence of veratridine (Meldrum et al., 1982). 10 gM D-tubocurarine completely abolished indirectly evoked twitches in those tissues. These results suggest that the potentiating effect of these agents on directly evoked twitch contractions occurs via mechanisms that are independent of the cholinergic system. Veratridine shifts the voltage dependence of activation of sodium channels and blocks inactivation of sodium channels to cause persistent activation of .H,
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sodium channels at the resting membrane potential (Narahashi, 1974), thereby increasing the influx of Na + (McKinney and Ratzlaff, 1987). Aconitine (Peper and Trautwein, 1967) and monensin (Pressman, 1976) have also been shown to increase in permeability of Na + across excitable membranes. The intracellular increase in levels of Na + stimulates the action o f N a + - K + pump (Creese and Mitchell, 1981). The N a + - K + pump is thought to be responsible for high hyperpolarization in nerve fibres (Rang and Ritchie, 1968). Activation of this pump system causes extrusion of Na + ions whose levels have increased inside the cell and restores intracellular homeostasis :
:
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I
1
Fig. 10. The absence of any effect of 5/~M ouabain or removal of K + from the bathing solution on twitch potentiation by l0 mM tetraethylammonium in mouse diaphragm muscles stimulated directly in solutions that contained l0/~M o-tubocurarine. (A) In a standard bathing solution. (B) In a K+-free bathing solution. Typical results, from I of 5 sets of similar observations, are presented. OUA: ouabain. TEA: tetraethylammonium.
Ouabain and Na channel activators ~-~
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0UA ..5/.~M
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B III~II~IIII~W~I~I~II~I~uIIt~I1III~IIIII~I~IIII~III1iiIIII1iIIm~IIIii~III~I~II1IIIIIIIiIIm~ImIIII~mII~IH~i~IuI~III~
~I[WUI~UUUUlUlIUlHU
I
4 - A P 200 y.M ;~ rain
Fig. 11. Potentiating effects of 200#M 4-aminopyridine, which are resistant to 5 #M ouabain and to removal of K + from the bathing solution, on directly-evoked twitches in mouse diaphragm muscles in solutions that contained I0 # M D-tubocurarine. Typical results, from 1 of 5 sets of similar observations, are presented. 4-AP: 4-aminopyridine. OUA: ouabain. (Clausen, 1986). This restoration may be related to the observed hyperpolarization (Rang and Ritchie, 1968). The present experiments indicate that veratridine, aconitine, and monensin increase the amplitude of twitch contractions in response to direct shocks. Thus, it is suggested that the twitch contractions, in the presence of these agents, must be coupled with an increased influx of Na t . Further more, the twitch contractions are most probably maintained by the activation of the Na+-K+-ATPase. The latter hypothesis is supported by the observation that treatment with ouabain and removal of K + from a bathing solution reduced the amplitude of the twitch contractions in the presence of these agents. Both ouabain and K÷-free solutions have been shown to inhibit the activity of Na+-K+-ATPase (Clausen, 1986). When the sodium pump system is blocked, Na + accumulates in the muscle fibres. In such a case, the twitch contractions were decreased in amplitude in spite of the presence of twitch potentiators such as veratridine, aconitine, and monensin. Thus, it is suggested that the excess and persistent accumulation of Na t inside the muscle fibres uncouples the excitation-contraction process. This hypothesis is supported by observations that the accumulation of Na ÷ inside the cell is associated with an inhibition of contractile responses of preparations of smooth muscle to different stimulants (Bose, 1974, 1975; Kishimoto et aL, 1980). Present experiments indicate that, only when ouabain was added to a solution containing veratridine, it significantly decreased all electrical parameters tested as the resting membrane potentials, action potentials, overshoot potentials, time to peak of the action potentials, and 10% duration of the action potentials. Thus, the whole reduction of the electrical activity could be related to the inhibition of twitch contraction. Such an alteration may be derived from the possible accumulation of internal Na t .
Caffeine (2mM) potentiated electrically driven twiches without producing any contraction on the base line. This effect was not reduced by ouabain or by ouabain in combination with veratridine which would be expected to cause accumulation of more Na + inside the muscle fibres. The site of action of caffeine is on the sarcoplasmic reticulum and caffeine requires Ca 2÷ for its action (Weber, 1966). Caffeine stimulates the Ca2+-releasing mechanism of this structure, but does not reduce its capacity to accumulate Ca 2+ (Endo et al., 1970). Thus, it is conceivable that the mechanism whereby Na t inhibits twitch contractions is independent of the process activated by caffeine on the sarcoplasmic reticulum. An Na--Ca exchange system exists in a variety of tissues (Blaustein, 1974). In vascular smooth muscle, this exchange system has been proposed to cause the development of tension when the concentration of intracellular Na t is elevated (Ozaki and Urakawa, 1979). The twitch potentiation by activators of sodium channels, demonstrated in the present experiments, may be related to such an exchange of internal Na t with external Ca 2÷. However, this possibility is unlikely, because the potentiating effects of such sodium channel activators occurred rather rapidly in a Ca2÷-free bathing solution and were not stimulated by the presence of ouabain, which should have increased the internal concentration of Na t . Nonetheless, Ca 2+ can compete with Na t at as yet unidentified sites that carry an anionic charge on the surface of the cell membrane, causing an inhibition of the influx of Na t (Luttgau and Niedergerke, 1958). If the same holds true in the diaphragm muscle, it is possible that removal of Ca 2+ from the bathing solution facilitates the effects of the activators of the sodium channels and also the proposed inhibitory effect of the accumulated internal Na t . Twitch potentiation was evident in the diaphragm muscles stimulated directly in the presence of tetraethylammonium and 4-aminopyridine. This type of
30
MASAKAZUNISHIMURAet al.
twitch potentiation was resistant to ouabain or removal o f K +. Tetraethylammonium (Stanfield, 1983) and 4-aminopyridine (Yeh et al., 1976) can inhibit permeability of membrane to K ÷, causing prolongation of the action potential. Thus, the antagonizing effect of ouabain and omission of K + from a bathing solution is specific for the activators of sodium channels. These results support the hypothesis that twitch contractions in the presence o f activators of sodium channels are maintained by the synchronous activation of N a ÷ - K ÷-ATPase. In conclusion, twitch contractions in the presence of blockers of sodium channels are probably maintained by the synchronous activation of N a + - K ÷ ATPase. Na ÷ accumulated in the muscle cells may reduce the amplitude o f twitch contractions by a mechanism other than that which is stimulated by caffeine and involves the release of Ca 2+ from the sarcoplasmic reticulum.
REFERENCES
Blaustein M. P. (1974) The interrelationship between sodium and calcium fluxes across cell membranes. Rev. Physiol. Biochem. Pharmac. 70, 33-82. Bose D. (1974) Mechanism of inhibition of smooth muscle tension in guinea-pig taenia coli by ouabain. Can. J. Physiol. Pharmac. 52, 898-901. Bose D. (1975) Mechanism of mechanical inhibition of smooth muscle by ouabain. Br. 3. Pharmac. 55, 111-116. Catterall W. A. (1975) Activation of the action potential Na ÷ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxin. J. biol. Chem. 250, 4053~,059. Clausen T. (1986) Regulation of active Na+-K ÷ transport in skeletal muscle. Physiol. Rev. 66, 542-580. Creese R., Head S. D. and Jenkinson D. F. (1987) The role of the sodium pump during prolonged end-plate currents in guinea-pig diaphragm. J. Physiol. 384, 377-403. Creese R. and Mitchell L. D. (1981) Spontaneous recovery from depolarizing drugs in rat diaphragm. J. Physiol. 313, 173-186. Endo M., Tanaka M. and Ogawa Y. (1970) Calcium induced release of calcium from the sarcoplasmic reticulum of skinned skeletal muscle fibres. Nature, Lond. 228, 34-36. Kishimoto T., Ozaki H. and Urakawa N. (1980) Quantitative relationship between cellular Na accumulation and relaxation produced by ouabain in depolarized smooth
muscle of guinea-pig taenia coli. Naunyn-Schmiedebergs Arch. Pharmac. 312, 199-207. Krueger B. K. and Blaustein M. P. (1980) Sodium channels in presynaptic nerve terminals: regulation by neurotoxins. J. gen. Physiol. 76, 287-313. Luttgau H. C. and Niedergerke R. (1958) The antagonism between Ca and Na ions on the frog's heart. J. Physiol. 143, 486-505. McKinney L. C. (1984) Effect of veratridine on membrane potential of sartorius muscle from Rana pipiens. Am. J. Physiol. 247, C309-C313. McKinney L. C. and Ratzlaff R. W. (1987) Sodium permeability of frog skeletal muscle in absence and presence of veratridine. Am. J. Physiol. 252, C190-C196. Meldrum M. J., Snider R. M. and Gerald M. D. (1982) Cholinergic-independent effects of amphetamine on mammalian skeletal muscle contractions. Neuropharmacology 21, 439-443. Meves H. (1966) The effect of veratridine on internally perfused giant axons. Pflfigers Arch. ges. Physiol. 290, 211-217. Narahashi T. (1974) Chemicals as tools in the study in excitable membranes. Physiol. Rev. 54, 813-889. Ohta M., Narahashi T. and Keeler R. F. (1973) Effects of veratrum alkaloids on membrane potential and conductance of squid and crayfish giant axons. J. Pharmac. exp. Ther. 184, 143-154. Ozaki H. and Urakawa N. (1979) Na42a exchange and tension development in guinea-pig aorta. NaunynSchmiedebergs Arch. Pharmac. 309, 171-178. Peper K. and Trautwein W. (1967) The effect of aconitine on the membrane current in cardiac muscle. Pflagers Arch. ges. Physiol. 296, 328-336. Pressman B. C. (1976) Biological applications of ionophores. A. Rev. Biochem. 45, 501-530. Rang H. P. and Ritchie J. M. (1968) On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations. J. Physiol. 196, 183-221. Stallcup W. B. (1977) Comparative pharmacology of voltagedependent sodium channels. Brain Res. 135, 37-53. Stanfield P. R. (1983) Tetraethylammonium ions and the potassium permeability of excitable ceils. Rev. Physiol. Biochem. Pharmac. 97, 1-67. Ulbricht W. (1969) The effect of veratridine on excitable membranes of nerve and muscle. Ergebn. Physiol. Biol. Chem. exp. Pharmak. 61, 18-71. Weber A. (1966) Energized calcium transport and relaxing factors. Curr. Topics Bioengng 1, 203-254. Yeh J. Z., Oxford G. S., Wu C. H. and Narahashi T. (1976) Dynamics of aminopyridine block of potassium channels in squid axon membrane. 3. gen. Physiol. 68, 519-535.