(Received 3 August 1979)

Transcription

1 J. Phymiol. (1980), 303, With 9 text-figure8 Printed in Great Britain THE RELATIONSHIP BETWEEN SODIUM PUMP ACTIVITY AND TWITCH TENSION IN CARDIAC PURKINJE FIBRES BY D. A. EISNER* AND W. J. LEDERERt From the University Laboratory of Physiology, Parks Road, Oxford OX1 3PT (Received 3 August 1979) SUMMARY 1. Sheep cardiac Purkinje fibres were studied under voltage clamp conditions to examine the relationship between the Na pump rate and twitch tension. 2. The Na pump activity was measured following a period of decreased Na pump rate and increased internal Na activity, Nai, by examining the electrogenic Na pump current transient produced by reactivating the Na pump (see Eisner & Lederer, 1980). 3. Various concentrations of Cs or Rb were used to reactivate the Na pump in the absence of extracellular K, K0. Results from such experiments showed that these 'activator cations' produced a monotonic increase in Na pump rate with increasing concentration while producing monotonic decreases in steady-state twitch tension. A given concentration of Rb was more potent than the same concentration of Cs in its effects on both Na pump rate and tension. Nevertheless, varying [Rb]0 or [Cs]o produced the same relationship between Na pump activity and tension. 4. After the preparation was exposed to low K0 (below 4 mm), the Na pump was reactivated with 10 mm-rbo (0-Ko). The area under the resulting electrogenic Na pump current transient gives a measure of the increase of [Na]i that occurred when the preparation was exposed to the test solution compared to the steady-state Nai in 10 mm-rbo. Increasing the duration of exposure to low Ko, further augments the twitch tension achieved at the end of the test period and the area under the electrogenic Na pump current transient. Similarly, for equal periods of exposure, the lower the [K]o in the test solution, the greater the increase of twitch tension at the end of the test period and the greater the area under the electrogenic Na pump current transient. 5. The relationship between tension and the area under the electrogenic Na pump current transient is the same for a variable duration exposure to 0-Ko or for a constant exposure to various low K0. It is concluded that a rise in [Na]i is the rate limiting step linking Na pump activity and twitch tension. 6. In an experiment similar to the one described in (4), the fibre was exposed to a test solution containing a variable concentration of one of the activator cations of the Na pump for a fixed period. The effects of different cations were compared with the effects of a test solution containing no activator cation. Increasing the * Present address: The Physiological Laboratory, Downing Street, Cambridge CB2 3EG. t Present address: Department of Physiology, University of Maryland School of Medicine, 660 West Redwood St., Baltimore, Maryland 21201, U.S.A. Reprint requests to W.J.L. at Baltimore /80/ $ The Physiological Society

2 476 D. A. EISNER AND W. J. LEDERER concentration of a particular activator cation from zero reduced the twitch tension augmentation in the test solution and also reduced the area under the electrogenic Na pump current transient on subsequently reactivating the Na pump with 10 mm- Rbo. It is concluded that the ability of a test solution to reduce the area of the electrogenic Na pump current transient reflects the ability of the test solution to activate the Na pump. Equivalent concentrations of the activator cations that are required to activate the Na pump are: 2 mm-k0 = 2 mm-rb0 = 6 mm-cs0 = 6 mm-nh~o = 22 mm-lio. TI0 was more effective than K0. The order of these cations to reduce twitch tension is found to be the same as that to reduce the area under the electrogenic Na pump current transient. 7. We conclude that the effects of the activator cations on twitch tension are determined by their effects on the Na pump and thereby on [Na]i. INTRODUCTION Reducing the bathing K concentration, [K]o, or applying cardiotonic steroids, increases the force of contraction of cardiac muscle (Ringer, 1883; Lee & Klaus, 1971). These manoeuvres are both known to inhibit the Na pump (Schatzmann, 1953; Glynn, 1956) and one theory of the subsequent inotropic effects depends on this observation. It has been suggested that a rise of [Na]i mediates the observed positive inotropy when agents that inhibit the Na pump are used (Baker, Blaustein, Hodgkin & Steinhardt, 1969; Langer, 1970; Glitsch, Reuter & Scholz, 1970). This rise of [Na]i may then lead to an increase of Cai via the Na-Ca exchange (Reuter & Seitz, 1968). However, there is considerable disagreement regarding the importance of Na pump inhibition in the inotropic actions of either reduced [K]o or cardiotonic steroids. Reduced [K]o has been reported to produce an increase in the heart's strength of contraction that could be dissociated from its effects on [Na]i (Gadsby, Niedergerke & Page, 1971). To account for such evidence, additional explanations of the inotropic effects of low K0 have been produced. Ehara (1974a, b) suggested that low Ko may increase twitch tension by a direct effect on Ca pumping perhaps similar to the K-Ca exchange described by Morad & Goldman (1973). Additionally, Goto, Tsuda & Yatani (1977) have found that low K0 may increase the slow inward current, I~I, and may therefore increase [Ca]i directly. Most workers allow, nevertheless, for a role of Na pump blockade in the inotropic effects of low K0. The point of interest is quantitative: Does the Na pump inhibition produced by low Ko account quantitatively for the observed increase of tension? The ionic basis of the inotropic action of cardiotonic steroids is controversial because of several recent findings. Several reports suggest that 'therapeutic' concentrations of cardiotonic steroids do not inhibit the Na pump. For example electrophysiological techniques (Cohen, Daut & Noble, 1976), measurements of tissue ionic content (Godfraind & Ghysel-Burton, 1977) and direct measurements of the intracellular Na activity (Deitmer & Ellis, 1978) suggest that the Na pump may even be stimulated by these low concentrations. Other workers have found, in contrast, that tension only increases when the Na pump has been inhibited (Hougen & Smith, 1978; Hougen, Lloyd & Smith, 1979; Michael, Pitts & Schwartz, 1978). Unfortu-

3 Na PUMP AND TENSION IN CARDIAC MUSCLE 477 nately few of these studies have simultaneously measured pump rate and tension in the same preparation. Although we do not investigate the actions of cardiotonic steroids in these experiments, the methods presented in this paper may resolve the controversy since we demonstrate simultaneous and repeated measurement of Na pump rate and tension. Eisner & Lederer (1979b) examined the role of the Na pump in the development of tension. They showed that low Ko produces an increase of twitch tension that can be reversed by cations known to activate the external (K) site of the Na pump. In the previous paper (Eisner & Lederer, 1980), we demonstrated that these activator cations produce an electrogenic Na pump current transient when added after a period of exposure to 0-K0. We have also shown that this pump current can be quantified to examine the Na pump activity. In this paper we will use this current transient to quantitatively investigate the relationship between twitch tension and Na pump rate. METHODS All experiments presented here were performed on isolated sheep cardiac Purkinje fibres. The general experimental methods have been described previously (Eisner & Lederer, 1980). Tension was measured with an Akers 803E piezoresistive element as described by Eisner & Lederer (1979a). The modified Tyrode solution consisted of: 145 mm-nacl, 4 mm-kcl, 5 mm-cacl2, 1 mm-mgcl2, 10 mm-tris HCl, 10 mm-glucose. It was oxygenated with 100% 02. Modifications to this standard solution will be indicated in the text. In describing superfusing solutions, we only specify alterations to the above, standard solution. Thus a solution described as 10 mm-rbo, 0-KI( is a solution in which all external K has been removed and 10 mm-rb added. Tl, Rb, Cs, NH4 and Li were added as chlorides. The ph of all solutions was kept at ph All experiments were done between 35 and 37 C and, during a given experiment, the temperature was maintained to C. RESULTS The relationship between Na pump rate and twitch tension In the previous paper (Eisner & Lederer, 1980) we demonstrated that the Na pump can be quantitatively investigated in the sheep cardiac Purkinje fibre. Furthermore we presented a simple model that permits interpretation of these results. Four major points are particularly relevant to the present work. (1) A fixed coupling ratio was found for the countertransport of Rb into the cell and Na out (cf. Gadsby & Cranefield, 1979). (2) At a given temperature, the Na pump rate depends on the external activator cation concentration (e.g. Tl, K, Rb, Cs, NH4, Li) and on the internal Na ion activity, Nal. (3) After a fixed period of Na loading, the decay rate constant (ke) of the electrogenic Na pump current transient reflects the degree of activation of the external Na pump site by the activator cation regardless of the exact level of Nai attained before reactivating the Na pump. (4) After a period of Nai loading, the magnitude of the electrogenic Na pump current transient (Ise) and the area under the current transient (Qe) depend on the degree to which Na, is elevated if the Na pump is reactivated with a fixed concentration of a given activator cation. Therefore, Qe can be used as a measure of the previous Na loading. We have used these results in order to analyse the importance of Na pump activity

4 478 D. A. EISNER AND W. J. LEDERER in regulating twitch tension. Fig. 1 shows the time course of twitch tension and membrane current under voltage clamp control. In this experiment, performed in O-K0, the superfusing solution was changed from the control (10 mm-rb0) to O-Rb0 for 10 min. Reducing external activator cation concentration decreases the Na pump rate and leads to a rise of [Na]i (Ellis, 1977). This lowered Na pump rate is associated with an increase of twitch tension as is shown in Fig. 1. After 10 min exposure to 50- A na 0-50_L J -_ 1 mgi 10-Rbo 50 na 0O -50 _- B 2-Rb. 5 min Fig. 1. Effect of Na pump activity on Purkinje fibre membrane current and tension. Ko was maintained at zero throughout the experiment. From a control solution of 10 mm-rbo, the preparation was exposed to 0-RbO for 10 min. Following this exposure to 0-RbO, [Rb]o was increased to 10 mm (A) or to 2 mm (B) at the time indicated by the vertical dashed line. In each panel the upper trace is the membrane current recorded at the holding potential of -61 mv. The lower trace is the tension record obtained simultaneously. Depolarizing voltage clamp pulses to -40 mv of 2 sec duration were applied at 0-1 Hz to elicit a twitch. These depolarizing pulses produce current steps that are not seen since they are off scale at this gain. Experiment Core volume pl. Cylindrical area cm2. O-Rb0, the solution was changed to 10 mm-rbo. This produces a large outward electrogenic Na pump current transient. The tension record shows that, during the period of the electrogenic Na pump current transient, the twitch tension also decreases. Similarly, the tonic component of tension at the holding potential also decays. Although difficult to resolve at the slow chart speed shown, an aftercontraction is present after exposure to O-RbO (see Eisner & Lederer, 1979a, b). This also disappears on adding 10 mm-rb0. Following an interval of recovery in the

5 Na PUMP AND TENSION IN CARDIAC MUSCLE 479 A o Rb Cs C ~ C 0 CO ~~ [M] (mm) B 1-5 E 10 C 0 C F- Rb O Cs [M] (mm) Fig. 2. Effect of [Rb]0 or [Cs]. on electrogenic Na pump current transient and steadystate twitch tension. From a control solution (4 mm-ko, 0-RbO, 0-Cs0) the preparation was initially exposed to 0-K0, 0-RbO, 0-Cso for 8 min followed by exposure to a test solution containing a variable concentration of either Rb0 or Cs0. Zero KO was present in the test solutions. A, the rate constant of decay of the electrogenic Na pump current transients measured at the holding potential (-53 mv) in the test solution as a function of RbN (Q) or Cs0 ( 0). These rate constants were obtained from semilogarithmic plots of the electrogenic Na pump currents. B, steady-state twitch tension. The points were obtained after 15 min in the test solution. The twitch was elicited by a 2-sec depolarizing voltage clamp pulse to -23 mv applied at 0 l Hz. Symbols: (0) Rb.; (@) Cs.. Experiment Core volume pl. Cylindrical area cm2.

6 480 D. A. EISNER AND W. J. LEDERER control solution (10 mm-rb.), the same procedure was repeated except that the Na pump was reactivated with 2 mm-rb0 following the 10 min exposure to O-Rbo. This lower concentration of Rb0 produces a smaller electrogenic Na pump current transient than does 10 mm-rbo. Similarly, the twitch decays to a smaller steadystate value in 10 mm-rb0 than in 2 mm-rbo. Furthermore, the decay of the current transient and the decay of tonic tension are clearly slower in 2 mm-rbo than in 10 mm-rbo. The results of this experiment provide direct evidence that a greater Na pump activity is associated with a smaller steady-state twitch tension. In the experiment shown in Fig. 2, we investigated the effects of a range of concentrations of two activator cations (RbO and Cso) on the electrogenic Na pump current transient and on twitch tension. The solution superfusing the Purkinje fibre preparation was changed from a control solution of 4 mm-ko, 0-Cso, 0-RbO to a solution of O-Ko, O-Cs0, O-RbO for 8 min. Following this fixed period of exposure, a test solution which contained O-Ko and a variable concentration of either Rbo or Cs, was superfused over the preparation. This produces an electrogenic Na pump current transient, the rate constant of decay (ke) of which depends on the relative degree of activation of the Na pump at the external (K) site (Eisner & Lederer, 1980). Fig. 2A shows a graph of the rate constant of decay of the electrogenic Na pump current transient as a function of Rbo or Cso in the test solution. Increasing the concentration of either Rbo or Cso in the test solution increases the Na pump activity as indicated by the increasing values of the rate constants shown in Fig. 2A. This figure also shows that Rbo is considerably more potent than Cso in activating the electrogenic Na pump current (cf. Eisner & Lederer, 1980). Fig. 2B shows how the activator cation concentration affects the steady-state twitch tension which was measured 15 min after adding the activator cation. Increasing the concentration of either Rbo or Cs0 decreases the twitch tension. A given concentration of Rbo produces a greater reduction of twitch tension than does the same concentration of Cs.. Thus Rbo, which at an equal concentration activates the Na pump to a greater extent than does CSk, produces a greater reduction of twitch tension. The results of the experiments shown in Figs. 1 and 2 substantiate the suggestion advanced by Eisner & Lederer (1979b) that the relative effects of activator cations on tension are correlated with their effects on the Na pump rate. If the Na pump rate is really the important factor in the effects of these activator cations on twitch tension, then equal steady-state twitch tensions should be associated with equal Na pump activities regardless of the activator cation used. In Fig. 3A the twitch tension is plotted as a function of the decay rate constant of the electrogenic Na pump current transient. To a first approximation, the relationship between twitch tension and Na pump rate is the same for Cso and Rbo. We conclude from this that, as far as twitch tension is concerned, it is the Na pump rate that is important and not the actual concentrations of Rbo or Cs0. It does not matter whether a given pump rate is produced by a small concentration of Rb or a larger concentration of Cs. It appears therefore that the inotropic effects of [Cs]o and [Rb]0 are determined by their effects on the Na pump rate. Fig. 3B presents data from five experiments similar to Fig. 3A. To permit comparison between different experiments (on different fibres), the rate constants of decay of the electrogenic Na pump current transients have been normalized with respect to the

7 Na PUMP AND TENSION IN CARDIAC MUSCLE 481 A 1*5 - Rb 0 Cs c 0 w -c0o B 1-0. u i I I I 1 ) Rate constant (sec-') a I- C-0 -S 11 0F U- e ,0- ' 0-5-~~~ Rate constant relative to 10 mm-rb. Fig. 3. Dependence of steady-state twitch tension on Na pump activity. A, data from single experiment. Abscissa: rate constant of electrogenic Na pump current transient. Ordinate: twitch tension. Results taken from Fig. 2. B, histogram from pooled results of five experiments similar to that shown in A. Ordinate: fractional inotropy. This is defined for a given experiment as (P. - PI0 Rb.)/(Po- PO0 Rbo)) where P., P10 Rb and PO are respectively the value of twitch tension in the test solution, in 10 mm-rbo and in 0-Rb0, 0-Cs0, 0-K0. Abscissa: the rate constant of the electrogenic Na pump current transient divided by the value in 10 mm-rb0. Results from Rbo and Cso have been mixed and used to construct the histogram. The heights of the extreme left and right bins are defined by the above tension normalization procedure. The other bins show the average (+ s.e. of mean) of the fractional inotropy over the range of normalized pump activity covered by the particular bin. i6 PHY 303

8 482 D. A. EISNER AND W. J. LEDERER value in 10 mm-rb0 (see Fig. 16B, Eisner & Lederer, 1980). The twitch tension has been expressed as the fractional degree of inotropy. This is defined in the Figure legend and is the increase in tension above the value in the control solution (10 mm- Rbo) divided by the increase in tension in O-Ko. Results from different activator cations have been mixed. The pooled results were then used to construct a histogram. The bin on the extreme left is narrow and the value is defined by the tension normalization procedure as is the value of the bin on the extreme right. Each of the five middle bins shows the average value of the 'fractional inotropy' over the range of normalized Na pump rate that each spans. Although the variation between fibres leads to the error bars shown in the Figure, the histogram shows a clearly nonlinear but monotonic relationship between twitch tension and Na pump rate. The relationship is steepest at low values of Na pump rate. The role of Nai in determining the magnitude of twitch tension Since the Na pump rate appears to determine the level of twitch tension when other elements are kept constant, we have tried to determine the relative importance of possible intermediate steps. One consequence of Na pump blockade is a rise of [Na]1. If one were able to use some aspect of the electrogenic Na pump current as a bio-assay for [Na]i, then one would be able to correlate changes in [Na]1 with changes in twitch tension. The open circles in Fig. 4 show the time course of the increase of twitch tension on decreasing [K]0 from 4 to 0 mm for 20 min during a single run. The twitch tension increases monotonically during this period of exposure to O-K0. We then exposed the fibre to O-K0 for different periods. The open triangles show the twitch tension measured at the end of each period of exposure to 0-K0 and are paired with the solid circles. The small variations between the open circles and open triangles are presumably due to slight variations in the state of the preparation between different runs. Nevertheless, the similarity between the development of tension during the 20 min exposure and the final twitch tension reached at the end of other runs demonstrates that the inotropic effects of O-K0 are reproducible. Each exposure to 0-K0 is terminated by reactivating the Na pump with 10 mm-rbo. This Na pump reactivation produces an electrogenic Na pump current transient. The filled circles show the area under the electrogenic Na pump current transient as a function of the duration of exposure to 0-K0, O-Rb0. The area increases with longer durations of exposure to 0-K0, 0-Rbo just as tension increases. On the basis of the model presented by Eisner & Lederer (1980) (cf. Rang & Ritchie, 1968) the area under the current transient represents the net charge extruded from the intracellular compartment by the Na pump in excess of the charge extruded in the steady-state in 10 mm-rbo. Since the final conditions in 10 mm-rbo are the same, the difference in the areas as a function of the time in 0-KI, O-Rb0 represents the different amounts by which [Na]i had risen after different durations of exposure to O-K0, O-Rb0. The straight line is a least-squares fit to the area data and has a slope of 0X 17 1C min-. From the measured core volume of this particular Purkinje fibre (0-015 /4l.) and assuming a 3 Na/2 K coupling ratio for the Na pump, one can calculate an apparent rate of rise of [Na]i of 0-35 mm min-. Results from three other experiments give calculated increases

9 Na PUMP AND TENSION IN CARDIAC MUSCLE 483 of [Na]i of mm min-' (+ S.E. of mean). Ellis (1977) reports a mean rate of rise of [Na]i following KO removal of 0-3 mm min-' in the sheep Purkinje fibre. This is sufficiently close to the values estimated from these experiments for one to be able to use the area of the electrogenic Na pump current transient as a bioassay for changes in [Na]1. The open triangles in Fig. 4 show the twitch tension reached at the end of each of the periods of exposure to O-Ko, O-Rbo. Thus the open triangles give the amount of positive inotropy corresponding to a certain increase of [Na]i as demonstrated by the area data (filled circles) ; 0,~~~~~~~~~~ S 0'2510 o f I-. ~ ~ ~ ~ ~ ~ ~ ~ ~~~ I5 0~~~~~~~~~ Time in 0-K0 (min) Fig. 4. Time course of development of positive inotropy and electrogenic Na pump current during exposure to O-K0, O-Rb0. From a control solution (4 mm-k0, 0-Rb,,), the preparation was exposed to a solution containing 0-K0, 0-Rb0 for a variable period of time as shown on the abscissa. [Rb]0 was then increased to 10 mm in order to produce an electrogenic Na pump current transient. The area under the current transient obtained at a holding potential of -60 mv was measured as described by Eisner & Lederer (1980). Twitch tension was measured continuously during the course of the experiment. The twitch was elicited by a 2 see depolarizing pulse from the holding potential of -60 mv to -39 mv at 0-1 Hz. 0, the time course of the increase of twitch tension during a single 20 min exposure to O-KO, O-RbO; *0, the area under the electrogenic Na pump current transient measured at the holding potential (-60 mv) following different duration exposures to O-K0, O-RbO; A, twitch tension at the end of different duration exposures to O-KO, O-RbO obtained just prior to reactivating the Na pump with 10 mm-rbo. Experiment Core volume 0*015 /ud. Cylindrical area cm2. In order to show twitch tension as a function of [Na]i, we plot tension against the area under the Na pump current transient in Fig. 5. The open circles are points obtained from different duration exposures to O-Ko. As in the experiment illustrated in Fig. 4, the twitch tension is measured just before reactivating the Na pump while 'area' represents the area under the electrogenic Na pump current transient obtained when the Na pump is reactivated. The relationship between these parameters appears to be roughly linear although this has no simple physical significance. Given that the inotropic effects of Na pump inhibition are mediated by an increase of [Na]i (e.g. Baker et al. 1969), one can formulate a sequence of events in which there are i6-2

10 484 D. A. EISNER AND W. J. LEDERER two possible rate limiting steps. (1) Na pump blockade leads to a rise of [Na]1. (2) The increased [Na]1 produces an increased [Ca]1 by some Na pump blockade I (1) tnat I (2) (possibly by Na-Ca exchange) tca, means, possibly via the Na-Ca exchange mechanism. In such a scheme either step (1) or step (2) could be rate limiting. The experiments shown in Figs. 4 and 5 allow one to distinguish between these two possibilities. If step (1) were rate limiting, then the relation between [Na]1 and tension should be unique. It should not matter whether a given rise of [Na]1 is produced by a brief exposure to 0-Ko or a longer exposure to 1 mm-ko (for example). If the elevation of [Na]1 by these two different procedures is the same, then the increase of twitch tension should be identical. If, however, step (2) is rate limiting or, if both steps are of comparable speed, a given increase of [Na]1 produced by prolonged exposure to 1 mm-ko will have a greater associated increase of tension than the same rise of [Na]i produced by a brief exposure to O-Ko. Furthermore, if one were to suppose that the inotropic effects of Na pump inhibition were mediated by some mechanism entirely independent of the rise of [Na]1, then there is no reason to expect a unique relationship between [Na]i and twitch tension. In addition to the points for different durations of exposure to 0-KO, Fig. 5 shows the effect of a fixed duration exposure (15 min) to solutions of varying [K]o (0.25, 0 5, 1 0 mm). Clearly the relationship between twitch tension and the area under the electrogenic Na pump current transient is the same for the exposure to different [K]o as it is for the different periods of exposure to O-Ko. In particular, a 15 min exposure to 1 mm-ko produces the same increase of twitch tension as a 3 min exposure to 0-K0. The areas under the electrogenic Na pump current transients following these exposures are identical. From this we conclude that the increase of [Na]1 produced by 15 min in 1 mm-ko is the same as that following 3 min in 0-Ko. This unique relationship between [Na]1 and twitch tension argues that step (1) is rate limiting and that the time course of positive inotropy reflects the time taken for [Na]1 to rise. It suggests furthermore that, if some mechanism entirely independent of [Na]1 were responsible for the observed inotropy, then this mechanism would have to develop its action fortuitously with the same time course as the rise of [Na]1. The rise of [Na]i depends (amongst other factors) on the relation between the Na permeability of the cell membrane and the cell volume. It is therefore unlikely that any other mechanism entirely unconnected with [Na]1 should have similar kinetics. The evidence of Fig. 5 therefore strongly supports an essential link between the rise of [Na]i and the inotropic effects of Na pump inhibition. Ehara (1974b) has suggested that the inotropic effects of low K0 are too rapid in onset to be due to a rise of [Na]1. Eisner & Lederer (1979b) showed that the onset of inotropy was not inconsistent with a rise of [Na]j. The experiment of Fig. 5 shows

11 Na PUMP AND TENSION IN CARDIAC MUSCLE 485 that an increase of [Na]1 can quantitatively account for the time course of the inotropic effects. The conclusion that step (2) is fast is consistent with the results of Niedergerke (1963) who changed the Na gradient across the plasma membrane directly by changing the extracellular Na concentration and found that the change of tension in frog ventricle was only limited by diffusion of Na through the extracellular space. Similarly, Vassort (1973) found that the effects on tension of lowering [Na]o were very rapid in frog atrium. Our results support these findings in showing that the inotropic effects subsequent to the increase of [Na]1 produced by low K. are also very rapid E 08_ I Area (MC) Fig. 5. Relationship between the increase of twitch tension and the rise in [Na]1. From a control solution (4 mm-k., 0-Rb0), the preparation was exposed to O-K., O-Rb, for a variable period of time. Twitch tension was determined and then [Rb]0 was increased to 10 mm in order to obtain an electrogenic Na pump current transient. The area under this current transient gives a measure of the change of [Na]i (see text). The magnitude of the twitch tension is plotted as a function of the area and is indicated by the open circles (0). The time of exposure to 0-K., 0-Rb0 is indicated above each point (min). The other symbols show the results of fixed periods of exposure (15 min) to test solutions containing variable concentrations of K0 before reactivating the Na pump with 10 mm-rb0. +, 0.25 mm-k.; V, 0.5 mm-k.; *, 1 mm-k.. The contractions were obtained by applying a 2 sec pulse to -35 mv from a holding potential of -61 mv at 0 1 Hz. The straight line is fit by eye. Experiment Core volume #1d. Cylindrical area cm2. Relative potencies of 'activator cation8' of the Na pump Electrogenic current. Eisner & Lederer (1980) showed the relation between the Na pump rate and the concentration of Rb or Cs. It is difficult to perform similar experiments with other ions known to activate the Na pump. For example, marked changes in membrane current produced by extracellular depletion of K obscure the electrogenic Na pump current transient. Experiments using Lio to reactivate the Na pump suffer from a different problem. Li is such a weak activator of the Na pump that, in order to obtain a measurable electrogenic Na pump current transient, very large concentrations (e.g. 70 mm) are required. The resulting change in [Na]o required to maintain a constant osmolality then becomes important.

12 486 D. A. EISNER AND W. J. LEDERER The experiments shown in Figs. 6, 7 and 8 were designed to measure the relative potencies of all the activator cations to activate the Na pump. In these experiments, the control solution contained 10 mm-rb., O-K0. The superfusing solution was changed to a test solution containing a variable concentration of one of the activator cations (see inset Fig. 7). After 10 min in the test solution, the preparation was returned to the control solution (10 mm-rb.). This produced an electrogenic Na pump current transient (Fig. 6A). In the previous section, we described how the area under the electrogenic Na pump current transient can give a measure of the rise of [Na]1. Since the steady-state Na1 in thc control solution (10 mmrbo) is A 10-Rb0 B (a) o-ko0 0o na -25 I -,mv ii i (b) 0-5-K \, Oj (C) 1-Ko mg I \ \o-k I1 0 (d) 2-K0-0 \ 10-Rbo (e) 1-Rb0-0 5min I Fig. 6. The ability of different [K]0 or [Rb]0 to sustain the activity of the electrogenic Na pump and prevent the increase oftwitch tension. From a control solution (IOmm-Rb0, 0-K0), the superfusing solution was changed to one containing a variable concentration of either Rb0 or Ko for 10 min and then the preparation was returned to the control solution (see inset Fig. 7). A, electrogenic Na pump current transients. These were obtained at a holding potential of -65 mv on switching to 10 mm-rb0, 0-K0 from the indicated test solutions. The test solutions were (a) 0-KO, 0-Rb0; (b) 0 5 mm-k0, 0-Rb0; (c) 1 mm-ko, 0-Rb0; (d) 2 mm-k0, 0-Rb0; (e) 0-K0, 1 mm-rb0. B, tension records obtained after to min exposure to the different test solutions. The upper trace shows membrane potential. The lower traces show superimposed oscilloscope tension records. Depolarizing voltage clamp pulses to -25 mv were applied for one sec from the holding potential of -65 mv at 0 1 Hz. The record shows a twitch in the control solution (10 mm-rbo, 0-K0) and after 10 min exposures to 0-K0, 1 mm-k0 and 2 mm-k0. The voltage clamp command potential was rounded (tn5 = 10 msec) to increase clamp stability. Experiment Core volume /41. Cylindrical area cm2.

13 Na PUMP AND TENSION IN CARDIAC MUSCLE 487 A 15 rarea 10-Rb0 + a (U m Li MO t Twitch 5. [MI0 (mm) A 40 B 4 E ca3 0 Li 0, I [M] (mm) Fig. 7. The relative potencies of different activator cations to sustain the activity of the electrogenic Na pump and prevent an increase of twitch tension. From a control solution (10 mm-rbo, 0-K0) the superfusing solution was changed to one containing a variable concentration of an activator cation (MO) for 10 min and then returned to the control solution. A, area under the electrogenic Na pump current transient that results from changing to a solution containing 10 mm-rb0 and no other activator cation after a period of 10 min in a test solution containing an activator cation of concentration [M]0. 0, TI0; 0O K.; 0, Rb0; +, Cso; A, NH4.; A Li0. B, twitch tension at the end of, a 10 min exposure to a test solution containing MO of an activator cation. A contraction was produced by applying a 1 sec depolarizing pulse to -25 mv at 0-1 Hz from a holding potential of -65 mv. Same experiment as in A. Experiment Core volume A1. Cylindrical area cm2.

14 488 D. A. EISNER AND W. J. LEDERER constant, changes in the measured 'area' reflect the level to which [Na]i rises by the end of exposure to the test solution. Therefore, the greater the area under the electrogenic Na pump current transient, the greater was the rise of [Na]1 produced during the period of exposure to the test solution. At a given concentration of an activator cation, the larger the rise in [Na]1 during the test period, the less the cation activates the external site of the Na pump. Unlike the more direct measurements of Na pump activity shown in Fig. 2, this experiment is not perturbed by variations in extracellular cation depletion effects during reactivation. Depletion of the extracellular Rb will still occur when the Na pump is reactivated. However, the amount of depletion should depend only on the Na pump activity which in turn will depend only on the increase of [Na]1 during the test period. Thus if two different test solutions activate the Na pump to the same extent, the areas under the electrogenic Na pump current transients that are seen on reactivating the Na pump with 10 mm-rbo should be equal. Fig. 6A shows the electrogenic Na pump current transients following 10 min exposures to different test solutions. Fig. 6A a shows the effect of a O-K0, 0-Rbo test solution. A large electrogenic Na pump current transient is seen. The effects of 0 5 mm-ko, 1 mm-ko, and 2 mm-k0 are shown in Fig. 6A b, c, d. As expected, the greater [K]o in the test solution, the smaller is the electrogenic Na pump current transient on subsequently reactivating the Na pump with 10 mm-rbo. Fig. 6Ae shows the effects of a test solution of 1 mm-rb0. The electrogenic Na pump current seen on reactivating the Na pump with 10 mm-rbo after exposure to 1 mm-rbo is similar in size to that produced after a test solution of 1 mm-k0. Fig. 7A shows a graph of the area under the electrogenic Na pump current transient (Qe) as a function of the concentration of activator cation concentration, [M]o. There appear to be roughly four different relationships between Qe and [M]o indicating that the order of potency to activate the Na pump is (1) Tlo (2) K0 and Rbo (3) Cso and NH4, (4) Lio. This order of potency of these cations to reactivate the Na pump is similar to that seen in numerous preparations such as the red blood cell (Whittam & Ager, 1964), crab nerve (Baker & Connelly, 1966) and mammalian C fibres (Rang & Ritchie, 1968). In particular this Figure emphasizes that Rbo and K0 have quantitatively similar effects in activating the external site of the Na pump. This finding allows one to take results obtained using Rbo to activate the Na pump in order to estimate the behaviour that K0 would have on the Na pump. Furthermore, from this experiment, one can quantify the relative potencies of the different activator cations to activate the Na pump. Approximately equivalent concentrations of these cations are 2 mm-k0 = 2 mm-rbo = 6 mv-cso = 6 mm-nh40 = 22 mm-lio. The relative values for Rbo and CsO fit fairly well with those from direct measurements of the activation curve shown by Eisner & Lederer (1980) where 2 mm-rbo = 6-6 mm-cso. The agreement between the results shown in Fig. 7A and the direct measurements of the activation curves suggest that the latter are not too badly contaminated by depletion effects. Fig. 8 shows a more quantitative comparison of the effects of Rbo, Cso and Lio on the electrogenic Na pump current transient. The model described in the previous paper (Eisner & Lederer, 1980) suggests an additional way to analyse this data. The relationship studied is the one that exists between [M]o (the concentration of an activator cation in the test solution that superfuses the preparation for a fixed

15 Na PUMP AND TENSION IN CARDIAC MUSCLE period) and the area under the electrogenic Na pump current transient (that develops when the preparation is subsequently superfused with 10 mm-rbo). From eqn. (7) of Eisner & Lederer, 1980, area = Qe = VFr(NaMo- Na 2 bo), 5A 489 E2 0 Rb [Ml-, (mm)-, Fig. 8. Quantitative comparison of the ability of different concentrations of Rb, Cs or Li to activate the electrogenic Na pump. The experiment is similar to that shown in Fig. 7. [K]O was zero throughout the experiment. From a control solution (10 mm-rbo), the superfusing solution was changed to one containing a variable concentration of an activator cation (MO) for 10 min and then the preparation was returned to the control solution. The graph shows: Abscissa, 1/MO; Ordinate, the area under the electrogenic pump current transient that results from changing to a solution containing 10 mm-rbo and no other activator cation after a period of 10 min in the test solution containing Mo. *, Rb; 0O Cs; A, Li. The straight lines were fitted to the data with a linear regression procedure giving the following parameters. Rb: slope, 2-5,uC mm; coefficient of determination, Cs: slope, 8 75,C mm; coefficient of determination, Li: slope, 40 38,uC mm; coefficient of determination, Experiment Core volume p1. Cylindrical area cm2. where Nam, is the level of [Na]1 at the end of the exposure to [M]. and Nat_=bO is the steady-state level of [Na]1 in 10 mm-rb.. V, F and r are respectively, the preparation intracellular volume, the Faraday and the electrogenic pump coupling ratio. To simplify the analysis we assume that the exposure to the test solution is long enough for [Na]i to reach an approximately steady-state. This assumption will be least valid for a test solution containing no activator cation in which [Na]i equilibrates slowly (Deitmer & Ellis, 1978). However a steady-state level of [Na]1 will be reached more quickly in test solutions which produce a greater activation of the Na pump. Using eqn. (1) from Eisner & Lederer (1980) and substituting for NaMO yields Qe = VFr [(MO) - Nat_ ], (1)

16 490 D. A. EISNER AND W. J. LEDERER the proportionality factor relating the Na pump rate to Nat. Using where f(m.) is the Michaelis-Menten formalism, we describe the dependence of f(m.) on M.: =V7M f (MO) MO KMO. + MVO' where VM- and KmO represent the maximum rate of the Na pump when fully activated by MO and the value of M. that produces half-maximal activation respectively. Substituting for f(mo) in eqn. (1) (above) leads to 1[T 10. [ lrbn Qe = [VFrJ VM + VrF MO Nat0]. Plotting Qe against 1/Mo should lead to a straight line of slope FrJ V(KOmo/ Vm). Since FrJV is constant, the slope should be proportional to (K"O/IVmox) and should be different for different activator cations. Since activator cations with greater affinities for the external site of the Na pump have lower KmO values, these activator cations will generate lines with smaller slopes. The data in Fig. 8 were obtained from an experiment similar to the one illustrated in Fig. 7. The graph shows a plot of Qe against 1/M0. The data from each activator cation lies on a straight line suggesting that the model is a reasonable approximation to the data. The slope is greatest for the Lio data and smallest for the experiments in RbO while the Cso results produce a line of intermediate slope. These results support those of Eisner & Lederer (1980) where the order of potency to activate the Na pump was found to be Rb > Cs > Li. Furthermore, quantitative comparison can be made with the Ko.5 and Vm.. values determined by Eisner & Lederer (1980). The ratio of the slopes of the lines for Rbo and Cso in Fig. 8 gives (K021 VRbo)/(KRb. VCso ) equal to Direct substitution of the values of K0.5 and Vmax from Fig. 16B of Eisner & Lederer (1980) yields a similar value of Furthermore, Fig. 8 shows that 2 mm-rbo and 25 mm-lio are equipotent in their ability to decrease the area of the subsequent electrogenic Na pump current transient. In the same preparation we compared the ability of various concentrations of Li and Rb to activate the Na pump current transient following a period of exposure to 0-K0, 0-RbO, 0-Lio in the manner illustrated in Fig. 14 of Eisner & Lederer (1980). The rate constant of decay of the electrogenic current transient on activation with 2 mm-rbo was sec-1 and this value is bracketed by the values for 20 mm-lio and 40 mm-lio of and sec-1 respectively. The two methods of estimating the relative potencies of the activator cations therefore lead to similar results. There are two problems with the estimates made for the relative potency of Lio. First, in order to maintain a constant osmotic and ionic strength in the superfusing solutions in these experiments, we have decreased [Na]0 when increasing [Li]o above 10 mm. Therefore the 20 mm-lio solution contained 125 mm-nao rather than 145 mm-nao. This will decrease the Na influx during the period of exposure to the test solution and therefore decrease the area of the subsequent electrogenic Na pump current transient. A more serious problem is that internal Li may actually inhibit the Na pump (Ku, Akera, Olgaard & Brody, 1978) and thus decrease the size of the subsequent electrogenic Na pump current transient. Both of these factors would decrease the area of the electrogenic Na pump current transient seen on subsequently reactivating the Na pump with 10 mm-rbo. Therefore the apparent ability of Lio to activate the Na pump may be greater than its real value. Thus the relative potency of Lio compared to Rbo may be even less than estimated here.

17 Na PUMP AND TENSION IN CARDIAC MUSCLE Further complicating these experiments is the fact that the activator cation concentration in the restricted extracellular spaces or clefts, [M]cieft, may be different from [M]O in the steady state. At high [M]o, [M]ciet will be less than MO because the Na pump will transport activator cation into the preparation. At lower [M]o, the effective [M]cIett will be greater than [M]O because of the accumulation of K in the clefts due to escape from the intracellular compartment. The particular experiments described above will be less perturbed by [M]o depletion effects during the test period than were the simple activator cation reactivation experiments of the type illustrated in Fig. 14 of Eisner & Lederer (1980). This is due to the fact that, at a given [M]o, the absolute pump rate is less during the test period. The lower pump rate reflects the lower [Na]l E 0~~~~~~~ C04 AA l Area (MC) Fig. 9. The relationship between the area under the electrogenic Na pump current transient and twitch tension. The experimental protocol is identical to that described for Fig. 7. The twitches were elicited by a 1 see depolarizing pulse to -25 mv from a holding potential of -54 mv applied tt 0-1 Hz. Abscissa: the area under the electrogenic Na pump current transient that results from changing to a solution containing 10 mm-rbo and no other activator cation after a period of 10 min in a test solution containing an activator cation of concentration Mo. Ordinate: twitch tension at the end of a 10 min exposure to the test solution. 0, Rb; 0O Cs; A, Li. The data are from the same experiment as Fig. 8. Experiment Core volume 0-037,ul. Cylindrical area 0'0099 em2. Tension. As expected, exposure to the various test solutions produces an increase of twitch tension. Fig. 6B shows the tension records in 10 mm-rbo (control) and after 10 min exposures to test solutions containing various [K]0 and O-Rb0. The greater [K]0 in the test solution, the smaller the increase of tension over the tension seen in the control solution (10 mm-rbo). Note that, as expected from the results of Eisner & Lederer (1979a), exposure to O-Ko results in the development of a marked component of voltage dependent tonic tension. Twitch tension measurements from a greater variety of test solutions are demonstrated in Fig. 7B. The points relating twitch tension to activator cation concentration fall into the same four groups as those relating the areas under the electrogenic Na pump current transient to the concentration of activator cation. Data from an experiment similar to that shown in Fig. 7 has been replotted in Fig. 9. Twitch tension is plotted as a function of the

18 492 D. A. EISNER AND W. J. LEDERER electrogenic Na pump current transient area. The points were obtained after exposure to various concentrations of Rb, Cs and Li. Allowing for the scatter, the result of this experiment is consistent with the hypothesis that twitch tension is uniquely related to the area under the electrogenic Na pump current transient. The same result is obtained from analysis of the experiment shown in Fig. 7. It is important to note that the 'area' measurement depends only on the final level of [Na]i achieved during exposure to the test solution. As such this result is additional evidence suggesting that a rise of [Na]1 is an important intermediate in the inotropic effects produced by the decreased pump rate on reducing activator cation concentration. DISCUSSION Activator cations. Eisner & Lederer (1979b) showed that activator cations of the Na pump abolish the inotropic effects of K depleted solutions. In this paper we have shown that they do this with the same relative potency with which they activate the Na pump. Furthermore it has been quantitatively shown that the effects of these activator cations on tension are uniquely determined by their action on the Na pump. This conclusion makes it possible to use external activator cations to examine the relationship between Na pump activity and tension and the role of Nat in the positive inotropic effects seen with Na pump inhibition. Previous workers have suggested effects other than Na pump blockade to explain the inotropic effects of K depleted solutions (Morad & Goldman, 1973; Ehara, 1974b; Goto et al. 1977). However, at least in the sheep Purkinje fibre, the activator cations provide a specific means to probe the relationship between Na pump rate and twitch tension. The role of Naj. Having shown that the effects of activator cations on twitch tension are mediated via their effects on Na pump activity, it is worth considering whether the Na pump rate affects tension via a change in [Na]1 or whether the Na pump affects tension by some other means. Besch & Schwartz (1970) have suggested that the binding of ouabain to the Na pump can have direct actions on Ca binding to membrane stores. This work has been supported by Gervais, Lane, Anner, Lindenmayer & Schwartz (1977). Similarly, Gadsby et al. (1971) have suggested that the inotropic effects of acetylstrophanthidin and low [K]o are not mediated via an increase of [Na]1. Fig. 5 shows, however, that the inotropic effects of a given solution are uniquely related to the area of the electrogenic Na pump current transient on subsequently reactivating the Na pump. As pointed out previously, this is consistent with the rise of [Na]1 being the rate limiting factor in the development of inotropy. Although our experiments offer no evidence, it has been shown that a change of [Na]i will affect [Ca]i by a Na-Ca exchange mechanism (Reuter & Seitz, 1968; Baker et al. 1969; Glitsch et al. 1970). The effects of activator cations have therefore defined the relationship between Na pump rate and twitch tension. This provides the basis for future work on the effects of inotropic drugs such as the cardiotonic steroids. Our results have shown that a significant degree of pump inhibition is required to increase twitch tension when lowering the extracellular activator cation concentration. Therefore any other inotropic manoeuvre which acts by Na pump inhibition must also produce a measureable degree of Na pump inhibition. It might previously have been argued that the

19 Na PUMP AND TENSION IN CARDIAC MUSCLE 493 relationship between Na pump activity and tension was so steep that a large degree of positive inotropy could be produced by a vanishingly small degree of Na pump inhibition. However, the results from the activator cation experiments show that, if a drug produces an inotropic response but no measurable Na pump inhibition, its action cannot depend on Na pump inhibition. We thank P. Flatman, I. M. Glynn, J. J. B. Jack, D. Noble, J. M. Ritchie, A. J. Spindler and R. W. Tsien for valuable discussion. We are grateful to the British Beef Co., Witney, and especially to Mr Sid Lovejoy for supplying sheep hearts. The work was supported by a grant from the British Heart Foundation to Dr D. Noble. D.A. E. is an M.R.C. scholar. This work was done during the tenure of a British-American research fellowship of the American Heart Association and the British Heart Foundation (W.J. L.). REFERENCES BAKER, P. F., BLAusTEIN, M. P., HODGKIN, A. L. & STEINHARDT, R. A. (1969). The influence of calcium on sodium efflux in squid axons. J. Physiol. 200, BAKER, P. F. & CONNELLY, C. M. (1966). Some properties of the external activation site of the sodium pump in crab nerve. J. PhymAol. 185, BEscH, H. R. & SCHWARTZ, A. (1970). On the mechanism of action of digitalis. J. mol. cell. Cardiol. 1, COHEN, I., DAUT, J. & NOBLE, D. (1976). An analysis of the actions of low concentrations of ouabain on membrane currents in Purkinje fibres. J. Phy8iol. 260, DEITMER, J. W. & ELLIS, D. (1978). The intracellular sodium activity of cardiac Purkinje fibres during inhibition and re-activation of the Na-K pump. J. Phy8iol. 284, EHARA, T. (1974a). Effects of adrenaline, ouabain, and some ionic conditions on the electrical activity of bullfrog ventricle. Jap. J. Physiol. 24, EHARA, T. (1974b). Late potentiating effect of low-k Ringer solution on the contractility of the bullfrog ventricle. Jap. J. Physiol. 24, EISNER, D. A. & LEDERER, W. J. (1979a). Inotropic and arrhythmogenic effects of potassium depleted solutions on mammalian cardiac muscle. J.Physiol. 294, EISNER, D. A. & LEDERER, W. J. (1979 b). The role of the sodium pump in the effects of potassium depleted solutions on mammalian cardiac muscle. J. Physiol. 294, EISNER, D. A. & LEDERER, W. J. (1980). Characterization of the electrogenic sodium pump in cardiac Purkinje fibres. J. Physiol. 303, ELLIS, D. (1977). The effects of external cations and ouabain on the intracellular sodium activity of sheep heart Purkinje fibres. J. Physiol. 273, GADSBY, D. C. & CRANEFIELD, P. F. (1979). Direct measurement of changes in sodium pump current in canine cardiac Purkinje fibres. Proc. natn. Acad. Sci. U.S.A. 76, GADSBY, D. C., NIEDERGERKE, R. & PAGE, S. (1971). Do intracellular concentrations of potassium or sodium regulate the strength of the heart beat? Nature, Lond. 232, GERVAIS, A., LANE, L. K., ANNER, B. M., LINDENMAYER, G. E. & SCHWARTZ, A. (1977). A possible mechanism of the action of digitalis. Ouabain action on cation binding to sites associated with a purified sodium-potassium-activated adenosinetriphosphatase from kidney. Circulation Res. 40, GLITSCH, H. G., REUTER, H. & SCHOLZ, H. (1970). The effect of the internal sodium concentration on calcium fluxes in isolated guinea-pig auricles. J. Physiol. 209, GLYNN, I. M. (1956). Sodium and potassium movements in human red cells. J. Physiol. 134, GODFRAIND, T. & GHYSEL-BURTON, J. (1977). Binding sites related to ouabain induced stimulation or inhibition of the sodium pump. Nature, Lond. 265, GOTO, M., TSUDA, Y. & YATANI, A. (1977). Two mechanisms for positive inotropism of low-k Ringer solution in bullfrog atrium. Nature, Lond. 268, HOUGEN, T. J., LLOYD, B. L. & SMITH, T. W. (1979). Effects of inotropic and arrhythm6genic digoxin doses and of digoxin-specific antibody on myocardial monovalent cation transport in the dog. Circulation Res. 44,