J. Physiol. (1976), 260, pp. 75-103
75
With 16 text-figure. Printed in Great Britain
AN ANALYSIS OF THE ACTIONS OF LOW CONCENTRATIONS OF OUABAIN ON MEMBRANE CURRENTS IN PURKINJE FIBRES
BY IRA COHEN, J(VRGEN DAUT AND DENTS NOBLE From the University Laboratory of Physiology, Parks Road, Oxford
(Received 26 November 1975) SUMMARY
1. The influence of low concentrations (5 x 10-8 to 5 x 10-7 M) of ouabain on the K gradient in sheep cardiac Purkinje fibres was observed by measuring changes in the reversal potential for a K specific current, iKs, and by measuring total steady-state current-voltage relations. 2. Provided that the bathing solution K concentration, [K]o was not too low, these doses of ouabain were often observed to increase the K gradient, i.e. the reversal potential was shifted in a negative direction. 3. The change in the reversal potential and in the current-voltage relation could be mimicked by reducing the value of [K]o in the absence of ouabain. It is therefore suggested that ouabain may stimulate the NabK+ exchange pump and so reduce the K concentration, [K]e, in the clefts of the preparation. 4. At sufficiently low values of [K]0, a dose of ouabain that was stimulatory may become inhibitory. The reversal potential for i~K then shifts in a positive direction. 5. During either stimulation or inhibition, the speed of change of reversal potential is consistent with a change in [K]e, which may change fairly rapidly. It is not possible to account for the results solely by changes in intracellular concentration, [K]1. 6. Low concentrations of ouabain were found to have no effect on the activation curve, 8, (Em), controlling iK,. It is concluded that the changes in iKsare solely attributable to changes in reversal potential. 7. Since net stimulation of the Na+-K+ exchange pump was observed to occur at doses of ouabain that exert a strong positive inotropic action on Purkinje fibres (Blood, 1975), it is not likely that the inotropic action is causally related to net pump inhibition.
76
I. COHEN, J. DA UT AND D. NOBLE INTRODUCTION
The cardiac glycosides are used in a variety of clinical conditions to improve the performance of the heart (see, for example, Smith & Haber, 1974). The desired therapeutic actions are to increase the strength of the beat and to suppress certain disturbances of rhythm. However, at dose levels that are not much larger than the therapeutic level, the glycosides may themselves induce arrhythmias (see, for example, Scherf & Schott, 1973, chapter 12). The therapeutic and toxic doses vary between individuals and depend so greatly on a variety of factors, such as plasma Ca2+ or K+ concentration, plasma acidity and anoxia, that a dose that is therapeutic under some conditions may easily prove toxic under other conditions. It is therefore important to study the influence of these factors on glycoside action. One of the aims of this paper is to investigate the way in which ouabain action on membrane currents in Purkinje fibres is dependent on the extracellular K concentration. The mechanism of the therapeutic action of ouabain is not yet known. One possibility that has been suggested recently (see Baker, Blaustein, Hodgkin & Steinhardt, 1969; Langer, 1970; Langer & Serena 1970) is that the therapeutic action may arise as a secondary consequence of inhibition of the sodium pump. According to this theory, enhanced intracellular Na following inhibition may allow Ca ions to accumulate intracellularly via an action on the Na+-Ca2+ exchange mechanism. A positive inotropic action, attributable to greater availability of intracellular Ca, should therefore always be linked to a rise in intracellular Na and a fall in intracellular K. In studies using radioactive tracers to measure changes in the distribution of ions following application of cardiac glycosides some workers have indeed found the results expected from inhibition of the Na-K exchange pump, namely an increase in intracellular Na (e.g. Langer & Serena, 1970) and a loss of intracellular potassium (e.g. Muller, 1965; for further references see Lee & Klaus, 1971). However, other work on the K content of cardiac muscle does not confirm these results and there are indications that a therapeutic effect may even be accompanied by an increase in intracellular K (Hagen, 1939; Boyer & Poindexter, 1940; Godfraind, 1973; Godfraind & Ghysel-Burton, 1976) or a retardation of K+ loss (Holland, Grieg & Dunn, 1954; Tuttle, Wit & Farah, 1961; Brown, Acheson & Grupp, 1970; for further references see Lee & Klaus, 1971). Another finding that suggested a link between inhibition of the pump and the positive inotropic effect was the parallelism found between the sensitivity of Na/K-ATPase preparations to cardiac glycosides and the magnitude of the positive inotropic effect in various animal species
77 ACTIONS OF OUABAIN ON PURKINJE FIBRES (Repke, 1965; Portius & Repke, 1962). In addition, a parallelism between
the degree of inhibition of ATPase and the magnitude of the positive inotropic effect produced by various doses of cardiac glycoside was found (Besch, Allen, Glick & Schwartz, 1970; Akera, Larsen & Brody, 1970). Once again, however, there is no consensus of opinion on this question since other experimenters have found a clear dissociation of the positive inotropic effect and inhibition of Na/K-ATPase (Okita, Richardson & Roth-Schechter, 1973; Peters, Raben & Wasserman, 1974). They found that the onset and the washout of the positive inotropic effect was much faster than the onset and especially the very slow washout of enzyme inhibition by cardiac glycosides. Moreover, stimulation rather than inhibition of the Na/K-ATPase by therapeutic concentrations of ouabain has been found by various groups (Okita et al. 1973; Peters et al. 1974) which argues against the view that blockage of the Na-K pump is causally related to the increase in strength of contraction. The use of the voltage-clamp method to measure ionic currents in cardiac muscle has provided a new way of tackling the problem. Purkinje fibres are particularly useful in this case for there is evidence that the reversal potential for the pace-maker current, iK,2 is at or close to the K equilibrium potential, EK (Noble & Tsien, 1968; Peper & Trautwein, 1969). Changes in the value of EK, and changes in the shape of the membrane current-voltage relations, may be used to detect changes in the K gradient across the cell membrane. Although in a preceding paper (Cohen, Daut & Noble, 1976) we have shown that the use of the 'K2 reversal potential to estimate EK is more complex than previously suspected, we have found consistent changes under the influence of ouabain which depended on the K concentration in the medium and on the dose of ouabain used. Several other groups have used the voltage clamp technique to study the actions of cardiotonic steroids (Aronson, Gelles & Hoffman, 1973; Isenberg & Trautwein, 1974; Lederer & Tsien, 1975). With the exception of Aronson et al. (1973) these studies have used concentrations of cardiotonic drug that are known to produce marked inhibition of the pump, e.g. 106 M ouabain. Similar doses were also used in the radioactive tracer studies of Langer & Serena (1970) and in the biochemical studies of Besch et al. (1970). Such concentrations are invariably associated with toxic effects (Sanyal & Saunders, 1961; Tuttle et al. 1961; Farah & Wit, 1963). Therapeutic doses of ouabain, on the other hand, produce plasma concentrations in the range 2-10-8 to 5 x 10-7 M (Smith & Haber, 1974). We therefore decided to investigate the effects of low concentrations of ouabain with the voltage-clamp technique. We found that doses in the therapeutic range can produce changes in the K+ gradient that reflect
78 I. COHEN, J. DA UT AND D. NOBLE stimulation of the Na+-K+ exchange pump, whereas 'toxic' effects such as membrane depolarization, potential oscillations and spontaneous activity were invariably associated with inhibition of the pump. We have not analysed the 'toxic' actions in this paper since we have concentrated primarily on understanding the effects of relatively low ouabain doses. Some of this work has appeared in abstract form (Cohen, Daut and Noble, 1975). -74
-82 mV 0 25
PA
-84
-86
10 sec
Fig. 1. Membrane currents recorded during voltage clamp pulses from the resting potential (-78 mV) to the potentials indicated. The fibre was placed in Tyrode solution containing 2-7 mM-K+. METHODS The methods were the same as in the preceding paper (Cohen et at. 1976). Ouabain stock solutions were prepared by dissolving 250 mg ouabain (Sigma, London) in 3 ml. methanol and diluting it with 4 mM-K+ Tyrode to 1 x 104 or 1 x 10-5M ouabain. These stock solutions were then further diluted with the appropriate Tyrode solution to give final concentrations of ouabain between 5 x 10-8 and 5X 10-7M. In most of the experiments described in this paper we have measured steadystate currents over a wide range of membrane potentials. Some records from one of our experiments are shown in Fig. 1. It can be seen that it may require up to 10 see for steady-state currents to be achieved and that, since recovery is also quite slow, it is necessary to allow 20 see or more between pulses. The time taken to construct a complete current-voltage diagram is therefore several minutes. It is nevertheless important to use steady-state conditions since experiments using much shorter pulses of constant duration may not be used to distinguish between effects on total membrane current and effects on the kinetics of current change. Where it is necessary to observe effects on the reversal potential for iK2 over a relatively short time scale, we have simply used short pulses near Ere, without
ACTTOATS OF OUABAIN ON PURKINJE FIBRES
79
constructing full current-voltage diagrams in every case. For similar reasons, the estimates of Er,, are usually more accurate than the steady-state current-voltage diagrams suggest since we usually used a fine grid of short pulses to determine Er., to within 1 or 2 mV (cf. Cohen et al. 1976, fig. 2). Such pulses were not used to plot points on the current-voltage diagram unless it was clear that a steady-state current level had been achieved. RESULTS
Changes in the steady-state current-voltage diagram produced by ouabain may arise from several possible actions: (a) changes in electrogenic pump current; (b) changes in ion equilibrium potentials; (c) changes in ionic conductances; and (d) changes in conductance kinetics. The changes we have observed may involve (a), as described also by Isenberg & Trautwein (1974). We have also observed substantial changes in the K equilibrium potential and there may be consequential changes in ionic conductance. The over-all effects are therefore quite complex particularly since the direction of an effect may also vary as a function of ion and drug concentrations. Our initial results were therefore confusing and difficult to interpret. We found that a key factor in interpreting the results was to determine the effect of extracellular K+ concentration on the iZK reversal potential and steady-state current-voltage diagrams in the same experiment as the effects of ouabain. This was found to be important for two major reasons. First, as we have shown in a preceding paper (Cohen et al. 1976) the influence of [K]0 on the reversal potential for iK2 is not necessarily well predicted by the Nernst equation since the immediate extracellular K+ concentration, [K]e, is rarely equal to [K]0, on the bathing solution concentration. Secondly, the influence of extracellular K± on the current-voltage diagram is somewhat variable from a quantitative point of view. A decrease in [K]o reduces the K+ conductance to depolarizing currents (Carmeliet, 1961; Hall & Noble, 1963). If this effect is large enough, the current-voltage diagrams may cross each other (see Noble, 1965). This is frequently, but not always, the case. Similar results have been obtained in the present experiments. In all cases, reduced [K]o decreases the conductance to depolarizing currents, sometimes giving rise to a cross-over effect (see Figs. 7 and 8) and sometimes just failing to do so (see Fig. 2). In order to interpret our results using ouabain in terms of changes in ionic gradients we therefore found it essential to first measure the effect of [K]o alone in each experiment.
Influence of extracellular K+ in absence of ouabain Fig. 2 shows the result of an experiment in which the effects of 4, 5.4 and 8 mM-[K]o were compared. The effects of ouabain at these various [K]o values in this experiment will be presented in Figs. 3-6.
I. COHEN, J. DA UT AND D. NOBLE As expected, the reversal potential for iKs changes substantially in a positive direction as [K]0 is increased. It is also clear that, in this experiment, the cross-over effect is virtually absent. Instead, the steady-state curves approach a similar current value at about -40 mV. The values of current at which Erev for i~K occur are of some importance since they enable us to determine whether [K]0 influences currents other than iK. The total steady-state membrane current, ii, is given by
80
ti
=
(1)
iK+iinB+ip,
where iINB is the background inward current responsible for maintaining the resting potential positive to EK and ip is any current carried by the Na-K exchange pump. At Erev, iK is zero and we have (iinB)Br + Yp)Rrevto Hence the current required hyperpolarize the membrane to net current carried by $INB and ip at that potential.
ii (Erev)
(2)
=
Erev is the
+3 -+1
mV -100
/
-50
-1 -3
//
75
With 16 text-figure. Printed in Great Britain
AN ANALYSIS OF THE ACTIONS OF LOW CONCENTRATIONS OF OUABAIN ON MEMBRANE CURRENTS IN PURKINJE FIBRES
BY IRA COHEN, J(VRGEN DAUT AND DENTS NOBLE From the University Laboratory of Physiology, Parks Road, Oxford
(Received 26 November 1975) SUMMARY
1. The influence of low concentrations (5 x 10-8 to 5 x 10-7 M) of ouabain on the K gradient in sheep cardiac Purkinje fibres was observed by measuring changes in the reversal potential for a K specific current, iKs, and by measuring total steady-state current-voltage relations. 2. Provided that the bathing solution K concentration, [K]o was not too low, these doses of ouabain were often observed to increase the K gradient, i.e. the reversal potential was shifted in a negative direction. 3. The change in the reversal potential and in the current-voltage relation could be mimicked by reducing the value of [K]o in the absence of ouabain. It is therefore suggested that ouabain may stimulate the NabK+ exchange pump and so reduce the K concentration, [K]e, in the clefts of the preparation. 4. At sufficiently low values of [K]0, a dose of ouabain that was stimulatory may become inhibitory. The reversal potential for i~K then shifts in a positive direction. 5. During either stimulation or inhibition, the speed of change of reversal potential is consistent with a change in [K]e, which may change fairly rapidly. It is not possible to account for the results solely by changes in intracellular concentration, [K]1. 6. Low concentrations of ouabain were found to have no effect on the activation curve, 8, (Em), controlling iK,. It is concluded that the changes in iKsare solely attributable to changes in reversal potential. 7. Since net stimulation of the Na+-K+ exchange pump was observed to occur at doses of ouabain that exert a strong positive inotropic action on Purkinje fibres (Blood, 1975), it is not likely that the inotropic action is causally related to net pump inhibition.
76
I. COHEN, J. DA UT AND D. NOBLE INTRODUCTION
The cardiac glycosides are used in a variety of clinical conditions to improve the performance of the heart (see, for example, Smith & Haber, 1974). The desired therapeutic actions are to increase the strength of the beat and to suppress certain disturbances of rhythm. However, at dose levels that are not much larger than the therapeutic level, the glycosides may themselves induce arrhythmias (see, for example, Scherf & Schott, 1973, chapter 12). The therapeutic and toxic doses vary between individuals and depend so greatly on a variety of factors, such as plasma Ca2+ or K+ concentration, plasma acidity and anoxia, that a dose that is therapeutic under some conditions may easily prove toxic under other conditions. It is therefore important to study the influence of these factors on glycoside action. One of the aims of this paper is to investigate the way in which ouabain action on membrane currents in Purkinje fibres is dependent on the extracellular K concentration. The mechanism of the therapeutic action of ouabain is not yet known. One possibility that has been suggested recently (see Baker, Blaustein, Hodgkin & Steinhardt, 1969; Langer, 1970; Langer & Serena 1970) is that the therapeutic action may arise as a secondary consequence of inhibition of the sodium pump. According to this theory, enhanced intracellular Na following inhibition may allow Ca ions to accumulate intracellularly via an action on the Na+-Ca2+ exchange mechanism. A positive inotropic action, attributable to greater availability of intracellular Ca, should therefore always be linked to a rise in intracellular Na and a fall in intracellular K. In studies using radioactive tracers to measure changes in the distribution of ions following application of cardiac glycosides some workers have indeed found the results expected from inhibition of the Na-K exchange pump, namely an increase in intracellular Na (e.g. Langer & Serena, 1970) and a loss of intracellular potassium (e.g. Muller, 1965; for further references see Lee & Klaus, 1971). However, other work on the K content of cardiac muscle does not confirm these results and there are indications that a therapeutic effect may even be accompanied by an increase in intracellular K (Hagen, 1939; Boyer & Poindexter, 1940; Godfraind, 1973; Godfraind & Ghysel-Burton, 1976) or a retardation of K+ loss (Holland, Grieg & Dunn, 1954; Tuttle, Wit & Farah, 1961; Brown, Acheson & Grupp, 1970; for further references see Lee & Klaus, 1971). Another finding that suggested a link between inhibition of the pump and the positive inotropic effect was the parallelism found between the sensitivity of Na/K-ATPase preparations to cardiac glycosides and the magnitude of the positive inotropic effect in various animal species
77 ACTIONS OF OUABAIN ON PURKINJE FIBRES (Repke, 1965; Portius & Repke, 1962). In addition, a parallelism between
the degree of inhibition of ATPase and the magnitude of the positive inotropic effect produced by various doses of cardiac glycoside was found (Besch, Allen, Glick & Schwartz, 1970; Akera, Larsen & Brody, 1970). Once again, however, there is no consensus of opinion on this question since other experimenters have found a clear dissociation of the positive inotropic effect and inhibition of Na/K-ATPase (Okita, Richardson & Roth-Schechter, 1973; Peters, Raben & Wasserman, 1974). They found that the onset and the washout of the positive inotropic effect was much faster than the onset and especially the very slow washout of enzyme inhibition by cardiac glycosides. Moreover, stimulation rather than inhibition of the Na/K-ATPase by therapeutic concentrations of ouabain has been found by various groups (Okita et al. 1973; Peters et al. 1974) which argues against the view that blockage of the Na-K pump is causally related to the increase in strength of contraction. The use of the voltage-clamp method to measure ionic currents in cardiac muscle has provided a new way of tackling the problem. Purkinje fibres are particularly useful in this case for there is evidence that the reversal potential for the pace-maker current, iK,2 is at or close to the K equilibrium potential, EK (Noble & Tsien, 1968; Peper & Trautwein, 1969). Changes in the value of EK, and changes in the shape of the membrane current-voltage relations, may be used to detect changes in the K gradient across the cell membrane. Although in a preceding paper (Cohen, Daut & Noble, 1976) we have shown that the use of the 'K2 reversal potential to estimate EK is more complex than previously suspected, we have found consistent changes under the influence of ouabain which depended on the K concentration in the medium and on the dose of ouabain used. Several other groups have used the voltage clamp technique to study the actions of cardiotonic steroids (Aronson, Gelles & Hoffman, 1973; Isenberg & Trautwein, 1974; Lederer & Tsien, 1975). With the exception of Aronson et al. (1973) these studies have used concentrations of cardiotonic drug that are known to produce marked inhibition of the pump, e.g. 106 M ouabain. Similar doses were also used in the radioactive tracer studies of Langer & Serena (1970) and in the biochemical studies of Besch et al. (1970). Such concentrations are invariably associated with toxic effects (Sanyal & Saunders, 1961; Tuttle et al. 1961; Farah & Wit, 1963). Therapeutic doses of ouabain, on the other hand, produce plasma concentrations in the range 2-10-8 to 5 x 10-7 M (Smith & Haber, 1974). We therefore decided to investigate the effects of low concentrations of ouabain with the voltage-clamp technique. We found that doses in the therapeutic range can produce changes in the K+ gradient that reflect
78 I. COHEN, J. DA UT AND D. NOBLE stimulation of the Na+-K+ exchange pump, whereas 'toxic' effects such as membrane depolarization, potential oscillations and spontaneous activity were invariably associated with inhibition of the pump. We have not analysed the 'toxic' actions in this paper since we have concentrated primarily on understanding the effects of relatively low ouabain doses. Some of this work has appeared in abstract form (Cohen, Daut and Noble, 1975). -74
-82 mV 0 25
PA
-84
-86
10 sec
Fig. 1. Membrane currents recorded during voltage clamp pulses from the resting potential (-78 mV) to the potentials indicated. The fibre was placed in Tyrode solution containing 2-7 mM-K+. METHODS The methods were the same as in the preceding paper (Cohen et at. 1976). Ouabain stock solutions were prepared by dissolving 250 mg ouabain (Sigma, London) in 3 ml. methanol and diluting it with 4 mM-K+ Tyrode to 1 x 104 or 1 x 10-5M ouabain. These stock solutions were then further diluted with the appropriate Tyrode solution to give final concentrations of ouabain between 5 x 10-8 and 5X 10-7M. In most of the experiments described in this paper we have measured steadystate currents over a wide range of membrane potentials. Some records from one of our experiments are shown in Fig. 1. It can be seen that it may require up to 10 see for steady-state currents to be achieved and that, since recovery is also quite slow, it is necessary to allow 20 see or more between pulses. The time taken to construct a complete current-voltage diagram is therefore several minutes. It is nevertheless important to use steady-state conditions since experiments using much shorter pulses of constant duration may not be used to distinguish between effects on total membrane current and effects on the kinetics of current change. Where it is necessary to observe effects on the reversal potential for iK2 over a relatively short time scale, we have simply used short pulses near Ere, without
ACTTOATS OF OUABAIN ON PURKINJE FIBRES
79
constructing full current-voltage diagrams in every case. For similar reasons, the estimates of Er,, are usually more accurate than the steady-state current-voltage diagrams suggest since we usually used a fine grid of short pulses to determine Er., to within 1 or 2 mV (cf. Cohen et al. 1976, fig. 2). Such pulses were not used to plot points on the current-voltage diagram unless it was clear that a steady-state current level had been achieved. RESULTS
Changes in the steady-state current-voltage diagram produced by ouabain may arise from several possible actions: (a) changes in electrogenic pump current; (b) changes in ion equilibrium potentials; (c) changes in ionic conductances; and (d) changes in conductance kinetics. The changes we have observed may involve (a), as described also by Isenberg & Trautwein (1974). We have also observed substantial changes in the K equilibrium potential and there may be consequential changes in ionic conductance. The over-all effects are therefore quite complex particularly since the direction of an effect may also vary as a function of ion and drug concentrations. Our initial results were therefore confusing and difficult to interpret. We found that a key factor in interpreting the results was to determine the effect of extracellular K+ concentration on the iZK reversal potential and steady-state current-voltage diagrams in the same experiment as the effects of ouabain. This was found to be important for two major reasons. First, as we have shown in a preceding paper (Cohen et al. 1976) the influence of [K]0 on the reversal potential for iK2 is not necessarily well predicted by the Nernst equation since the immediate extracellular K+ concentration, [K]e, is rarely equal to [K]0, on the bathing solution concentration. Secondly, the influence of extracellular K± on the current-voltage diagram is somewhat variable from a quantitative point of view. A decrease in [K]o reduces the K+ conductance to depolarizing currents (Carmeliet, 1961; Hall & Noble, 1963). If this effect is large enough, the current-voltage diagrams may cross each other (see Noble, 1965). This is frequently, but not always, the case. Similar results have been obtained in the present experiments. In all cases, reduced [K]o decreases the conductance to depolarizing currents, sometimes giving rise to a cross-over effect (see Figs. 7 and 8) and sometimes just failing to do so (see Fig. 2). In order to interpret our results using ouabain in terms of changes in ionic gradients we therefore found it essential to first measure the effect of [K]o alone in each experiment.
Influence of extracellular K+ in absence of ouabain Fig. 2 shows the result of an experiment in which the effects of 4, 5.4 and 8 mM-[K]o were compared. The effects of ouabain at these various [K]o values in this experiment will be presented in Figs. 3-6.
I. COHEN, J. DA UT AND D. NOBLE As expected, the reversal potential for iKs changes substantially in a positive direction as [K]0 is increased. It is also clear that, in this experiment, the cross-over effect is virtually absent. Instead, the steady-state curves approach a similar current value at about -40 mV. The values of current at which Erev for i~K occur are of some importance since they enable us to determine whether [K]0 influences currents other than iK. The total steady-state membrane current, ii, is given by
80
ti
=
(1)
iK+iinB+ip,
where iINB is the background inward current responsible for maintaining the resting potential positive to EK and ip is any current carried by the Na-K exchange pump. At Erev, iK is zero and we have (iinB)Br + Yp)Rrevto Hence the current required hyperpolarize the membrane to net current carried by $INB and ip at that potential.
ii (Erev)
(2)
=
Erev is the
+3 -+1
mV -100
/
-50
-1 -3
//
