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The antiarrhythmic effect of potassium and rubidium in strophanthidine toxicity
European Journal of Pharmacology, 62 (1980) 1--15
1
© Elsevier/North-Holland Biomedical Press
THE ANTIARRHYTHMIC EFFECT OF POTASSIUM AND RUBIDIUM IN STROPHANTHIDIN TOXICITY * CHENG I. LIN ** and MARIO VASSALLE ***
Departments of Physiology and Pharmacology, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, U.S.A. Received 22 November 1979, accepted 29 November 1979
C.I. LIN and M. VASSALLE, The antiarrhythmic effect of potassium and rubidium in strophanthidin toxicity, European J. Pharmacol. 62 (1980) 1--15. The effect of potassium and rubidium on the electrical and mechanical activity of canine Purkinje fibers were studied in vitro in the presence and absence of strophanthidin. High (5.4 mM) K or 2.7 Rb decreased the force of contraction. In the presence of these ions, strophanthidin increased the force of contraction as usual but the onset of arrhythmias was delayed. During the toxic stage of strophanthidin, high K or Rb increased the force of contraction, abolished the arrhythmias and improved markedly the action potential. In the presence of calcium overload induced by exposure to a K-poor or Na-free solution, K and Rb induced an increase in force of contraction. And in ventricular muscle these ions relaxed the contracture induced by strophanthidin. It is concluded that K and Rb (in addition to other mechanisms) exert an antiarrhythmic action by increasing potassium conductance and by reducing the calcium overload induced by strophanthidin. Cardiac glycosides
Cardiac arrhythmias
Calcium overload
1. Introduction It is well known that potassium antagonizes the arrhythmogenic effects of cardiac glycosides (Sampson et al., 1943) and depresses the digitalis-enhanced idioventricular automaticity (Vassalle and Greenspan, 1963). The mechanism of the depression of spontaneous discharge by potassium is a flattening of diastolic depolarization (Vassalle et al., 1962). Recently, it has been shown that cardiac glycosides induce a transient oscillation superimposed on diastolic depolarization (see Ferrier, * Supported by a grant (No. HL17451) from the National Institutes of Health, Heart and Lung Institute. ** Dr. Lin was a Janet and Philip Bard Fund Fellow while carrying out this work in partial fulfillment of a Ph.D. degree in Pharmacology. *** Send correspondence to: Dr. M.V., Department of Physiology, Box 31, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, U.S.A.
Potassium
Rubidium
1977) and that the transient oscillation becomes flatter when [K]o is increased (Ferrier and Moe, 1973). Since the transient oscillations are somehow related to calcium accumulation in the cell, the question arises as to whether K leads to a decrease in [Call. This would make the antiarrhythmic action of potassium specifically suited for cardiac glycosides. The decline of [Call could be brought about by K by restoring the function of the transport ATPase. For this reason it was rather interesting to compare the effects of potassium with those of rubidium (Rb). Rubidium has been reported to counteract glycoside-induced arrhythmias in vivo (Osman et al., 1976) as potassium does. Yet, in the presence of Na and K, rubidium inhibits the activity of Na÷,K÷-ATPase and increases the force of contraction (Ku et al., 1974, 1975) in contrast to potassium. It is possible, therefore, that potassium and rubidium have an antiar-
2 rhythmic effect that is not related to their action on the pump. Alternatively, the stimulatory action of rubidium on the Na ÷, K÷-ATPase (Skou, 1965; Post et al., 1972; Tobin et al., 1974) may prevail over its inhibitory action depending on the experimental conditions. The aim of the present study was to investigate the antiarrhythmic effects of potassium and rubidium in canine Purkinje fibers exposed in vitro to strophanthidin. Both electrical and mechanical activities were recorded. The results show that potassium and rubidium have similar effects on the electrical and mechanical activities in the absence and in the presence of strophanthidin and that both ions decrease the calcium overload induced by strophanthidin. A preliminary report has appeared in abstract form (Lin and Vassalle, 1978b).
2. Materials and methods Mongrel dogs of either sex weighing 12--20 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and the heart quickly excised. Strands of Purkinje fibers were removed from the ventricles and placed in a tissue bath perfused with oxygenated (97% 02 and 3% CO2) Tyrode solution at 37°C. The composition of the Tyrode solution in mM was as follows: NaC1 137; KC1 2.7; NaHCO3 11.9; NaH2PO4 0.45; MgC12 0.5; CaC12 2.7; dextrose 5.5. Rubidium was added to the Tyrode solution or was substituted for potassium, as specified where appropriate. Also, the concentration of other ions (calcium, potassium) was altered in different experiments. In the Na-free solution, Na was substituted by tetraethylammonium (see Lin and Vassalle, 1979). The preparations were either spontaneously active or driven (usually at 60/min) in different experiments. Suprathreshold electrical stimuli of 2 msec duration were provided by a Grass $4 Stimulator through a Grass SIU 4678 Stimulus Isolation Unit and were
c.I. LIN, M. VASSALLE delivered to the preparation by means of steel pins insulated except for the tip. One end of the preparation was fixed with one of the steel pins and the other end was connected by means of a short silk thread to a rigid rod attached to a Grass Model FTO3C Force
3. Results
3.1. The effect o f 5.4 mM K on the electrical and mechanical activity of driven Purhinje fibers The effect of K on the electrical activity of Purkinje fiber has been described (see Vassalle, 1977) but little is known about its effect on mechanical activity. The point is of
K ÷ A N D Rb ÷ O N S T R O P H A N T H I D I N TOXICITY
3
500 ms
500 ms
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I CONTROL ][K]Q5.4 rnM RECOVERY I Fig. 1. Effect of [K]o on electrical and mechanical events of Purkinje fiber. Upper panels were recorded before, during and after high [K]o (5.4 raM) perfusion. The time and voltage calibrations for the top strip are at the right upper hand comer. The time and force calibrations for the bottom strip are at the right upper hand corner of that strip. The dots above the slow speed trace show the time at which the top panels have been recorded. Preparation &iven at 60/min.
obvious interest with respect to calcium metabolism. In fig. 1, the top strip shows the action potentials and the force curves in Tyrode solution (first panel), in the presence of T y r o d e containing 5.4 mM K (second panel) and during the recovery (third panel). The b o t t o m strip shows the contractile force recorded at a low speed. On increasing K from 2.7 to 5.4 raM, the duration of the action potential decreased and Ema x became less negative b y 8.8 mV. At the same time, the contractile force decreased by 50%. These changes were rapidly reversible as shown in the last upper panel. In 8 experiments, exposure to 5.4 mM K consistently caused the changes illustrated in fig. 1. The average decrease in force in 5.4 mM K was 43 + 2% (P < 0.001).
3.2. Effects o f rubidium on the electrical and mechanical activity o f driven Purkinje fibers Rubidium shortens the action potential and decreases the maximum diastolic potential, as
CONTROL
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Fig. 2. Effect of rubidium on the electrical and mechanical events of a Purkinje fiber. In the top strip, the panels were recorded before, during and after adding 2.7 mM Rb. The bottom strip is a slow speed recording of mechanical events. Preparation driven at 60/min. Other explanations as in the legend of fig. 1.
K does (Mfiller, 1965). The effect of Rb on force in Purkinje fibers is unknown. In fig. 2, the t o p strip shows the action potentials and contractile force curves in Tyrode solution (first panel), in the presence of Tyrode solution containing 2.7 mM Rb (second panel) and during recovery (third panel). The bott o m strip shows the contractile force recorded at slow speed. On adding R b to the Tyrode solution, the action potential duration decreased somewhat at the plateau b u t not during the late phase 3 and the maximum diastolic potential (Era=x) became less negative b y 5 mV. However, the contractile force n o t only did n o t increase but, instead, decreased b y 25%. The changes induced b y Rb were rapidly reversible as all parameters returned to control values within 4 rain (third t o p panel). In 14 experiments, exposure to 2.7 mM R b consistently caused the changes illustrated in fig. 2. The average decrease in contractile force during 2--5 rain exposure to Rb was 24 -+ 3% below control. In one of these experiments, the fiber was exposed to 2.7 mM RbC1 f o r 20 min: the contractile force reached a steady value (--25%) within 5 min. Thus, adding 2.7 mM K or 2.7 mM Rb to
4
C.I. LIN, M. VASSALLE
20sec> 11111!111111111'
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3.4. The effect of strophanthidin in the presence of high potassium
I I IIIIIIIIllll
RECOVERY
1
Fig. 3. Effect of r u b i d i u m o n spontaneous discharge of a Purkinje fiber. The spontaneous rate of the preparation decreased from 15 beats/min to about 4 beats/min o n exposure to RbC1 (2.7 mM). The time, voltage and force calibration a r e a t the right hand of the traces.
the Tyrode solution decreased the force of contraction but more so (+79%) when K was added.
3.3. The effect of Rb on spontaneous actwity of Purkinje fibers Rubidium increases potassium conductance (Miiller, 1965) as K does, and therefore it should slow the discharge of spontaneous fibers, as K does (Vassalle, 1965). This is illustrated in fig. 3. The preparation was allowed to discharge spontaneously in 2.7 mM K Tyrode. At the first break in the contractile force record, 2.7 mM Rb was added to the Tyrode solution. As a consequence, within a few beats, the upper curvature of the diastolic depolarization which precedes the upstroke of the action potential became considerably less pronounced and the threshold was missed. With successive beats, Em~ decreased and the initial diastolic depolarization became flatter. Within 1 rain, the cycle length of the spontaneous beat increased from 4.1 to 13.9 sec. During the recovery, the rate of the spontaneous rhythm exceeded the control (+50%) for a short period of time and then gradually returned to control within 5 min. In 4 experiments, the spontaneous rate decreased from a control value of 20 + 4 to 5 + 4 beats/rain (--75%) during Rb exposure. The results described are similar to the effects induced by increasing K (Vassalle, 1965; see fig. 7 in Vassalle, 1977).
The administration of strophanthidin to Purkinje fibers causes an initial increase in force ('therapeutic effect') followed by a decrease ('toxic effect') (Lin and Vassalle, 1978a; Vassalle and Lin, 1979). The action potential decreases in amplitude, the slope of diastolic depolarization increases, spontaneous rhythms develop and inexcitability eventually ensues ('electrical toxicity'). The decrease in force of contraction ('mechanical toxicity') appears to be due to an excessive accumulation of calcium (calcium overload) and conditions which decrease this overload cause an increase in force ('rebound phenomenon', Vassalle and Lin, 1979). In the protocol adopted, the Purkinje fibers were first exposed to strophanthidin until inexcitability developed. After full recovery, the preparations were exposed to 5.4 mM K until a new steady state was attained and then strophanthidin was added. In fig. 4, strip A shows the action potentials and the contractile force curves in Tyrode solution (first panel) and in the presence of strophanthidin (second, third and fourth panel). Strip B shows a low-speed recording of the mechanical activity. As usual, strophanthidin at first increased the duration of the action potential (second A panel) and then Em~x decreased and an oscillatory potential was present early in diastole (third A panel). Eventually, the diastolic depolarization became very steep, spontaneous activity developed at a depolarized level and force increased during diastole (fourth A panel). The strip B shows the usual increase in force (+233%) followed by a decline. In the lower part of the figure, strip C shows the electrical records in Tyrode solution (first panel), in 5.4 mM K (second panel) and in 5.4 mM K plus strophanthidin (subsequent panels). Strip D shows a low-speed mechanical record. The higher K induced the usual changes in the action potential and force of contraction. Strophanthidin
K ÷ AND Rb + ON STROPHANTHIDIN TOXICITY
5 500ms
A _
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Fig. 4. The effect of strophanthidin in the presence of high K. In strip A, the first panel was recorded before and the other panels during strophanthidin exposure. In strip B, the slow speed mechanical record is shown. In strip C, the first panel was recorded in Tyrode solution, the second in the presence of high (5.4 raM) K, and the other panels in the presence of high K and strophanthidin. In strip D, a slow speed mechanical recording is shown. Preparation driven at 60/min. Other explanations as in the legend of fig. 1.
increased the force of contraction (+350%) but more slowly. The decline in force was also present but it took much longer for the arrhythmias to appear. It is apparent that there were no oscillatory potentials during early diastole and that the rate of the spontaneous rhythm was slower in the presence than in the absence of high potassium. In 8 experiments in fibers driven at 60/rain, the time required for strophanthidin to induce spontaneous activity was 425 ± 61 sec in 2.7 mM K and was much longer when the strophanthidin was administered to the same preparations in the presence of 5.4 mM K (1051 ± 68 sec, +147%). In actuality, in 5 preparations no spontaneous discharge had developed by the end of 20 min strophanthi-
din perfusion. In the remaining 3 preparations, the time to spontaneous discharge was 347 ± 54 in 2.7 and 798 ± 92 sec in 5.4 mM K (+130%). In 5 experiments, the time to peak inotropic effect was 190 ± 22 sec in 2.7 mM K solution and it was significantly longer in 5.4 mM K solution (327 ± 31, +79%, P < 0.05). However, the peak strophanthidin inotropy developed in 5.4 mM K was not significantly different from that developed in 2.7 mM K (+8%, P > 0.05). 3.5. The effects o f strophanthidin in the presence o f rubidium In view of the fact that both K and Rb decrease the force of contraction (figs. 1 and
C.I. LIN, M. VASSALLE 5o0m$
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.
. Rb
2.7ram
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Fig. 5. The effect of strophanthidin in the presence of Rb. In the top strip, the first panel was recorded in Tyrode solution, the second in the presence of Rb, and the last three panels in the presence of Rb and strophanthidin. Preparation driven at 60/rain. Other explanations as in the legend of fig. 1.
2), Rb may also antagonize the strophanthidin arrhythmias in a manner similar to that of potassium. The results shown in fig. 5 were obtained in the same preparation as fig. 4. The top strip shows the electrical and mechanical events in Tyrode solution (first panel), in the presence of 2.7 Rb (second panel) and in the presence of Rb and strophanthidin (last 3 panels). Rubidium decreased the maximum diastolic potential and the force (--10%). Strophanthidin caused the usual changes in the presence of Rb as it did in the presence of higher K. Also, in the presence of Rb (as compared to its absence), the oscillatory potential was absent in early diastole, the slope of diastolic depolarization was less, the spontaneous rhythm was slower, the force in diastole was steady and the time to the onset of spontaneous rhythm was longer (+33%). These effects resemble those of K, although were less pronounced in the presence of Rb than of K. In 7 experiments, the time required for the development of the spontaneous rhythm was 437 + 62 sec in the absence and 835 + 96 sec in the presence of Rb (+91%, P < 0.05). In one experiment, the concentration of Rb was increased to 5 mM and strophanthidin did not cause spontaneous discharge during 1620 sec of exposure. In the same preparation, in the
absence of Rb, strophanthidin induced a spontaneous rhythm in 580 sec. The magnitude of the inotropic effect of strophanthidin was somewhat (but insignificantly) less in the presence of Rb (--11 -+ 5%, P > 0.05, n = 6). The time to the peak inotropic effect was 157 -+ 26 sec in Tyrode solution and was significantly longer in Rb solution (322 +- 90 sec, +105%, P < 0.05).
3.6. The effect of high potassium on strophanthidin toxicity It has been proposed that strophanthidin increases exchangeable calcium to an optimal value during its positive (therapeutic) inotropic effect and to an excessive value (calcium overload) during the toxic phase of declining force (Lin and VassaUe, 1978a; Vassalle and Lin, 1979). If the antagonism of strophanthidin toxicity by K reflects a lowering [Ca]i, K should decrease force during the therapeutic and increase it during the toxic stage of strophanthidin action (when calcium overload is present). In fig. 6, the top strip shows the action potentials and contractile curves in the presence of strophanthidin (first and third panels) and in the presence of strophanthidin plus higher (5.4 raM) K (second, fourth and fifth panels). The bottom strip shows a slow
K ÷ AND Rb ÷ ON STROPHANTHIDIN TOXICITY
7 5 0 0 ms
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Fig. 6. Reversal of strophanthidin toxicity by potassium. Traces in upper panels were recorded before, during and after raising [K] o from 2.7 mM to 5.4 mM (still in the presence of strophanthidin). The preparation was driven at 60/min in the first, second and fifth panel. Other explanations as in the legend of fig. 1.
speed recording of mechanical activity. As a consequence of strophanthidin administration, the contractile force increased. During the 'therapeutic' action of strophanthidin, increasing [K]o from 2.7 to 5.4 mM for 100 sec decreased the force by 13% while at the same time it shortened the action potential and decreased Emax further. That the decrease in force was not due to the onset of the strophanthidin-induced mechanical toxicity is shown by the fact that the force transiently increased when [K]o was returned to 2.7 mM (bottom strip). Toxicity occurred shortly thereafter as the fiber developed spontaneous action potentials, a markedly steepened diastolic depolarization and a pronounced decrease in force (third top panel}. While strophanthidin administration was continued, K was increased to 5.4 mM once more. This resulted in an increase in Emax, a flattening of diastolic depolarization, a slowing of the rate of discharge and an increase in force (fourth top panel). Shortly thereafter, the spontaneous activity had decreased sufficiently for the fiber to be driven again at 60/min. The action potential and the force were almost as large as in the second panel, but the action potential was shorter and the diastolic depolarization steeper. When K was reduced to 2.7 raM, arrhythmias occurred within 1 rain.
In 7 experiments, increasing K from 2.7 to 5.4 mM during the stage of mechanical toxicity increased the force of contraction by 92 -+ 37% (P < 0.05).
3. 7. The effect of rubidium on strophanthidin toxicity If Rb acts as potassium does, in the toxic stage Rb should also improve the action potential and increase the force of contraction. In fig. 7, the first panel shows the spontaneous action potentials and the contractile force of a fiber intoxicated with strophanthidin. The small amplitude of the spontaneous action potentials, the steep diastolic depolarization, the small force of contraction and the progressive increase in force during diastole are evidence of an advanced stage of toxicity. When shortly thereafter Rb was added to the strophanthidin solution (second panel recorded at a lower speed), it is apparent that within 1 min the maximum diastolic potential became more negative, the diastolic depolarization flatter, the rate of discharge slower, the amplitude of the action potential and that of the force larger. The last panel (recorded at the same speed as the first) shows the marked improvement of the action potential in spite of the fact that strophanthidin exposure was continued.
8
C.I. LIN, M. VASSALLE 10~
~ 2.-~m~
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STROPHANTHIDIN
I0-6 M
_
Fig. 7. The reversal of strophanthidin toxicity by rubidium. The first panel was recorded during late stage of strophanthidin intoxication and the preparation was discharging spontaneously at 83 beats/rain. At the beginning of the second panel, RbC1 (2.7 mM) was added to the strophanthidin-Tyrode solution. The second panel was recorded at a slower speed as indicated by the different time calibration. The third panel shows the partial reversal of strophanthidin toxicity by rubidium (the preparation responded again to a 60/min drive).
Therefore, Rb decreases the force of contraction in Tyrode solution but increases the force when the fiber is calcium-overloaded in the presence of strophanthidin. In 8 experiments, the contractile force declined to 15 + 7% of control value during late strophanthidin exposure and tLb increased the force of contraction by 141 + 21% in these fibers (P < 0.001).
3.8. The effect of low K on force of contraction and the rebound phenomenon The decrease in force induced by both Rb and K in Purkinje fibers was postulated above to be mediated through a decrease in [Ca]i. If this is correct, then decreasing [K]o should increase [Ca]i and the force of contraction. In fact, a very low [K]o may cause a calcium
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Fig. 8. Effect of potassium ana rubidium on the contractile force of Purkinje fiber exposed to low [K]o. In the upper panels, [K]o was decreased from 2.7 mM to 0.54 mM for 20 min and then returned to 2.7 mM again (RECOVERY). In the lower panels, the fiber was perfused for 20 min in 0.54 mM [K]o Tyrode solution and then with K-free 2.7 Rb Tyrode solution. The first break in trace was 14 min and the second 5 min. The fiber was driven at 90 per min.
K ÷ AND
Rb ÷ ON STROPHANTHIDIN
TOXICITY
overload, not unlikely strophanthidin and high calcium (Lin and Vassalle, 1978a; Vassalle and Lin, 1979). In fig. 8, the top strip shows the effects of 0.54 mM K (first panel) and the recovery in 2.7 mM K Tyrode (second and third panels). The bottom strip shows the effects of low K (first panel) and the recovery in 2.7 mM Rb (K-free) Tyrode (second and third panels). In the top strip, when the K was decreased to 0.54 mM, the force increased by 180% within 3 min and then declined to a value of 72% above control by the end of the 20 min perfusion. That the decline of force after the peak was due to calcium overload is suggested by the fact that returning to 2.7 mM K caused a transient increase in force (+132%) before the force returned to the original value. Such a rebound is found each time a fiber overloaded with calcium is allowed to decrease its excessive calcium load (Vassalle and Lin, 1979). It implies that in low K, the internal calcium increases to an optimal value (peak force) and then to an excessive value (subsequent decline in force). The rebound then results from a decrease in cellular calcium toward an optimal value. Naturally, as [Ca]i returns to its normal value in Tyrode solution, the force then declines to the original control value. If Rb also decreases the internal [Ca], it should also cause a rebound increase in force as K does. The bottom strip in fig. 8 shows that switching from a low K solution to a 2.7 Rb K-free Tyrode solution resulted in a rebound increase in force similar to that observed in 2.7 K Tyrode solution. In 3 experiments, in low K (0.54--1.08 mM K) the force initially increased (+114 + 33%) and then stabilized at a somewhat lower level (+35+20%). A rebound increase in force occurred on returning from 0.54 mM K to either 2.7 mM K (+106%) or 2.7 mM Rb
(+96%).
3.9. The effect o f rubidium on the events recorded in Na-free solution
When Na is substituted with tetraethylammonium, Purkinje fibers depolarize and
9
develop spontaneous slow responses (Aronson and Cranefield, 1973). The excitatory current under these conditions is carried by calcium (Aronson and Cranefield, 1973) and this leads to calcium overload (Lin and Vassalle, 1979). It was of interest to test rubidium in this preparation, since it would matter little whether Rb stimulates or inhibits the Na ÷, K÷-ATPase (there is no sodium in the solution). But Rb should increase the force of contraction if it decreases calcium overload and should depress spontaneous activity if it increases potassiu m conductance. The effect of rubidium was tested on Purkinje fibers perfused in Na-free solution in 2 experiments. In fig. 9, the top strip shows the electrical and mechanical records in Na-free solution (first panel), in Na-free solution plus rubidium (second panel) and during the recovery (third panel). In the bottom strip, the electrical events from the other experiment in the absence and presence of rubidium I sec
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Fig. 9. Effect of Rb on the electrical and mechanical activity of fibers perfused in Na-free TEA (tetraethylammonium) solution. In the top strip, the panels were recorded before, during and after exposure to 1.5 mM Rb. In the lower strip, Rb (3 raM) was perfused during the time indicated in the first panel and was discontinued at the beginning of the second panel. Fibers were spontaneously active (59 beats/ min at the beginning of the top panel and 47 beats/ min at the beginning of the bottom panel). [Ca]o = 4 raM.
10
C.I. LIN, M. VASSALLE
are shown. In the first top panel, the fiber was discharging spontaneously at 59 beats/min. As usually seen under this condition (Lin and Vassalle, 1979), there was a contraction during the action potential and an aftercontraction during diastole which were of approximately the same magnitude. After contractions are seen in several tissues when the calcium load is excessive (see Ferrier, 1976). In the presence of Rb, the action potential was somewhat smaller, the initial slope of diastolic depolarization was flatter and the rate slower (48 beats/rain). Furthermore, the force of the contraction was larger (+43%) and the development of the aftercontraction slower. During the recovery in Na-free solution, the diastolic depolarization accelerated once more and the force of the contraction was smaller than that of the aftercontraction. In this fiber increasing Rb to 3 mM slowed the rate of discharge more (to 26/rain) and increased again the force of contraction. In another fiber (bottom strip) 3 mM Rb decreased the rate of discharge (47 beats/min) somewhat before abruptly suppressing it and causing hyperpolarization. The effect was reversible, as shown in the second bottom panel. The decrease in the amplitude of the action potential was minimal before the abrupt quiescence.
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3.10. The effect o f high calcium in the presence o f rubidium The depressant effect of rubidium on the force of contraction might have involved several mechanisms. One possibility is that Rb antagonizes the calcium entry. To gain some information on this point, experiments were carried out to find out how the positive inotropic effect of high calcium is affected by rubidium. In fig. 10, strip A shows the action potentials and the contractile force in Tyrode solution (first panel), in the presence of Rb (second panel), of Rb plus high calcium (third panel) and of rubidium alone (fourth panel). Strip B shows a slow speed recording of the positive inotropic effect of calcium in the
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Fig. 10. Inotropic effect of calcium in the presence and in the absence of Rb. In strip A, the first panel was recorded in Tyrode, the second in the presence of Rb, The third in the presence of Rb and high calcium and the fourth in the presence of Rb. Strip B is a record of mechanical activity in the presence of Rb and high Ca and strip C in the presence of high Ca. Preparation driven at 60/rain.
presence of rubidium. And strip C shows the recording of the positive inotropic effect of calcium in Tyrode solution. As shown in strip A, Rb had the usual effect on the electrical and mechanical events. When calcium was increased from 2.7 to 5.4 mM in the presence of Rb, the force increased considerably (+270%) and the effect was reversible. The positive inotropic effect of high Ca in the absence of Rb was about the same (+207%, strip C) but the peak effect was attained sooner in the absence than in the presence of rubidium. In 2 additional experiments high (5.4 mM) Ca increased force by 120.3% in the absence and by 123.3% in the presence of Rb.
3.11. The effect o f rubidium in ventricular muscle fibers Since it has been reported (Ku et al., 1974, 1975) that in atrial muscle rubidium increases force (at least under certain conditions), Rb and K were tested also on ventricular muscle fibers. In fig. 11, strip A shows the effect of increasing K to 5.4 mM, strip B the effect of
K* A N D Rb* O N S T R O P H A N T H I D I N TOXICITY
L
B
K 5.4 mM
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11
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Rb 2.'7 mM
I
Rb 5.4 mM
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]
Fig. 11. Effect of high K and Rb on mechanical activity of ventricular muscle fibers. In strips A, B and C, a ventricular muscle was exposed to 5.4 mM K, 2.7 mM Rb and 5.4 mM Rb, respectively. Preparation was driven at 60/rain.
adding 2.7 mM Rb and strip C the effect of adding 5.4 mM Rb to the Tyrode solution. In all these instances, the force fell, although to a smaller extent than in Purkinje fibers. In 4 experiments, 2.7 mM Rb decreased the contractile force of ventricular muscle by 14 + 3% (P < 0.05). There was another reason to test potassium and rubidium in ventricular muscle fibers. In contrast to Purkinje fibers (Lin and Vassalle, 1978a; Vassalle and Lin, 1979), strophanthidin causes contracture in ventricular muscle (see Lee and Klaus, 1971). In fig. 12, the contracture developed on exposure to 10 -4 M strophanthidin is shown in the top and in the bottom strips. When K was increased to 5.4 mM (top strip) or 2.7 mM Rb
I
STROPH. I0-~ M
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STROPK I O - 6 M
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Fig. 12. Effect of high K and Rb on contracture by strophanthidin in ventricular muscle fibers. The preparation was driven at 60/rain and allowed to develop contracture. In the top strip, second panel, K was doubled to 5.4 mM; in the b o t t o m strip, second panel, 2.7 mM Rb was added to the s o l u t i o n . Rate of drive was 60/min. induced
(bottom strip) was added to the solution, the resting force decreased markedly in both cases. Since contracture is agreed to reflect an increase in [Ca]i, the experiment strongly supports the concept that K and Rb decrease the calcium overload induced by strophanthidin.
4. Discussion The present results show that in Purkinje fibers: (1) K decreases the force of contraction; (2) Rb has similar although less pronounced effects; (3) both K and Rb antagonize strophanthidin toxicity and increase force in strophanthidin intoxicated fibers; (4) both ions induce a force rebound in fibers previously exposed to low [K]o; (5) Rb decreases the rate of discharge and increases the force of contraction in fibers perfused in a sodium-free solution; (6) Rb does not prevent or antagonize the inotropic effect of either strophanthidin or high calcium; (7) both K and Rb decrease the force of contraction more than in ventricular muscle fibers and antagonize the strophanthidin-induced contracture in muscle fibers. These results suggest the conclusion that the mechanism of the antiarrhythmic action of K and Rb includes a decrease in the' calcium overload induced by strophanthidin and that this same mechanism is responsible for the relaxation of contracture in ventricular muscle fibers. The antiarrhythmic action of potassium on digitalis toxicity is likely to involve several mechanisms in addition to those studied in the present report. One mechanism is the reduction of the amount of cardiac glycosides bound to cardiac cells. This is shown by the findings that digoxin content of heart muscle decreases when serum potassium increases (Ebert et al., 1963) and that the uptake of 3H-ouabain in guinea-pig hearts is inversely related to potassium concentration (Dutta and Marks, 1969). Also, the myocardial accumulation of 3H
12 animals with potassium deficiency (Cohn et al., 1967). A second mechanism which may be of importance in the abolition of the arrhythmias is a stimulation of Na÷,K÷-ATPase by potassium. Such a stimulation (Skou, 1957) would result in hyperpolarization by restoring the ionic gradients and (if the sodium pumping is electrogenic) by increasing the net outward current. Some effects of rubidium reported in the literature seem at variance with a role of Na÷,K÷-ATPase activation in terminating the arrhythmia since K and Rb antagonize digitalis-induced arrhythmias both in vivo (Osman et al., 1976) and in vitro (figs. 4, 5, 6 and 7), while having an opposite effect on the activity of Na÷,K÷-ATPase (Ku et al., 1974, 1975). One explanation for this discrepancy between actions on arrhythmias and on Na ÷, K÷-ATPase could be that other effects of K and Rb are more important in antagonizing the arrhythmias than whatever effect these ions have on Na÷,K÷-ATPase. However, an alternative explanation is that the apparent discrepancy between K and Rb is due only to different experimental conditions. Rubidium is an effective substitute for potassium in activating the Na÷,K÷-ATPase (Skou, 1960; Post et al., 1972; Tobin et al., 1974), but, in a medium containing high (5.8 m M ) K, Rb (2-5 m M ) inhibits cardiac ATPase activity (Ku et al., 1974, 1975). In the presence of high K, the activity of the enzyme is already maximal as adding 2--5 mM K failed to alter the Na ÷, K+-ATPase activity (Ku et al., 1975). Rubidium has a higher affinity for the enzyme than K has, but slows down the turnover of the enzyme by the formation of a relatively stable bond with the dephospho-enzyme which hinders its rapid rephophorylation (Post et al., 1972; Tobin et al., 1974). It is possible therefore that Rb may inhibit the Na÷,K÷-ATPase when it is fully activated in the presence of high K, but may stimulate the activity at the usual [K]o. At 2.7 mM K, the ATPase may n o t be fully activated and adding Rb may increase the activity of the enzyme (but less than adding an equivalent amount of K due to
C.I.LIN, M. VASSALLE the slower turnover). When the activation is already maximal in the presence of high (5.8 mM) K, only the inhibitory action of Rb would become manifest, as suggested by the findings of Ku et al. (1975). In a K-free solution, Rb can substitute for K but irreversible deterioration develops in about 1 h in Purkinje fibers (Miiller, 1965), possibly in relation to the hindered rephophorylation of the enzyme. It is true that Rb increased the force of contraction in atrial tissues (Ku et al., 1974, 1975) and decreased it in both Purkinje (fig. 2) and ventricular muscle fibers (fig. 12). However, this discrepancy may be only apparent since the [K]o in the atrial strips experiments was 5.8 mM (Na÷,K÷-ATPase is already maximally stimulated) and in the present experiments was 2.7 mM. It should be noted that ~f a stimulation of the Na÷,K÷-ATPase activity by K and Rb is likely to contribute to the removal of strophanthidin toxicity, the rapid increase in membrane potential induced by potassium and rubidium in intoxicated fibers is likely to reflect an increase in potassium conductance (see below) as such hyperpolarization was induced by Rb even in the absence of Na (fig. 9). The present experiments suggest that two additional mechanisms are of importance in the antiarrhythmic action of both potassium and rubidium. One is the increase in membrane conductance to potassium. An increase in[K]o from 2.7 to 5.4 mM halves the resting membrane resistance (VassaUe, 1965, 1966) and increases the movements of radioactive potassium (Carmeliet, 1961). An increase in potassium conductance flattens diastolic depolarization and brings about the cessation of spontaneous discharge (Vassalle, 1965). In the presence of cardiac glycosides, the diastolic depolarization is also flattened by K (Vassalle, Karis and Hoffman, 1962; see fig. 12 in Vassalle and Musso, 1976; and fig. 7 in Vassalle, 1977) and spontaneous activity reduced or suppressed. In fact, in the late stage of toxicity high K hyperpolarizes the fibers (Anderson et al., 1976; fig. 6 in the present experiments). And Emax of the fibers
K+ AND Rb ÷ ON STROPHANTHIDIN
TOXICITY
intoxicated by strophanthidin is more negative in the presence of high K (fig. 4). Rubidiu m has been shown to increase potassium conductance (Miiller, 1965) and in the present experiments induced changes similar to those induced by potassium (e.g. see effects on spontaneous discharge, figs. 3 and 9). The other mechanism by which potassium and rubidium appear to specifically antagonize digitalis arrhythmias is an interference with calcium metabolism whereby the calciu m overload induced by glycosides is reduced. The calcium overload induced by cardiac glycosides results in a decrease in force of contraction, as several lines of evidence support (Lin and Vassalle, 1978a; Vassalle and Lin, 1979}. The existence of calcium overload in the present experiment is indicated by: (1) the decline in force and afterdepolarizations during the late stage of strophanthidin exposure (figs. 4 and 6); (2) the fall in force during low K perfusion (fig. 8); (3) the aftercontractions in the Na-free calcium solution (fig. 9); and (4) the contracture in ventricular muscle fibers exposed to strophanthidin (fig. 12). In these situations, there was force develo p m e n t or aftercontractions during diastole and this is generally attributed to an excessive calcium load. In all of these situations, potassium or rubidium increased the force of contraction or decreased the diastolic force (figs. 6, 7, 8, 9 and 12) as one would expect. Other events also show that the calcium overload was decreased by K and Rb. Thus, when strophanthidin was given in the presence of higher K or Rb there were no obvious oscillations in diastole and aftercontractions were absent (figs. 6 and 7). The conclusion that both K and Rb decrease the calcium overload in the presence of toxic concentrations of cardiac glycosides is strengthened by the finding that both ions decrease calcium in cardiac tissues of animals treated with cardiac glycosides (Osman et al., 1976). That indeed K and Rb can decrease the cellular calcium released in systole is shown by the fact that in the absence of overload both ions decreased (instead of increasing) the
13
force of contraction (figs. 1, 2 and 11), and this occurred also in the therapeutic stage of strophanthidin administration (fig. 6). In sheep Purkinje fibers, a reduction of [K]o 5.4 to 4.05 mM increases the force of contraction (as a low dose of ouabain does) and an increase of K to 6.75 mM induces 'aftercontractions' (as a high dose of ouabain does) (Blood, 1975). In the present experiments the force decreased when [K]o was increased from 2.7 to 5.4 mM (fig. 1) and increased when K was decreased to 0.54 mM (fig. 8). The force decreased also in ventricular muscle with higher K (fig. 11) as reported by Cohn et al. (1967), but far less than in Purkinje fibers (compare figs. 1 and 11). These results and those with Rb (figs. 2 and 11) suggest that these two ions interfere with calcium metabolism in reducing contractile force in the absence of or in the therapeutic stage of cardiac glycosides and in increasing force when the calcium load is excessive. One mechanism by which force is changed could be the reduction of the slow inward current by these two ions as this current is found to increase when [K]o is decreased, indicating a degree of K-Ca antagonism (Goto et al., 1978). The fact that force increased when calcium was increased in the presence of rubidium (fig. 10) shows that calcium can antagonize the effect of rubidium, as rubidium antagonizes the effect of calcium, but that there is no block of the slow channel. The reduction in the rate of discharge by K and Rb during strophanthidin toxicity should also result in a decreased binding of the strophanthidin. This beneficial effect would be expected on the basis of the finding that the binding of cardiac glycosides to tissues is less at lower rates ( B e n t f e l d e t al., 1977). In conclusion, it appears both K and Rb (figs. 3 and 9) increase K conductance and this is important in reducing spontaneous discharge in the absence and in the presence of cardiac glycosides. The evidence that these two ions reduce the calcium overload in the presence of strophanthidin points to a new antiarrhythmic mechanism which appears to be of a specific nature.
14 References
Anderson, G.J., F.C. Bailey, J. Reiser and A. Freeman, 1976, Electrophysiological observations on the digitalis-potassium interaction in canine Purkinje fibers,Circulation Res. 39,717. Aronson, R.S. and P.F. Cranefield, 1973, The electrical activity of canine cardiac Purkinje fibers in sodium-free, calcium-rich solutions, J. Gen. Physiol. 61,786. Bentfeld, M., H, Liillmann, T. Peters and D. Proppe, 1977, Interdependence of ion transport and the action of ouabain in heart muscle, Br. J. Pharmacol. 61, 19. Blood, B.E., 1975, The influence of low doses of ouabain and potassium ions on sheep Purkinje fibres contractility,J. Physiol. (London) 251, 69P. Carmeliet, E.E., 1961, Chloride and Potassium Permeability in Cardiac Purkinje Fibres (Arscia S.A. Presses Acad~miques Eurol~ennes, BruxeUes). Cohn, K.E., R.E. Kleiger and D.C. Harrison, 1967, Influence of potassium depletion on myocardial concentration of tritiateddigoxin, Circulation Res. 20, 473. Dutta, S. and B.H. Marks, 1969, Factors that regulate ouabain-H 3 accumulation by the isolated guineapig heart, J. Pharmacol. Exp. Ther. 170, 318. Ebert, P.A., L.J. Greenfield and W.C. Austen, 1963, The effect of increased serum potassium on the digoxin content of the canine heart, Bull. John Hopkins Hosp. 112, 151. Ferrier, G.R., 1976, The effects of tension on acetylstrophanthidin-induced transient depolarization and aftercontractions in canine myocardial and Purkinje tissues,Circulation Res. 38, 156. Ferrier, G.R., 1977, Digitalis arrhythmias: role of oscillatory afterpotentials,Progr. Cardiovasc. Dis. 19, 459. Ferrier, G.R. and G.K. Moe, 1973, Effect of calcium on acetylstrophanthidin-induced transient depolarizations in canine Purkinje tissue, Circulation Res. 33,508. Goto, M., A. Yatani and Y. Tsuda, 1978, Membrane calcium current in cardiac excitation: effects of ATP and related substances and sodium pump on bullfrog atrium, in: Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 11, Heart Function and Metabolism, eds. T. Kobayashi, T. Sano and N.S. Dhalla (University Park Press, Baltimore) pg. 3.7. Ku, D., T. Akara, T. Tobin and T.M. Brody, 1974, Effects of rubidium on cardiac tissue: inhibition of Na+,K÷-ATPase and stimulation of contractile force, Res. Commun. Chem. Pathol. Pharmacol. 9, 431. Ku, D., T. Akera, T. Tobin and T.M. Brody, 1975, Effects of monovalent cations on cardiac Na ÷,
C.I. LIN, M. VASSALLE
K*-ATPase activity and on contractile force. Nannyn-Schmiedeb. Arch. Pharmacol. 290, 113. Lee, K.S. and W. Klaus, 1971, The subcellular basis for the mechanism of inotropic action of cardiac glycosides, Pharmacol. Rev. 23, 193. Lin, C.I. and M. Vassalle, 1978a, Role of sodium in strophanthidin toxicity of Purkinje fibers,Am. J. Physiol. 234, H477. Lin, C.I. and M. Vassalle, 1978b, Rubidium effects in the presence and in the absence of strophanthidin in cardiac Purkinje fibers, Bull. N.Y. Acad. Med. 54,322 (abstract). Lin, C.I. and M. Vassalle,1979, Sodium lack prevents strophanthidin toxicity in cardiac Purkinje fibers, Cardiology 64,110. Miiller, P., 1965, Potassium and rubidium exchange across the surface membrane of cardiac Purkinje fibres,J. Physiol. (London) 177,453. Osman, F.H., E.M. Ammar, A.M. Afifi and N.M. Ahmed, 1976, Potentiation by lithium and protection by rubidium of digitalisintoxication, Jap.J. Exp. Med. 46, 1. Post, R.L., G. Hegyvary and S. Kume, 1972, Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium in transport adenosine triphosphatase, J. Biol. Chem. 247, 6530. Sampson, J.J., E.C. Albertson and B. Kondo, 1943, The effect on man of potassium administration in relation to digitalis glycosides, with special reference to blood serum potassium, the electrocardiogram, and ectopic beats, Am. Heart J. 26, 164. Skou, J.C., 1957, The influence of some cations on an adenosinetriphosphatase from peripheral nerves, Biochim. Biophys. Acta 23,394. Skou, J.C., 1960, Further investigation on a Mg÷÷+ Na÷-activated adenosinetriphosphatase, possibly related to the active linked transport of Na ÷ and K ÷ across the nerve membrane, Biochim. Biophys. Acta 42, 6. Skou, J.C., 1965, Enzymatic basis for active transport of Na ÷ and K ÷ across cell membrane, Physiol. Rev. 45, 596. Tobin, T., T. Akera, C.S. Hart and T.M. Brody, 1974, Lithium and rubidium interactions with sodiumand potassium-dependent adenosine triphosphatase: a molecular basis for the pharmacological actions of these ions, Mol. Pharmacol. 10, 501. Vassalle, M., 1965, Cardiac pacemaker potential at different extra- and intracellular K concentrations, Am. J. Physiol. 208,770. Vassalle, M., 1966, Analysis of cardiac pacemaker potential using a 'voltage clamp' technique' Am. J. Physiol. 210, 1335. Vassalle, M., 1977, Generation and conduction of impulses in the heart under physiological and pathological conditions, Pharmacol. Ther. B. 3, 1.
K ÷ AND
Rb ÷ ON STROPHANTHIDIN
TOXICITY
Vassalle, M. and K. Greenspan0 1963, Effects of potassium on ouabain-induced arrhythmias, Am. J. Cardiol. 12, 692. Vassalle, M., J. Karis and B.F. Hoffman, 1962, Toxic effects of ouabain on Purkinje fibers and ventricular muscle fibers, Am. J. Physiol. 203,433. Vassalle, M. and C.I. Lin, 1979, Effect of calcium on strophantnldin-induced electrical and mechanical
15
toxicity in cardiac Purkinje fibers, Am. J. Physiol. 235, H689. Vassalle, M. and E. Musso, 1976, On the mechanism underlying digitalis toxicity in cardiac Purkinje fibers, in: Recent Advances on Cardiac Structure and Metabolism, Vol. 9, The Sarcolemma, eds. P.-E. Roy and N.S. Dhalla (University Park Press, Baltimore) p. 355.
1
© Elsevier/North-Holland Biomedical Press
THE ANTIARRHYTHMIC EFFECT OF POTASSIUM AND RUBIDIUM IN STROPHANTHIDIN TOXICITY * CHENG I. LIN ** and MARIO VASSALLE ***
Departments of Physiology and Pharmacology, State University of New York, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, U.S.A. Received 22 November 1979, accepted 29 November 1979
C.I. LIN and M. VASSALLE, The antiarrhythmic effect of potassium and rubidium in strophanthidin toxicity, European J. Pharmacol. 62 (1980) 1--15. The effect of potassium and rubidium on the electrical and mechanical activity of canine Purkinje fibers were studied in vitro in the presence and absence of strophanthidin. High (5.4 mM) K or 2.7 Rb decreased the force of contraction. In the presence of these ions, strophanthidin increased the force of contraction as usual but the onset of arrhythmias was delayed. During the toxic stage of strophanthidin, high K or Rb increased the force of contraction, abolished the arrhythmias and improved markedly the action potential. In the presence of calcium overload induced by exposure to a K-poor or Na-free solution, K and Rb induced an increase in force of contraction. And in ventricular muscle these ions relaxed the contracture induced by strophanthidin. It is concluded that K and Rb (in addition to other mechanisms) exert an antiarrhythmic action by increasing potassium conductance and by reducing the calcium overload induced by strophanthidin. Cardiac glycosides
Cardiac arrhythmias
Calcium overload
1. Introduction It is well known that potassium antagonizes the arrhythmogenic effects of cardiac glycosides (Sampson et al., 1943) and depresses the digitalis-enhanced idioventricular automaticity (Vassalle and Greenspan, 1963). The mechanism of the depression of spontaneous discharge by potassium is a flattening of diastolic depolarization (Vassalle et al., 1962). Recently, it has been shown that cardiac glycosides induce a transient oscillation superimposed on diastolic depolarization (see Ferrier, * Supported by a grant (No. HL17451) from the National Institutes of Health, Heart and Lung Institute. ** Dr. Lin was a Janet and Philip Bard Fund Fellow while carrying out this work in partial fulfillment of a Ph.D. degree in Pharmacology. *** Send correspondence to: Dr. M.V., Department of Physiology, Box 31, Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, New York 11203, U.S.A.
Potassium
Rubidium
1977) and that the transient oscillation becomes flatter when [K]o is increased (Ferrier and Moe, 1973). Since the transient oscillations are somehow related to calcium accumulation in the cell, the question arises as to whether K leads to a decrease in [Call. This would make the antiarrhythmic action of potassium specifically suited for cardiac glycosides. The decline of [Call could be brought about by K by restoring the function of the transport ATPase. For this reason it was rather interesting to compare the effects of potassium with those of rubidium (Rb). Rubidium has been reported to counteract glycoside-induced arrhythmias in vivo (Osman et al., 1976) as potassium does. Yet, in the presence of Na and K, rubidium inhibits the activity of Na÷,K÷-ATPase and increases the force of contraction (Ku et al., 1974, 1975) in contrast to potassium. It is possible, therefore, that potassium and rubidium have an antiar-
2 rhythmic effect that is not related to their action on the pump. Alternatively, the stimulatory action of rubidium on the Na ÷, K÷-ATPase (Skou, 1965; Post et al., 1972; Tobin et al., 1974) may prevail over its inhibitory action depending on the experimental conditions. The aim of the present study was to investigate the antiarrhythmic effects of potassium and rubidium in canine Purkinje fibers exposed in vitro to strophanthidin. Both electrical and mechanical activities were recorded. The results show that potassium and rubidium have similar effects on the electrical and mechanical activities in the absence and in the presence of strophanthidin and that both ions decrease the calcium overload induced by strophanthidin. A preliminary report has appeared in abstract form (Lin and Vassalle, 1978b).
2. Materials and methods Mongrel dogs of either sex weighing 12--20 kg were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and the heart quickly excised. Strands of Purkinje fibers were removed from the ventricles and placed in a tissue bath perfused with oxygenated (97% 02 and 3% CO2) Tyrode solution at 37°C. The composition of the Tyrode solution in mM was as follows: NaC1 137; KC1 2.7; NaHCO3 11.9; NaH2PO4 0.45; MgC12 0.5; CaC12 2.7; dextrose 5.5. Rubidium was added to the Tyrode solution or was substituted for potassium, as specified where appropriate. Also, the concentration of other ions (calcium, potassium) was altered in different experiments. In the Na-free solution, Na was substituted by tetraethylammonium (see Lin and Vassalle, 1979). The preparations were either spontaneously active or driven (usually at 60/min) in different experiments. Suprathreshold electrical stimuli of 2 msec duration were provided by a Grass $4 Stimulator through a Grass SIU 4678 Stimulus Isolation Unit and were
c.I. LIN, M. VASSALLE delivered to the preparation by means of steel pins insulated except for the tip. One end of the preparation was fixed with one of the steel pins and the other end was connected by means of a short silk thread to a rigid rod attached to a Grass Model FTO3C Force
3. Results
3.1. The effect o f 5.4 mM K on the electrical and mechanical activity of driven Purhinje fibers The effect of K on the electrical activity of Purkinje fiber has been described (see Vassalle, 1977) but little is known about its effect on mechanical activity. The point is of
K ÷ A N D Rb ÷ O N S T R O P H A N T H I D I N TOXICITY
3
500 ms
500 ms
>
>
60~
60$ec •
I CONTROL ][K]Q5.4 rnM RECOVERY I Fig. 1. Effect of [K]o on electrical and mechanical events of Purkinje fiber. Upper panels were recorded before, during and after high [K]o (5.4 raM) perfusion. The time and voltage calibrations for the top strip are at the right upper hand comer. The time and force calibrations for the bottom strip are at the right upper hand corner of that strip. The dots above the slow speed trace show the time at which the top panels have been recorded. Preparation &iven at 60/min.
obvious interest with respect to calcium metabolism. In fig. 1, the top strip shows the action potentials and the force curves in Tyrode solution (first panel), in the presence of T y r o d e containing 5.4 mM K (second panel) and during the recovery (third panel). The b o t t o m strip shows the contractile force recorded at a low speed. On increasing K from 2.7 to 5.4 raM, the duration of the action potential decreased and Ema x became less negative b y 8.8 mV. At the same time, the contractile force decreased by 50%. These changes were rapidly reversible as shown in the last upper panel. In 8 experiments, exposure to 5.4 mM K consistently caused the changes illustrated in fig. 1. The average decrease in force in 5.4 mM K was 43 + 2% (P < 0.001).
3.2. Effects o f rubidium on the electrical and mechanical activity o f driven Purkinje fibers Rubidium shortens the action potential and decreases the maximum diastolic potential, as
CONTROL
.
I Rb2.7mMI RECOVERY
m~
~
.
I
Fig. 2. Effect of rubidium on the electrical and mechanical events of a Purkinje fiber. In the top strip, the panels were recorded before, during and after adding 2.7 mM Rb. The bottom strip is a slow speed recording of mechanical events. Preparation driven at 60/min. Other explanations as in the legend of fig. 1.
K does (Mfiller, 1965). The effect of Rb on force in Purkinje fibers is unknown. In fig. 2, the t o p strip shows the action potentials and contractile force curves in Tyrode solution (first panel), in the presence of Tyrode solution containing 2.7 mM Rb (second panel) and during recovery (third panel). The bott o m strip shows the contractile force recorded at slow speed. On adding R b to the Tyrode solution, the action potential duration decreased somewhat at the plateau b u t not during the late phase 3 and the maximum diastolic potential (Era=x) became less negative b y 5 mV. However, the contractile force n o t only did n o t increase but, instead, decreased b y 25%. The changes induced b y Rb were rapidly reversible as all parameters returned to control values within 4 rain (third t o p panel). In 14 experiments, exposure to 2.7 mM R b consistently caused the changes illustrated in fig. 2. The average decrease in contractile force during 2--5 rain exposure to Rb was 24 -+ 3% below control. In one of these experiments, the fiber was exposed to 2.7 mM RbC1 f o r 20 min: the contractile force reached a steady value (--25%) within 5 min. Thus, adding 2.7 mM K or 2.7 mM Rb to
4
C.I. LIN, M. VASSALLE
20sec> 11111!111111111'
i i
i
I i
I C'ONTROLI Rb 2.7raM ]
i
3.4. The effect of strophanthidin in the presence of high potassium
I I IIIIIIIIllll
RECOVERY
1
Fig. 3. Effect of r u b i d i u m o n spontaneous discharge of a Purkinje fiber. The spontaneous rate of the preparation decreased from 15 beats/min to about 4 beats/min o n exposure to RbC1 (2.7 mM). The time, voltage and force calibration a r e a t the right hand of the traces.
the Tyrode solution decreased the force of contraction but more so (+79%) when K was added.
3.3. The effect of Rb on spontaneous actwity of Purkinje fibers Rubidium increases potassium conductance (Miiller, 1965) as K does, and therefore it should slow the discharge of spontaneous fibers, as K does (Vassalle, 1965). This is illustrated in fig. 3. The preparation was allowed to discharge spontaneously in 2.7 mM K Tyrode. At the first break in the contractile force record, 2.7 mM Rb was added to the Tyrode solution. As a consequence, within a few beats, the upper curvature of the diastolic depolarization which precedes the upstroke of the action potential became considerably less pronounced and the threshold was missed. With successive beats, Em~ decreased and the initial diastolic depolarization became flatter. Within 1 rain, the cycle length of the spontaneous beat increased from 4.1 to 13.9 sec. During the recovery, the rate of the spontaneous rhythm exceeded the control (+50%) for a short period of time and then gradually returned to control within 5 min. In 4 experiments, the spontaneous rate decreased from a control value of 20 + 4 to 5 + 4 beats/rain (--75%) during Rb exposure. The results described are similar to the effects induced by increasing K (Vassalle, 1965; see fig. 7 in Vassalle, 1977).
The administration of strophanthidin to Purkinje fibers causes an initial increase in force ('therapeutic effect') followed by a decrease ('toxic effect') (Lin and Vassalle, 1978a; Vassalle and Lin, 1979). The action potential decreases in amplitude, the slope of diastolic depolarization increases, spontaneous rhythms develop and inexcitability eventually ensues ('electrical toxicity'). The decrease in force of contraction ('mechanical toxicity') appears to be due to an excessive accumulation of calcium (calcium overload) and conditions which decrease this overload cause an increase in force ('rebound phenomenon', Vassalle and Lin, 1979). In the protocol adopted, the Purkinje fibers were first exposed to strophanthidin until inexcitability developed. After full recovery, the preparations were exposed to 5.4 mM K until a new steady state was attained and then strophanthidin was added. In fig. 4, strip A shows the action potentials and the contractile force curves in Tyrode solution (first panel) and in the presence of strophanthidin (second, third and fourth panel). Strip B shows a low-speed recording of the mechanical activity. As usual, strophanthidin at first increased the duration of the action potential (second A panel) and then Em~x decreased and an oscillatory potential was present early in diastole (third A panel). Eventually, the diastolic depolarization became very steep, spontaneous activity developed at a depolarized level and force increased during diastole (fourth A panel). The strip B shows the usual increase in force (+233%) followed by a decline. In the lower part of the figure, strip C shows the electrical records in Tyrode solution (first panel), in 5.4 mM K (second panel) and in 5.4 mM K plus strophanthidin (subsequent panels). Strip D shows a low-speed mechanical record. The higher K induced the usual changes in the action potential and force of contraction. Strophanthidin
K ÷ AND Rb + ON STROPHANTHIDIN TOXICITY
5 500ms
A _
L_--
-
\
_
L..-.-
I rain
J
B I TYR.
D I
II
I
I TYR. I
STROPH.
10-6 M
, ,
K 5.4=M
' "
,'STROPH.
I0 "6 M
I
JJ
I Jlll Nil Jl
Jl
I
Fig. 4. The effect of strophanthidin in the presence of high K. In strip A, the first panel was recorded before and the other panels during strophanthidin exposure. In strip B, the slow speed mechanical record is shown. In strip C, the first panel was recorded in Tyrode solution, the second in the presence of high (5.4 raM) K, and the other panels in the presence of high K and strophanthidin. In strip D, a slow speed mechanical recording is shown. Preparation driven at 60/min. Other explanations as in the legend of fig. 1.
increased the force of contraction (+350%) but more slowly. The decline in force was also present but it took much longer for the arrhythmias to appear. It is apparent that there were no oscillatory potentials during early diastole and that the rate of the spontaneous rhythm was slower in the presence than in the absence of high potassium. In 8 experiments in fibers driven at 60/rain, the time required for strophanthidin to induce spontaneous activity was 425 ± 61 sec in 2.7 mM K and was much longer when the strophanthidin was administered to the same preparations in the presence of 5.4 mM K (1051 ± 68 sec, +147%). In actuality, in 5 preparations no spontaneous discharge had developed by the end of 20 min strophanthi-
din perfusion. In the remaining 3 preparations, the time to spontaneous discharge was 347 ± 54 in 2.7 and 798 ± 92 sec in 5.4 mM K (+130%). In 5 experiments, the time to peak inotropic effect was 190 ± 22 sec in 2.7 mM K solution and it was significantly longer in 5.4 mM K solution (327 ± 31, +79%, P < 0.05). However, the peak strophanthidin inotropy developed in 5.4 mM K was not significantly different from that developed in 2.7 mM K (+8%, P > 0.05). 3.5. The effects o f strophanthidin in the presence o f rubidium In view of the fact that both K and Rb decrease the force of contraction (figs. 1 and
C.I. LIN, M. VASSALLE 5o0m$
%j' . ICONTROL[
.
. Rb
2.7ram
. [
.
Ii
STROPHANTHIDIN I0-6 M
I i
Fig. 5. The effect of strophanthidin in the presence of Rb. In the top strip, the first panel was recorded in Tyrode solution, the second in the presence of Rb, and the last three panels in the presence of Rb and strophanthidin. Preparation driven at 60/rain. Other explanations as in the legend of fig. 1.
2), Rb may also antagonize the strophanthidin arrhythmias in a manner similar to that of potassium. The results shown in fig. 5 were obtained in the same preparation as fig. 4. The top strip shows the electrical and mechanical events in Tyrode solution (first panel), in the presence of 2.7 Rb (second panel) and in the presence of Rb and strophanthidin (last 3 panels). Rubidium decreased the maximum diastolic potential and the force (--10%). Strophanthidin caused the usual changes in the presence of Rb as it did in the presence of higher K. Also, in the presence of Rb (as compared to its absence), the oscillatory potential was absent in early diastole, the slope of diastolic depolarization was less, the spontaneous rhythm was slower, the force in diastole was steady and the time to the onset of spontaneous rhythm was longer (+33%). These effects resemble those of K, although were less pronounced in the presence of Rb than of K. In 7 experiments, the time required for the development of the spontaneous rhythm was 437 + 62 sec in the absence and 835 + 96 sec in the presence of Rb (+91%, P < 0.05). In one experiment, the concentration of Rb was increased to 5 mM and strophanthidin did not cause spontaneous discharge during 1620 sec of exposure. In the same preparation, in the
absence of Rb, strophanthidin induced a spontaneous rhythm in 580 sec. The magnitude of the inotropic effect of strophanthidin was somewhat (but insignificantly) less in the presence of Rb (--11 -+ 5%, P > 0.05, n = 6). The time to the peak inotropic effect was 157 -+ 26 sec in Tyrode solution and was significantly longer in Rb solution (322 +- 90 sec, +105%, P < 0.05).
3.6. The effect of high potassium on strophanthidin toxicity It has been proposed that strophanthidin increases exchangeable calcium to an optimal value during its positive (therapeutic) inotropic effect and to an excessive value (calcium overload) during the toxic phase of declining force (Lin and VassaUe, 1978a; Vassalle and Lin, 1979). If the antagonism of strophanthidin toxicity by K reflects a lowering [Ca]i, K should decrease force during the therapeutic and increase it during the toxic stage of strophanthidin action (when calcium overload is present). In fig. 6, the top strip shows the action potentials and contractile curves in the presence of strophanthidin (first and third panels) and in the presence of strophanthidin plus higher (5.4 raM) K (second, fourth and fifth panels). The bottom strip shows a slow
K ÷ AND Rb ÷ ON STROPHANTHIDIN TOXICITY
7 5 0 0 ms
\ b
"
]CONTROLI
jk__..._
] K 5.4raM STROPHANTHIDIN10-6 M
,'~
_4
[ J
Fig. 6. Reversal of strophanthidin toxicity by potassium. Traces in upper panels were recorded before, during and after raising [K] o from 2.7 mM to 5.4 mM (still in the presence of strophanthidin). The preparation was driven at 60/min in the first, second and fifth panel. Other explanations as in the legend of fig. 1.
speed recording of mechanical activity. As a consequence of strophanthidin administration, the contractile force increased. During the 'therapeutic' action of strophanthidin, increasing [K]o from 2.7 to 5.4 mM for 100 sec decreased the force by 13% while at the same time it shortened the action potential and decreased Emax further. That the decrease in force was not due to the onset of the strophanthidin-induced mechanical toxicity is shown by the fact that the force transiently increased when [K]o was returned to 2.7 mM (bottom strip). Toxicity occurred shortly thereafter as the fiber developed spontaneous action potentials, a markedly steepened diastolic depolarization and a pronounced decrease in force (third top panel}. While strophanthidin administration was continued, K was increased to 5.4 mM once more. This resulted in an increase in Emax, a flattening of diastolic depolarization, a slowing of the rate of discharge and an increase in force (fourth top panel). Shortly thereafter, the spontaneous activity had decreased sufficiently for the fiber to be driven again at 60/min. The action potential and the force were almost as large as in the second panel, but the action potential was shorter and the diastolic depolarization steeper. When K was reduced to 2.7 raM, arrhythmias occurred within 1 rain.
In 7 experiments, increasing K from 2.7 to 5.4 mM during the stage of mechanical toxicity increased the force of contraction by 92 -+ 37% (P < 0.05).
3. 7. The effect of rubidium on strophanthidin toxicity If Rb acts as potassium does, in the toxic stage Rb should also improve the action potential and increase the force of contraction. In fig. 7, the first panel shows the spontaneous action potentials and the contractile force of a fiber intoxicated with strophanthidin. The small amplitude of the spontaneous action potentials, the steep diastolic depolarization, the small force of contraction and the progressive increase in force during diastole are evidence of an advanced stage of toxicity. When shortly thereafter Rb was added to the strophanthidin solution (second panel recorded at a lower speed), it is apparent that within 1 min the maximum diastolic potential became more negative, the diastolic depolarization flatter, the rate of discharge slower, the amplitude of the action potential and that of the force larger. The last panel (recorded at the same speed as the first) shows the marked improvement of the action potential in spite of the fact that strophanthidin exposure was continued.
8
C.I. LIN, M. VASSALLE 10~
~ 2.-~m~
___l I
STROPHANTHIDIN
I0-6 M
_
Fig. 7. The reversal of strophanthidin toxicity by rubidium. The first panel was recorded during late stage of strophanthidin intoxication and the preparation was discharging spontaneously at 83 beats/rain. At the beginning of the second panel, RbC1 (2.7 mM) was added to the strophanthidin-Tyrode solution. The second panel was recorded at a slower speed as indicated by the different time calibration. The third panel shows the partial reversal of strophanthidin toxicity by rubidium (the preparation responded again to a 60/min drive).
Therefore, Rb decreases the force of contraction in Tyrode solution but increases the force when the fiber is calcium-overloaded in the presence of strophanthidin. In 8 experiments, the contractile force declined to 15 + 7% of control value during late strophanthidin exposure and tLb increased the force of contraction by 141 + 21% in these fibers (P < 0.001).
3.8. The effect of low K on force of contraction and the rebound phenomenon The decrease in force induced by both Rb and K in Purkinje fibers was postulated above to be mediated through a decrease in [Ca]i. If this is correct, then decreasing [K]o should increase [Ca]i and the force of contraction. In fact, a very low [K]o may cause a calcium
60111c
Ic I
,~ o.54
!
RECOVE,~
I c I
,, o.s4
I
,b 27
__
I
iii "
I
Fig. 8. Effect of potassium ana rubidium on the contractile force of Purkinje fiber exposed to low [K]o. In the upper panels, [K]o was decreased from 2.7 mM to 0.54 mM for 20 min and then returned to 2.7 mM again (RECOVERY). In the lower panels, the fiber was perfused for 20 min in 0.54 mM [K]o Tyrode solution and then with K-free 2.7 Rb Tyrode solution. The first break in trace was 14 min and the second 5 min. The fiber was driven at 90 per min.
K ÷ AND
Rb ÷ ON STROPHANTHIDIN
TOXICITY
overload, not unlikely strophanthidin and high calcium (Lin and Vassalle, 1978a; Vassalle and Lin, 1979). In fig. 8, the top strip shows the effects of 0.54 mM K (first panel) and the recovery in 2.7 mM K Tyrode (second and third panels). The bottom strip shows the effects of low K (first panel) and the recovery in 2.7 mM Rb (K-free) Tyrode (second and third panels). In the top strip, when the K was decreased to 0.54 mM, the force increased by 180% within 3 min and then declined to a value of 72% above control by the end of the 20 min perfusion. That the decline of force after the peak was due to calcium overload is suggested by the fact that returning to 2.7 mM K caused a transient increase in force (+132%) before the force returned to the original value. Such a rebound is found each time a fiber overloaded with calcium is allowed to decrease its excessive calcium load (Vassalle and Lin, 1979). It implies that in low K, the internal calcium increases to an optimal value (peak force) and then to an excessive value (subsequent decline in force). The rebound then results from a decrease in cellular calcium toward an optimal value. Naturally, as [Ca]i returns to its normal value in Tyrode solution, the force then declines to the original control value. If Rb also decreases the internal [Ca], it should also cause a rebound increase in force as K does. The bottom strip in fig. 8 shows that switching from a low K solution to a 2.7 Rb K-free Tyrode solution resulted in a rebound increase in force similar to that observed in 2.7 K Tyrode solution. In 3 experiments, in low K (0.54--1.08 mM K) the force initially increased (+114 + 33%) and then stabilized at a somewhat lower level (+35+20%). A rebound increase in force occurred on returning from 0.54 mM K to either 2.7 mM K (+106%) or 2.7 mM Rb
(+96%).
3.9. The effect o f rubidium on the events recorded in Na-free solution
When Na is substituted with tetraethylammonium, Purkinje fibers depolarize and
9
develop spontaneous slow responses (Aronson and Cranefield, 1973). The excitatory current under these conditions is carried by calcium (Aronson and Cranefield, 1973) and this leads to calcium overload (Lin and Vassalle, 1979). It was of interest to test rubidium in this preparation, since it would matter little whether Rb stimulates or inhibits the Na ÷, K÷-ATPase (there is no sodium in the solution). But Rb should increase the force of contraction if it decreases calcium overload and should depress spontaneous activity if it increases potassiu m conductance. The effect of rubidium was tested on Purkinje fibers perfused in Na-free solution in 2 experiments. In fig. 9, the top strip shows the electrical and mechanical records in Na-free solution (first panel), in Na-free solution plus rubidium (second panel) and during the recovery (third panel). In the bottom strip, the electrical events from the other experiment in the absence and presence of rubidium I sec
.LLLL ,L.,L..L,LLL L
Rb l.SmM
J
No-FREE SOLUTION IHHI"'''",,'H ..... ,
r I
Rb
3 mM
I Na- FREE SOLUTION
j
Fig. 9. Effect of Rb on the electrical and mechanical activity of fibers perfused in Na-free TEA (tetraethylammonium) solution. In the top strip, the panels were recorded before, during and after exposure to 1.5 mM Rb. In the lower strip, Rb (3 raM) was perfused during the time indicated in the first panel and was discontinued at the beginning of the second panel. Fibers were spontaneously active (59 beats/ min at the beginning of the top panel and 47 beats/ min at the beginning of the bottom panel). [Ca]o = 4 raM.
10
C.I. LIN, M. VASSALLE
are shown. In the first top panel, the fiber was discharging spontaneously at 59 beats/min. As usually seen under this condition (Lin and Vassalle, 1979), there was a contraction during the action potential and an aftercontraction during diastole which were of approximately the same magnitude. After contractions are seen in several tissues when the calcium load is excessive (see Ferrier, 1976). In the presence of Rb, the action potential was somewhat smaller, the initial slope of diastolic depolarization was flatter and the rate slower (48 beats/rain). Furthermore, the force of the contraction was larger (+43%) and the development of the aftercontraction slower. During the recovery in Na-free solution, the diastolic depolarization accelerated once more and the force of the contraction was smaller than that of the aftercontraction. In this fiber increasing Rb to 3 mM slowed the rate of discharge more (to 26/rain) and increased again the force of contraction. In another fiber (bottom strip) 3 mM Rb decreased the rate of discharge (47 beats/min) somewhat before abruptly suppressing it and causing hyperpolarization. The effect was reversible, as shown in the second bottom panel. The decrease in the amplitude of the action potential was minimal before the abrupt quiescence.
\ \+
3.10. The effect o f high calcium in the presence o f rubidium The depressant effect of rubidium on the force of contraction might have involved several mechanisms. One possibility is that Rb antagonizes the calcium entry. To gain some information on this point, experiments were carried out to find out how the positive inotropic effect of high calcium is affected by rubidium. In fig. 10, strip A shows the action potentials and the contractile force in Tyrode solution (first panel), in the presence of Rb (second panel), of Rb plus high calcium (third panel) and of rubidium alone (fourth panel). Strip B shows a slow speed recording of the positive inotropic effect of calcium in the
~ms
\
\
60sec
I
I_
II
I
I
c
I
'
I
Fig. 10. Inotropic effect of calcium in the presence and in the absence of Rb. In strip A, the first panel was recorded in Tyrode, the second in the presence of Rb, The third in the presence of Rb and high calcium and the fourth in the presence of Rb. Strip B is a record of mechanical activity in the presence of Rb and high Ca and strip C in the presence of high Ca. Preparation driven at 60/rain.
presence of rubidium. And strip C shows the recording of the positive inotropic effect of calcium in Tyrode solution. As shown in strip A, Rb had the usual effect on the electrical and mechanical events. When calcium was increased from 2.7 to 5.4 mM in the presence of Rb, the force increased considerably (+270%) and the effect was reversible. The positive inotropic effect of high Ca in the absence of Rb was about the same (+207%, strip C) but the peak effect was attained sooner in the absence than in the presence of rubidium. In 2 additional experiments high (5.4 mM) Ca increased force by 120.3% in the absence and by 123.3% in the presence of Rb.
3.11. The effect o f rubidium in ventricular muscle fibers Since it has been reported (Ku et al., 1974, 1975) that in atrial muscle rubidium increases force (at least under certain conditions), Rb and K were tested also on ventricular muscle fibers. In fig. 11, strip A shows the effect of increasing K to 5.4 mM, strip B the effect of
K* A N D Rb* O N S T R O P H A N T H I D I N TOXICITY
L
B
K 5.4 mM
..J
11
Imln
I ~.
Rb 2.'7 mM
I
Rb 5.4 mM
..[
]
Fig. 11. Effect of high K and Rb on mechanical activity of ventricular muscle fibers. In strips A, B and C, a ventricular muscle was exposed to 5.4 mM K, 2.7 mM Rb and 5.4 mM Rb, respectively. Preparation was driven at 60/rain.
adding 2.7 mM Rb and strip C the effect of adding 5.4 mM Rb to the Tyrode solution. In all these instances, the force fell, although to a smaller extent than in Purkinje fibers. In 4 experiments, 2.7 mM Rb decreased the contractile force of ventricular muscle by 14 + 3% (P < 0.05). There was another reason to test potassium and rubidium in ventricular muscle fibers. In contrast to Purkinje fibers (Lin and Vassalle, 1978a; Vassalle and Lin, 1979), strophanthidin causes contracture in ventricular muscle (see Lee and Klaus, 1971). In fig. 12, the contracture developed on exposure to 10 -4 M strophanthidin is shown in the top and in the bottom strips. When K was increased to 5.4 mM (top strip) or 2.7 mM Rb
I
STROPH. I0-~ M
[
STROPK I O - 6 M
.............
i
Fig. 12. Effect of high K and Rb on contracture by strophanthidin in ventricular muscle fibers. The preparation was driven at 60/rain and allowed to develop contracture. In the top strip, second panel, K was doubled to 5.4 mM; in the b o t t o m strip, second panel, 2.7 mM Rb was added to the s o l u t i o n . Rate of drive was 60/min. induced
(bottom strip) was added to the solution, the resting force decreased markedly in both cases. Since contracture is agreed to reflect an increase in [Ca]i, the experiment strongly supports the concept that K and Rb decrease the calcium overload induced by strophanthidin.
4. Discussion The present results show that in Purkinje fibers: (1) K decreases the force of contraction; (2) Rb has similar although less pronounced effects; (3) both K and Rb antagonize strophanthidin toxicity and increase force in strophanthidin intoxicated fibers; (4) both ions induce a force rebound in fibers previously exposed to low [K]o; (5) Rb decreases the rate of discharge and increases the force of contraction in fibers perfused in a sodium-free solution; (6) Rb does not prevent or antagonize the inotropic effect of either strophanthidin or high calcium; (7) both K and Rb decrease the force of contraction more than in ventricular muscle fibers and antagonize the strophanthidin-induced contracture in muscle fibers. These results suggest the conclusion that the mechanism of the antiarrhythmic action of K and Rb includes a decrease in the' calcium overload induced by strophanthidin and that this same mechanism is responsible for the relaxation of contracture in ventricular muscle fibers. The antiarrhythmic action of potassium on digitalis toxicity is likely to involve several mechanisms in addition to those studied in the present report. One mechanism is the reduction of the amount of cardiac glycosides bound to cardiac cells. This is shown by the findings that digoxin content of heart muscle decreases when serum potassium increases (Ebert et al., 1963) and that the uptake of 3H-ouabain in guinea-pig hearts is inversely related to potassium concentration (Dutta and Marks, 1969). Also, the myocardial accumulation of 3H
12 animals with potassium deficiency (Cohn et al., 1967). A second mechanism which may be of importance in the abolition of the arrhythmias is a stimulation of Na÷,K÷-ATPase by potassium. Such a stimulation (Skou, 1957) would result in hyperpolarization by restoring the ionic gradients and (if the sodium pumping is electrogenic) by increasing the net outward current. Some effects of rubidium reported in the literature seem at variance with a role of Na÷,K÷-ATPase activation in terminating the arrhythmia since K and Rb antagonize digitalis-induced arrhythmias both in vivo (Osman et al., 1976) and in vitro (figs. 4, 5, 6 and 7), while having an opposite effect on the activity of Na÷,K÷-ATPase (Ku et al., 1974, 1975). One explanation for this discrepancy between actions on arrhythmias and on Na ÷, K÷-ATPase could be that other effects of K and Rb are more important in antagonizing the arrhythmias than whatever effect these ions have on Na÷,K÷-ATPase. However, an alternative explanation is that the apparent discrepancy between K and Rb is due only to different experimental conditions. Rubidium is an effective substitute for potassium in activating the Na÷,K÷-ATPase (Skou, 1960; Post et al., 1972; Tobin et al., 1974), but, in a medium containing high (5.8 m M ) K, Rb (2-5 m M ) inhibits cardiac ATPase activity (Ku et al., 1974, 1975). In the presence of high K, the activity of the enzyme is already maximal as adding 2--5 mM K failed to alter the Na ÷, K+-ATPase activity (Ku et al., 1975). Rubidium has a higher affinity for the enzyme than K has, but slows down the turnover of the enzyme by the formation of a relatively stable bond with the dephospho-enzyme which hinders its rapid rephophorylation (Post et al., 1972; Tobin et al., 1974). It is possible therefore that Rb may inhibit the Na÷,K÷-ATPase when it is fully activated in the presence of high K, but may stimulate the activity at the usual [K]o. At 2.7 mM K, the ATPase may n o t be fully activated and adding Rb may increase the activity of the enzyme (but less than adding an equivalent amount of K due to
C.I.LIN, M. VASSALLE the slower turnover). When the activation is already maximal in the presence of high (5.8 mM) K, only the inhibitory action of Rb would become manifest, as suggested by the findings of Ku et al. (1975). In a K-free solution, Rb can substitute for K but irreversible deterioration develops in about 1 h in Purkinje fibers (Miiller, 1965), possibly in relation to the hindered rephophorylation of the enzyme. It is true that Rb increased the force of contraction in atrial tissues (Ku et al., 1974, 1975) and decreased it in both Purkinje (fig. 2) and ventricular muscle fibers (fig. 12). However, this discrepancy may be only apparent since the [K]o in the atrial strips experiments was 5.8 mM (Na÷,K÷-ATPase is already maximally stimulated) and in the present experiments was 2.7 mM. It should be noted that ~f a stimulation of the Na÷,K÷-ATPase activity by K and Rb is likely to contribute to the removal of strophanthidin toxicity, the rapid increase in membrane potential induced by potassium and rubidium in intoxicated fibers is likely to reflect an increase in potassium conductance (see below) as such hyperpolarization was induced by Rb even in the absence of Na (fig. 9). The present experiments suggest that two additional mechanisms are of importance in the antiarrhythmic action of both potassium and rubidium. One is the increase in membrane conductance to potassium. An increase in[K]o from 2.7 to 5.4 mM halves the resting membrane resistance (VassaUe, 1965, 1966) and increases the movements of radioactive potassium (Carmeliet, 1961). An increase in potassium conductance flattens diastolic depolarization and brings about the cessation of spontaneous discharge (Vassalle, 1965). In the presence of cardiac glycosides, the diastolic depolarization is also flattened by K (Vassalle, Karis and Hoffman, 1962; see fig. 12 in Vassalle and Musso, 1976; and fig. 7 in Vassalle, 1977) and spontaneous activity reduced or suppressed. In fact, in the late stage of toxicity high K hyperpolarizes the fibers (Anderson et al., 1976; fig. 6 in the present experiments). And Emax of the fibers
K+ AND Rb ÷ ON STROPHANTHIDIN
TOXICITY
intoxicated by strophanthidin is more negative in the presence of high K (fig. 4). Rubidiu m has been shown to increase potassium conductance (Miiller, 1965) and in the present experiments induced changes similar to those induced by potassium (e.g. see effects on spontaneous discharge, figs. 3 and 9). The other mechanism by which potassium and rubidium appear to specifically antagonize digitalis arrhythmias is an interference with calcium metabolism whereby the calciu m overload induced by glycosides is reduced. The calcium overload induced by cardiac glycosides results in a decrease in force of contraction, as several lines of evidence support (Lin and Vassalle, 1978a; Vassalle and Lin, 1979}. The existence of calcium overload in the present experiment is indicated by: (1) the decline in force and afterdepolarizations during the late stage of strophanthidin exposure (figs. 4 and 6); (2) the fall in force during low K perfusion (fig. 8); (3) the aftercontractions in the Na-free calcium solution (fig. 9); and (4) the contracture in ventricular muscle fibers exposed to strophanthidin (fig. 12). In these situations, there was force develo p m e n t or aftercontractions during diastole and this is generally attributed to an excessive calcium load. In all of these situations, potassium or rubidium increased the force of contraction or decreased the diastolic force (figs. 6, 7, 8, 9 and 12) as one would expect. Other events also show that the calcium overload was decreased by K and Rb. Thus, when strophanthidin was given in the presence of higher K or Rb there were no obvious oscillations in diastole and aftercontractions were absent (figs. 6 and 7). The conclusion that both K and Rb decrease the calcium overload in the presence of toxic concentrations of cardiac glycosides is strengthened by the finding that both ions decrease calcium in cardiac tissues of animals treated with cardiac glycosides (Osman et al., 1976). That indeed K and Rb can decrease the cellular calcium released in systole is shown by the fact that in the absence of overload both ions decreased (instead of increasing) the
13
force of contraction (figs. 1, 2 and 11), and this occurred also in the therapeutic stage of strophanthidin administration (fig. 6). In sheep Purkinje fibers, a reduction of [K]o 5.4 to 4.05 mM increases the force of contraction (as a low dose of ouabain does) and an increase of K to 6.75 mM induces 'aftercontractions' (as a high dose of ouabain does) (Blood, 1975). In the present experiments the force decreased when [K]o was increased from 2.7 to 5.4 mM (fig. 1) and increased when K was decreased to 0.54 mM (fig. 8). The force decreased also in ventricular muscle with higher K (fig. 11) as reported by Cohn et al. (1967), but far less than in Purkinje fibers (compare figs. 1 and 11). These results and those with Rb (figs. 2 and 11) suggest that these two ions interfere with calcium metabolism in reducing contractile force in the absence of or in the therapeutic stage of cardiac glycosides and in increasing force when the calcium load is excessive. One mechanism by which force is changed could be the reduction of the slow inward current by these two ions as this current is found to increase when [K]o is decreased, indicating a degree of K-Ca antagonism (Goto et al., 1978). The fact that force increased when calcium was increased in the presence of rubidium (fig. 10) shows that calcium can antagonize the effect of rubidium, as rubidium antagonizes the effect of calcium, but that there is no block of the slow channel. The reduction in the rate of discharge by K and Rb during strophanthidin toxicity should also result in a decreased binding of the strophanthidin. This beneficial effect would be expected on the basis of the finding that the binding of cardiac glycosides to tissues is less at lower rates ( B e n t f e l d e t al., 1977). In conclusion, it appears both K and Rb (figs. 3 and 9) increase K conductance and this is important in reducing spontaneous discharge in the absence and in the presence of cardiac glycosides. The evidence that these two ions reduce the calcium overload in the presence of strophanthidin points to a new antiarrhythmic mechanism which appears to be of a specific nature.
14 References
Anderson, G.J., F.C. Bailey, J. Reiser and A. Freeman, 1976, Electrophysiological observations on the digitalis-potassium interaction in canine Purkinje fibers,Circulation Res. 39,717. Aronson, R.S. and P.F. Cranefield, 1973, The electrical activity of canine cardiac Purkinje fibers in sodium-free, calcium-rich solutions, J. Gen. Physiol. 61,786. Bentfeld, M., H, Liillmann, T. Peters and D. Proppe, 1977, Interdependence of ion transport and the action of ouabain in heart muscle, Br. J. Pharmacol. 61, 19. Blood, B.E., 1975, The influence of low doses of ouabain and potassium ions on sheep Purkinje fibres contractility,J. Physiol. (London) 251, 69P. Carmeliet, E.E., 1961, Chloride and Potassium Permeability in Cardiac Purkinje Fibres (Arscia S.A. Presses Acad~miques Eurol~ennes, BruxeUes). Cohn, K.E., R.E. Kleiger and D.C. Harrison, 1967, Influence of potassium depletion on myocardial concentration of tritiateddigoxin, Circulation Res. 20, 473. Dutta, S. and B.H. Marks, 1969, Factors that regulate ouabain-H 3 accumulation by the isolated guineapig heart, J. Pharmacol. Exp. Ther. 170, 318. Ebert, P.A., L.J. Greenfield and W.C. Austen, 1963, The effect of increased serum potassium on the digoxin content of the canine heart, Bull. John Hopkins Hosp. 112, 151. Ferrier, G.R., 1976, The effects of tension on acetylstrophanthidin-induced transient depolarization and aftercontractions in canine myocardial and Purkinje tissues,Circulation Res. 38, 156. Ferrier, G.R., 1977, Digitalis arrhythmias: role of oscillatory afterpotentials,Progr. Cardiovasc. Dis. 19, 459. Ferrier, G.R. and G.K. Moe, 1973, Effect of calcium on acetylstrophanthidin-induced transient depolarizations in canine Purkinje tissue, Circulation Res. 33,508. Goto, M., A. Yatani and Y. Tsuda, 1978, Membrane calcium current in cardiac excitation: effects of ATP and related substances and sodium pump on bullfrog atrium, in: Recent Advances in Studies on Cardiac Structure and Metabolism, Vol. 11, Heart Function and Metabolism, eds. T. Kobayashi, T. Sano and N.S. Dhalla (University Park Press, Baltimore) pg. 3.7. Ku, D., T. Akara, T. Tobin and T.M. Brody, 1974, Effects of rubidium on cardiac tissue: inhibition of Na+,K÷-ATPase and stimulation of contractile force, Res. Commun. Chem. Pathol. Pharmacol. 9, 431. Ku, D., T. Akera, T. Tobin and T.M. Brody, 1975, Effects of monovalent cations on cardiac Na ÷,
C.I. LIN, M. VASSALLE
K*-ATPase activity and on contractile force. Nannyn-Schmiedeb. Arch. Pharmacol. 290, 113. Lee, K.S. and W. Klaus, 1971, The subcellular basis for the mechanism of inotropic action of cardiac glycosides, Pharmacol. Rev. 23, 193. Lin, C.I. and M. Vassalle, 1978a, Role of sodium in strophanthidin toxicity of Purkinje fibers,Am. J. Physiol. 234, H477. Lin, C.I. and M. Vassalle, 1978b, Rubidium effects in the presence and in the absence of strophanthidin in cardiac Purkinje fibers, Bull. N.Y. Acad. Med. 54,322 (abstract). Lin, C.I. and M. Vassalle,1979, Sodium lack prevents strophanthidin toxicity in cardiac Purkinje fibers, Cardiology 64,110. Miiller, P., 1965, Potassium and rubidium exchange across the surface membrane of cardiac Purkinje fibres,J. Physiol. (London) 177,453. Osman, F.H., E.M. Ammar, A.M. Afifi and N.M. Ahmed, 1976, Potentiation by lithium and protection by rubidium of digitalisintoxication, Jap.J. Exp. Med. 46, 1. Post, R.L., G. Hegyvary and S. Kume, 1972, Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium in transport adenosine triphosphatase, J. Biol. Chem. 247, 6530. Sampson, J.J., E.C. Albertson and B. Kondo, 1943, The effect on man of potassium administration in relation to digitalis glycosides, with special reference to blood serum potassium, the electrocardiogram, and ectopic beats, Am. Heart J. 26, 164. Skou, J.C., 1957, The influence of some cations on an adenosinetriphosphatase from peripheral nerves, Biochim. Biophys. Acta 23,394. Skou, J.C., 1960, Further investigation on a Mg÷÷+ Na÷-activated adenosinetriphosphatase, possibly related to the active linked transport of Na ÷ and K ÷ across the nerve membrane, Biochim. Biophys. Acta 42, 6. Skou, J.C., 1965, Enzymatic basis for active transport of Na ÷ and K ÷ across cell membrane, Physiol. Rev. 45, 596. Tobin, T., T. Akera, C.S. Hart and T.M. Brody, 1974, Lithium and rubidium interactions with sodiumand potassium-dependent adenosine triphosphatase: a molecular basis for the pharmacological actions of these ions, Mol. Pharmacol. 10, 501. Vassalle, M., 1965, Cardiac pacemaker potential at different extra- and intracellular K concentrations, Am. J. Physiol. 208,770. Vassalle, M., 1966, Analysis of cardiac pacemaker potential using a 'voltage clamp' technique' Am. J. Physiol. 210, 1335. Vassalle, M., 1977, Generation and conduction of impulses in the heart under physiological and pathological conditions, Pharmacol. Ther. B. 3, 1.
K ÷ AND
Rb ÷ ON STROPHANTHIDIN
TOXICITY
Vassalle, M. and K. Greenspan0 1963, Effects of potassium on ouabain-induced arrhythmias, Am. J. Cardiol. 12, 692. Vassalle, M., J. Karis and B.F. Hoffman, 1962, Toxic effects of ouabain on Purkinje fibers and ventricular muscle fibers, Am. J. Physiol. 203,433. Vassalle, M. and C.I. Lin, 1979, Effect of calcium on strophantnldin-induced electrical and mechanical
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