J. Phyoiol. (1978), 284, pp. 241-259 241With 10 text-fgurePrinted in Grea Britain

THE INTRACELLULAR SODIUM ACTIVITY OFCARDIAC PURKINJE FIBRES DURING INHIBITION AND

RE-ACTIVATION OF THE Na-K PUMP

By JOACHIM W. DEITMER* AND DAVID ELLIStFrom the Department of Physiology, University of Bristol, Bristol, BS8 1TD

(Received 3 March 1978)

SUMMARY

1. The intracellular Na activity, a'j, of sheep heart Purkinje fibres was continu-ously monitored using Na+-sensitive glass micro-electrodes. The effects of removaland restoration of external K, and of application and removal of various cardioactivesteroids, were investigated.

2. The ala increased in K-free solutions and rapidly recovered on addition ofexternal K. The rate of this recovery depended on both the external K concen-tration, [K]O, and the aka. The rate of a' a recovery was found to be half maximallyactivated at a [K]o of about 10 mm. If corrections are applied to allow for changes inthe net passive Na influx at various [K]o, then this value is increased to approxi-mately 12 5 mM.

3. At a given [K]0, there appeared to be a linear relationship between the rate ofaia recovery and the level to which a'a had increased in K-free solution (over therange of aia from 7-5 to 31 mM).

4. Addition of the cardioactive steroids strophanthidin, acetylstrophanthidin,actodigin (AY 22,241) or dihydro-ouabain produced rapid changes of a' a. At lowconcentrations, these compounds sometimes produced a small decrease in akNa, whileat concentrations above 10-7 M they produced a dose-dependent increase.

5. The effects on aINa of both low and high concentrations of all these cardioactivesteroids were readily reversible within 120 min. The time course of the a'a recoverymainly depended on the concentration of the cardioactive steroid applied, and on thelevel to which aia had increased.

6. Upon addition of a cardioactive steroid (above 10-7 M), aiNa at first increasedalmost linearly with time. The rates of such an increase were measured during thisperiod at various cardioactive steroid concentrations and used to produce dose-response curves. The concentrations that produced a half-maximum rate of a'aincrease were near to 10-6 M for strophanthidin and acetylstrophanthidin, but nearto 10-5 M for actodigin and dihydro-ouabain.

7. The mean maximum rate of a'a increase produced by the addition of a highcardioactive steroid concentration was 0f49 + 0f17 mM/min (± S.D., n = 21). Thiswould indicate a net passive Na influx into the cells of approximately 2x8 p-mole/cm2 sec.

* Present address: LS Allgemeine Zoologie, Ruhr-Universitat, D-4630 Bochum, WestGermany.

t Present address: Physiology Department, University of Edinburgh, Edinburgh EH 8 9AG.

J. W. DEITMER AND D. ELLIS8. This maximum rate of a'a increase could be achieved by the addition of 10-5 M-

strophanthidin or acetylstrophanthidin, but 10-4 to 10-3 M-actodigin or dihydro-ouabain was required to produce a similar rate of increase.

9. The addition of these high cardioactive steroid concentrations produced aninitially rapid increase of a' a. After 15-30 min this a a increase slowed considerably.The ata appeared to reach a 'plateau' within 2-4 hr at levels much below thosepredicted for a Na electrochemical equilibrium across the cell membrane.

INTRODUCTION

The intracellular Na activity of cells is generally believed to be controlled primarilyby the activity of the Na-K pump. In cardiac muscle the ionic dependence of thepumping mechanism is difficult to investigate due to the complexity of the tissue, i.e.the cells are often small with large and/or tortuous extracellular spaces. Flame photo-metry has been used to study changes in the Na content produced by changing the[K]o in cat ventricular muscle (Page, Goerke & Storm, 1964; Page & Storm, 1965) andin guinea-pig auricle (Glitsch, Pusch & Venetz, 1976). Active Na transport appearedto depend upon the concentration of both intracellular Na and extracellular K. InPurkinje fibres, however, only a small effect of the [K]o on active Na extrusion wasfound, using radioactive Na measurements (Bosteels & Carmeliet, 1972).

Na+-sensitive micro-electrodes have been used to measure directly changes in theinternal Na activity, at a. The aka rapidly changes to anew steady-state level when the[K]0 is altered (Ellis, 1977b). In the present study we have investigated the depen-dence of the rate of a'~arecovery from K-free solution on both the [K]o and the a'a.

In the second part of this paper we have looked at the dose-dependent effects ofvarious cardioactive steroids on a'Na, and compared their effects with K-free solutions.These experiments were designed to study the effectiveness of these cardioactivesteroids as inhibitors of the Na-K pump, and the time course of the recovery of ata onremoval of the steroids.

Preliminary reports of some of this work have already been presented (Deitmer &Ellis, 1977a, 1978b).

METHODS

The experimental procedures were essentially the same as described previously (Ellis, 1977b;Deitmer & Ellis, 1978a), with isolated sheep heart Purkinje fibres being pinned into a smallsuperfusion chamber where the exchange rate of solutions was 6-10 bath volumes/min.

Micro-electrode8Conventional and Na+-sensitive micro-electrodes were produced as described by Thomas

(1970, 1978). They were used to penetrate cells between 0 5 and 1 mm apart along the fibre. AllNa+-sensitive electrodes used had a response of at least 54 mV (up to 61 mV) for a tenfold changein the external Na activity (at 35 "C), the response being more than 90% complete within30-60 sec.

SolutionsThe normal saline contained (mM) Na 140; K 6 (4); Ca 2; Mg 1; Cl 152 (150); glucose 10; and

was buffered with 10 mM-Hepes (N-2-hydroxyethylpiperazine-N'-2 ethansulphonic acid) to givea pH of 7-4 (± 0-1), and was gassed with 100% 02. The temperature of the solutions was main-tained at 35 'C by a heating unit with a feed-back circuit operated by a sensing thermistor placedat the entrance to the chamber.

242

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Na 243When external K was reduced to zero it was replaced by either Tris (6 mM) or glucose (12 mM).

When high KO concentrations were used (12 mM), all solutions were made 6 mm hypertonic bythe addition of K, 6 mM-Tris or 12 mM-glucose.Ouabain (B.D.H.), strophanthidin (Boehringer Mannheim) and acetylstrophanthidin (Eli

Lilly) were normally dissolved in ethanol to produce a stock solution of 10-2 M. Thus, with a maxi-mum concentration of 10-4 M in the perfusion solution the amount of ethanol did not exceed 1 %.This quantity of ethanol had no observable effect on the response of the Na+-sensitive electrodes,but reduced the a&. by approximately 7%. The membrane potential hyperpolarized by 1-2 mV.These effects of 1% ethanol may be due to volume changes in the cell produced by an osmoticprocess. Lower concentrations of ethanol (0-1 %, as was present in 10-i M-strophanthidin oracetylstrophanthidin) had no effect on either a',, the response of the Na+-sensitive micro-electrode or the membrane potential (Deitmer & Ellis, 1978a). The 5 x 10-4 M-ouabain solutionwas produced by using a stock solution of 5 x 10-2 M, thus the ethanol did not exceed 1 % of thetest solution.

Actodigin (Ayerst 22, 241) and dihydro-ouabain (Boehringer Mannheim) appeared to be morereadily water soluble and were therefore dissolved in an ethanol Tyrode mixture (1: 10) to givestock solutions of 10-2 M.

Activity measurementThe amount of Na present in the cells has been expressed as the internal Na activity, aN,

both in the text and in the Figures. This contrasts with our previous publications (Ellis, 1977a,b; and Deitmer & Ellis, 1977a, b, 1978a) where the amount of internal Na was expressed in theFigures as the Na ion concentration, [Na+]i. The a' a value can be converted to [Na+]i by multi-plying by 1*33 (see Ellis, 1977b). This assumes that the internal activity coefficient for Na is thesame as that in the external solution (0-75). We have, in this paper, changed and used the term,at , in the Figures, primarily because (1) no assumptions concerning the Na activity coefficientare required and (2) to use the same expression as in the text. It has to be remembered, however,that any change in a'. will, in fact, be indicative of a larger change in the intracellular Na ionconcentration (expressed in absolute figures rather than as percentage changes). Both the aaand the intracellular Na ion concentration values will be lower than the total Na concentration ofthe cell (as measured by, e.g. flame photometry) which includes both bound and free Na. For afuller description of the advantages and disadvantages of various methods of expressing theamounts of ions measured in cells by ion-sensitive micro-electrodes, see Thomas (1978).

RESULTS

The effect of external K on the aka recovery from K-free solutionsThe a a in Purkinje fibres begins to increase almost immediately after the removal

of K0, presumably due to inhibition of the Na-K pump, while restoration of Koinitiates a rapid recovery (Ellis, 1977b). We have investigated the effect of differentextracellularK concentrations on the rate of a a recovery from its elevation in K-freesolutions. Fig. 1 shows an experiment of this type. The a' a was allowed to rise in theK-free solution to approximately the same level (14 mM) in each case, so that therecovery processes were initiated at a similar aba. The addition ofK then produced arapid decrease of ata to a new stable level. Both the rate of aka recovery and the newsteady-state level varied with the [K]O. The higher the [K]0, the faster aia recoveredand the lower the eventual steady-state level of aia.The membrane potential, Em, falls in K-free solutions to a new relatively stable

level (Weidmann, 1956; Carmeliet, 1961; Ellis, 1977b). In the experiment shown inFig. 1, Em 'switches' from -70 to about -30 mV in the K-free solution. When K(3, 6 or 12 mm) is added,Em 'switches' back to the original hyperpolarized state. ThisEm 'switch' was only transient on addition of 3 mM-K and did not occur at all in1 mM-K. Sometimes the addition of K was accompanied by spontaneous action poten-

J. W. DEITMER AND D. ELLIStials (e.g. in 3 and 6 mM-K). It is conspicuous that, on addition of KO, Em reached anew steady-state only after a transient hyperpolarization. The size of the transienthyperpolarization was dependent upon the [K]O added, the higher the [K]0, the largerwas this hyperpolarization. It seems likely that the transient hyperpolarization onaddition of K. was due to electrogenic Na pumping, as has been suggested for othercardiac tissues (Page & Storm, 1965; Glitsch, 1972).The recovery ofa 5 on addition ofK occurred with a single exponential time course

(coefficient of correlation greater than 0-98 in all experiments) from which the rateconstant can be determined. This rate constant has been used to calculate the rate ofair recovery (lAaIa/t). Fig. 2 shows the dependence of the rate of a'a recovery on

15 min

._z A ,4

E 6I6 1E10

-f 50

E9070- 12

Fig. 1. Pen recording of an experiment showing the effect of various [K]. on therecovery of a', (upper trace) and on Em (lower trace) following exposure to a K-freesolution. The a' was allowed to rise in K-free solution to approximately the same levelin each case before K was restored. Upon addition of 6 and 3 mm-[K]0 a few spontaneousaction potentials occurred (these are greatly diminished in size due to the slow response-time of the pen recorder).

both the aia and on the [K],. The rate of decrease of aia appears linearly related tothe a'5 at any time during the recovery from K-free solution, and is faster the higherthe [K]O.From experiments like that shown in Fig. 1 we have measured the maximum rate

of a a recovery on addition of various concentrations of K0 (Fig. 3). A line has beenfitted to the points on the assumption that the relationship indicates simple firstorder kinetics (of course our results do not exclude the existence of a more compli-cated relationship between [K]o and pump rate). The results suggest that a half-maximal activation of a'a recovery occurs at a [K]o of approximately 10 mM.The rate of aia recovery in these experiments would be influenced by the rate of

passive Na influx. This influx is dependent upon the membrane potential as part ofthe driving force for Na entry, and on the Na permeability of the membrane. The

244

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Na 245large depolarization in K-free solutions has been suggested to be due to an increase inthe relative permeability of Na to that ofK (e.g. Carmeliet, 1961; Gadsby & Crane-field, 1977). We have attempted to estimate the extent of changes in the net passiveNa influx at various K concentrations (1, 6 and 12 mm) by exposure of the prep-arations to 10-5 M strophanthidin (the rate of aNa increase being used as an estimateof the rate of net passive Na influx). The average depolarizations produced by 1 and12 mM-[K]O, strophanthidin containing solutions were 31-2 + 3 9 mV (mean + S.D.,n = 5) and 84+ P7 mV (n = 4) respectively from the normal Em (in 6 mM-[K]O,10-5 M-strophanthidin) of 64-1 + 4-0 mV. The rates of aia increase were measuredafter strophanthidin addition, with care being taken to measure the rate of increase

4 ,12K

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0 6 K

I~~ ~ ~ ~ ~ ~ ~~~~~~~~~K

.el 0~~~~

0 - o----~ ~ ~~- 1

5 7 5 10 12 5 15a'Na(mM)

Fig. 2. Graph (of data from Fig. 1) of the rate of a' recovery from K-free solution,Aal /t, plotted against the a' in the presence of the [K]0 (mM) indicated (filledcircles). The open circles represent the steady-state level ofC. reached in these differentK0 solutions. The straight lines through the points were drawn by eye. The rate of a'N&recovery was calculated from the product of the level of a' above its steady-statevalue and the rate constant of the recovery. These al recoveries had single exponentialtime courses, the rate constants of which were calculated by computer.

over the same a'a range in each [K]o (as the [K]o affects the steady-state levels ofa a). The average rate of aia increase, compared to that in 6 mM-[K]o was 90-7 +14-2 0/ (n = 5) in 1 mM-IK]o and 92-5 + 80 0/ (n = 4) in 12 mM-[K]o. If a correction isapplied for these changes in the net passive Na influx in the calculation of the half-maximal activation of aka recovery by K0, then the Km obtained is approximately12-5 mm. It should be noted, however, that the reduced rate of aiNa rise in 12 mm-K0could have been due partly to a decreased binding of strophanthidin to the membrane.Some other factors which might influence the reliability of the Km value as calcu-lated from our experiments will be discussed later.

246 J. W. DEITMER AND D. ELLIS

ata recovery from K-free solution initiated at various a a levels

So far experiments have been described where the ad a had been allowed to rise tothe same level, and where its recovery had been initiated by various [K]o. In anotherseries of experiments we have allowed akNa to rise to differentvalues, and then initiatedthe recovery by adding either 4 or 6 mm-K.

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6

0

0 2 4 6 8 10 12[K]0 (mM)

Fig. 3. Graph of data from three experiments (like that in Fig. 1) showing the relation-ship between the [K]0 and the maximum rate of a'5 recovery from K-free solution. Themaximum rate of decrease of a4 has been expressed as the decrease per minute as apercentage of the initial a', in the K-free solution. This procedure was used in order tonormalize the results from the three experiments as the a'. had been raised in the K-freesolution to levels varying between 13 and 18 mm. The line has been drawn according tothe equation.

Maximum rate of a' decrease = y[K]O/x+ [K]O,where x and y are constants. These constants were obtained from the intercepts of adouble reciprocal plot of the data (coefficient of correlation = 09996). The value x, i.e.the value of the K. for [K]O, was 10 1 mm; and y, the mean value of the calculatedmaximum rate of a4 decrease, was 52-4% per minute of the level to which a'. hadincreased. The bars indicate ± 1 standard deviation.

Fig. 4 shows an experiment of this kind. After removal of K. the a'a rises rapidlyinitially but then tends to reach a plateau level. In order to raise aka to higher levels,the divalent cations Ca and Mg were removed (replaced by 4-5 mM-Tris). This causedaia to rise very rapidly (see also Deitmer & Ellis, 1978a). When Ca and Mg were re-added, the rise of aika was stopped and there was even a slight decrease despite thecontinued superfusion with K-free solution. Re-addition of 4 mm-K produced animmediate and rapid recovery of a'ba. On the right of the Figure is shown the recovery

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Na 247

of aiNa from an even higher level produced by a more prolonged exposure to divalentcation and K-free solutions. The akN. recoveries reach a maximum rate within2-4 mm after addition ofK0 and this rate is maintained until a'N. has been reduced torelatively low levels.

-i20 A B 10min

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EE

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70 t I

OCa OMgp I _ _

OK OK

Fig. 4. Pen recordings of an experiment showing the recovery of a' from very highlevels. (A) the d. was raised to higher levels than could be achieved in K-free solutionbythe removal of Ca and Mg and the addition of 1 mM-EDTA. The restoration of Ca andMg produced a small, slow recovery ofa.. After 15 min 4m -K was added, producinga rapid a. decrease. In (B) the 4. was raised to an even higher level initially by alonger exposure to the Ca- and Mg-free solution.

In the partofthe experiment shown in Fig. 4 the membrane potential had stabilizedat a reduced level of about -40 mV even in the presence of 4 mM-K due to prolongedprevious exposures to divalent cation-free solutions. In experiments where exposuresto divalent cation-free solutions were fairly short, full recovery of the membranepotential occurred (see Deitmer & Ellis, 1978a). On removal of K. in the experimentshown, the Em decreased by another 15 mV followed by a slow repolarization. WhenCao and Mg0 were also removed, the membrane depolarized even further. This changewas reversible on restoration of the divalent cation concentration. Following theaddition ofK there was always a transient hyperpolarization, similar to that describedpreviously (for Fig. 1). The more rapid recovery of akNa observed when it wasallowed to rise to a higher value, was accompanied by a larger transient hyper-polarization.The results of eleven experiments of the type shown in Fig. 4 are shown in Fig. 5,

where the maximum rate of a'Na recovery from K-free solutions has been plottedagainst the level to which aka was allowed to rise. The a a had been raised to variouslevels with K-free, K- and Ca-free or K- and divalent cation-free solutions. Themaximum rate of akNa recovery was then measured when 6 mM-K was added. Thereappears to be a linear relationship between the rate of a' a recovery and the level towhich it had increased, with no saturation being apparent for this range of a'a up to31 mm This suggests that the Km for the activation of the aiNa recovery by akN. islikely to be greater than 31 m .

248 J. W. DEITMER AND D. ELLISThe slope of the least-squares line drawn through the experimental points gives a

rate constant of 04188 min' (in the presence of 6 mM-K). Thus, the rate constant of thea a recovery from K-free solutions appears to be proportional to the [K]0 (see Fig. 2),but independent of the ata over this range. The line intercepts the X-axis at an a1Navalue of 5-5 mm. This value is near to that of the normal a'Na measured in theseexperiments of 6-62 + 1P43 mm, with a mean Em of 72-8 + 4-3 mV in the presence of6 mM-K (values + S.D., n = 22). At this level of a1Na the Na extrusion system should,of course, be in a steady state with the net Na influx.

6

4-5 0

E 3

15

0 ~ ~ .

6 12 18 24 30 36al .(mM)

Fig. 5. Graph of data from eleven experiments (like that shown in Fig. 4) showing therelationship between the maximum rate of a' decrease, Aa I/t, in 6 mM-[K]. andthe level to which a' had increased in K-free solutions. The highest levels wereachieved by intermediate exposures to divalent cation-free solutions in a similar way tothe experiment shown in Fig. 4. A least-squares regression line was fitted to theexperimental points, coefficient of correlation 0904, slope 0-188. The extrapolated lineintercepts the X-axis at an a'. value of 5.5 mi.

Effects of cardioactive steroids on a a. Experiments with strophanthidin and ouabainOuabain at a concentration of about 10-7 M or higher causes a dose-dependent rise

in aka (Ellis, 1977b). The effects of this glycoside are very poorly reversible over atime span of hours. We have therefore looked at the effect of rapidly-reversible cardio-active steroids on aka. In Fig. 6 the effects ona5aof 10- M-strophanthidin and10-5 m-ouabain are compared. Both cardioactive steroids caused a rapid rise of akawithin about 2 min of their application (0.53 mM/min in 10-5 M-strophanthidin and0-45 mM/min in 10-5 M-ouabain). After removal of strophanthidin, aa still rose forsome 10 min before it began to decrease, and reached the control level after another40 min. Similarly, when ouabain was removed from the bathing solutions, a' a also

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Nacontinued to rise for 10-15 min, but after this the recovery of a'Na was very slow. Evenafter several hours aiNa was much higher than the control level.Both cardioactive steroids produced a membrane depolarization of about 5 mV

which was relatively rapidly reversed after the removal of strophanthidin, but onlyslowly reversed after the removal of ouabain. Experiments using higher concen-trations of strophanthidin indicated that the. maximum rate of akNa increase wasalready achieved with a strophanthidin concentration of 10-5 M. This suggests that10-5 M-strophanthidin is able to block the Na-K pump completely. In four experi-ments 10-5 M-strophanthidmn caused a maximum rate of aia rise of 101F3 % ± 9 5 %compared to that produced by 10-4 M-strophanthidin.

10 minm

20-

E 10

S _

600-

W 80

Strophanthidin Ouabain(10 5M) (10' M)

Fig. 6. Pen recording of an experiment showing the effect of 10 min applications of10-6 M-strophanthidin and 10-6 m-ouabain on a' and Em. The break in the recordingwas due to the fact that the voltage recorded by the conventional micro-electrodeappeared unstable so it was removed from the cell. A few minutes later another cell wasimpaled giving a more stable penetration. Equal and opposite voltage changes appearon the a4 recording because the signal measured by the conventional micro-electrode iselectronically subtracted from that of the Na+-sensitive micro-electrode.

Fig. 7 shows the effect on aka of a long exposure to 10-5 M-strophanthidin. The riseof a'a was fastest initially and linear with time for 10-15 mi, but then slowed con-siderably. After about 1-5 hr the a'Na rose only very slowly and appeared to approacha 'plateau'. The a1Na was however still surprisingly low (about 17 mM) so that therewas still a large electrochemical gradient for Na to enter the cell. The rise of aia isplotted on a linear scale in Fig. 7B to demonstrate the changes with time moreclearly.One possible reason for the maintenance of ada at a relatively low level under these

conditions is that the Na-K pump may become re-activated when a'a reaches highlevels. We therefore added a very high dose of ouabain (5 x 10-4 M) to try to ensurethat the Na-K pump was completely blocked. The aiNa rose slightly on the addition ofa x 10-4 M ouabain before again appearing to level off, still at only 18 mm. The Naequilibrium potential at this aiNa level (when the external Na activity is 105 mm)would be + 47 mV, but the membrane potential was greater than -60 mV. This

249

250 J. W. DEITMER AND D. ELLIS

'plateau' level of aka is not due to an effect on the Na+-sensitive micro-electrode asthe electrode is able to record much higher levels. Exposure of Purkinje fibres todivalent cation-free solutions for 20-30 mm can increase the aia to about 100 mM(with Em decreasing to about -10 mV).

10 min

50-- Em-E 60--E 70-_W 80-1_ 5 x leM -ouabain

10- M -strophanthidin

20 B 5x104AM -ouabain

15 0 000000

0~~~

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0 20 40 60 80 100 120 140t (min)

Fig. 7. A, experiment showing the effect of a long exposure to 10- M-strophanthidin ona4; and Em. After 110 min 5 x 10-4 M-ouabain was applied which caused a' to rise to aslightly higher ' plateau' level. B, graph of the experiment shown in A on a linear at.scale.

Do8e-re8pon8e curve for strophanthidinThe effects of relatively short exposures (15-30 min) to various concentrations of

strophanthidin on aia and on the rate of aka changes have been investigated asshown in Fig. SA. Strophanthidin was applied at concentrations between 1*5 x 10-8and 1.5 x 10-4 M. The first 15 min of each application are shown in the traces. With aconcentration of 1-5 x 10-8 M, akNa slowly decreased, as was also observed at low con-centrations of ouabain (Ellis, 1977b). The aka stabilized at a lower level which wasthen maintained for the rest of the period of strophanthidin exposure (10 min inthis case). With 1-5 x 10-7 M-strophanthidin, alkTa rose slowly, after a small trans-ient decrease. This transient decrease was often apparent on addition of higher

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Nastrophanthidin concentrations. With increasing concentrations however, the sub-sequent rate of rise of aiNa increased, and the latency of the onset of rise decreased.The a1Na at 1 mI intervals after strophanthidin application has been plotted on a

linear scale in Fig. 8B. The rise of alqa seems to be linear for the first 8-12 n. Themaximum rate increase was determined from this initial period and plotted against

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Fig. 8. The effect of strophanthidin on a'... A, pen-recordings showing the changes inaN.. (upper trace) and E. (lower trace) produced by strophanthidin concentrationsbetween 1-5 x 10-8 M and 1-5 x 1O-4M; [K]0 = 6 m . The recoveries of ah afterremoval of strophanthidin have been cut out of the Figure to shorten its length. Thebreaks between the traces were for periods of between 20 and 120 min. B, a linear plotof the rise in a' , during exposures to the strophanthidin concentrations indicated,measured from the experiment shown in A. The circles represent the initial, andthe squares the final, exposures to 1-5 x 10-5 M-strophanthidin, respectively. Theintermediate exposure to 1-5 x 10-4 M-strophanthidin is represented by triangles. C,the maximum rate of a,,. increase (measured from part B) has been plotted againstthe strophanthidin concentration.

the strophanthidin concentration in the form of a dose-response curve (Fig. 8C).In this and in three other experiments of this type the threshold concentration ofstrophanthidin required to produce a rise of a'a was between 108 and 10-7 M. Ahalf-maximal effect on the rate of at rise was achieved at a concentration near to10- M (Table 1).

The maximum rate at which a a could be increased by the addition of high

-iEz 8

6

4

251

J. W. DEITMER AND D. ELLISstrophanthidin concentrations (10-5 M and more) was close to 0-75 mM/mm (equiv-alent to an increasein the [Na+]i of 1 mM/m; Deitmer & Ellis, 1977a). The mean valuefor the maximum rate of aka rise was 0-49 + 0-17 (mean + S.D., n = 21). This rise ofaka probably reflects the net passive Na influx into the cells. Taking a value for thecell surface area/volume ratio of 0-39 /sm-1 (Mobley & Page, 1972) for sheep Purkinjefibres and the intracellular Na activity coefficient as 0-75, this increase in the a aindicates a net Na influx of 2-8 p-mole/cm2 sec.

15 min

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80LJ ~~Lj LJ _j

Actodigini Strophanthidin Acetylstrophanthidin Dihydro-ouabain OK(1O-Mml (10-5 M) (10 M) (105 M)

Fig. 9. Comparison of the effects of various cardioactive steroids (10- M) and of re-moval of external K on a'. and E,. Upon re-addition of external K (6 mM) there weresome spontaneous action potentials (the amplitude of which have been greatly reducedby the slow response-time of the pen recorder).

Comparison of various reversible cardioactive steroidsFig. 9 shows the effect on a1a of adding the cardioactive steroids actodigin, stroph-

anthidin, acetylstrophanthidin and dihydro-ouabain, at a concentration of 10-i Mand, at the end for comparison, an exposure to K-free solution. At this concentrationstrophanthidin and acetylstrophanthidin produced a considerably larger effect on a'5than actodigin, dihydro-ouabain or the removal of K0.When any of the cardioactive steroids were removed, aiNa continued to rise for

some 5-20 min, but eventually decreased and returned to the control level within afurther 20-60 min.The time required for ata to return to the control level appeared to be dependent

upon the extent to which it had increased. If concentrations of cardioactive steroidswere chosen to raise aka to the same level, in approximately the same time ofexposure,then the rate of recovery was very similar after removal of strophanthidin, acetyl-strophanthidin and dihydro-ouabain. However, the effect of actodigin on a1N, seemedto be reversed up to 25 % faster than that of the other compounds tested.Removal of K0 produced a rate of aiNa rise which ranged between 30 and 80 % of

that obtained with the maximum doses of the cardioactive steroids. This indicates, assuggested previously (Ellis, 1977b) that the Na-K pump is only partially inhibited inK-free solutions, presumably due to a maintenance of some pump activity by aleakage of K from the cells and its accumulation in the intercellular cleft system.

Fig. 10 shows the results of an experiment where a number of concentrations of

252

CARDIAC Na-K PUMP ACTIViTY AND INTERNAL Na

acetylstrophanthidin, actodigin and dihydro-ouabain were tested in the same experi-ment. The experimental points were obtained from the initial maximum rate of aisaincrease (in a similar manner to that shown in Fig. 8B). The dose-response curve foracetylstrophanthidin was very similar to that of strophanthidin (Fig. 8C; see alsoTable 1). The dose-response curves for actodigin and dihydro-ouabain were similarto each other but were shifted to approximately tenfold higher concentrations whencompared with acetylstrophanthidin (Table 1).

0*75 r

- 050

EE'-i

at 0.25I

0

Acetylstrophanthidin0

Actodigin (v)Dihydro-ouabain (C)

- 0-

10 7

I 1

10 6 lo-,[Cardioactive steroid] (M)

J10o-4

Fig. 10. Dose-response curves (produced by the procedure illustrated in Fig. 8) for thecardioactive steroids acetylstrophanthidin, actodigin and dihydro-ouabain. All theexperimental points were obtained from one continuous micro-electrode impalement ofa single cell. Only one line has been drawn through the points obtained with actodiginand dihydro-ouabain because they lie very close together.

TABLE 1. The concentration of the cardioactive steroids that produce an increase of a' at halfthe maximal rate that can be achieved with very high cardioactive steroid concentrations(normally 10-5 M-strophanthidin and acetylstrophanthidin but 10-4-10-3 M-dihydrO-Ouabainand actodigin). When insufficiently high concentrations of dihydro-ouabain or actodigin wereused to produce a maximal effect on a'a (e.g. Fig. 10) the concentration has been calculated withreference to the rate in 10-, M-acetylstrophanthidin which was assumed to be 100% effective.However, in each of the four experiments concerned four concentrations of dihydro-ouabain andactodigin were tested over the range 10-7-10-4 M.

Cardioactive steroid

StrophanthidinAcetylstrophanthidinDihydro-ouabainActodigin

Concentration of cardioactivesteroid (M) producing 50% max

rate of aNa increaseMean (+S.D.)

854(±3-9) x10-7 n = 48-1(±5-5) x10-7 n = 46-3(±2-3)x106 n = 592(±11-8)x106 n = 5

-

253

J. W. DEITMER AND D. ELLISAt concentrations near to 108 M-acetylstrophanthidin produced a decrease in a'Na

similar to that described for strophanthidin (Fig. 8). Dihydro-ouabain and actodigin,at concentrations between 108 and 10-7 M, however, did not appear to produce adecrease of a... This effect of dihydro-ouabain would be consistent with the obser-vation (Ghysel-Burton & Godfraind, 1977) that low doses are unable to produce theincrease in theK content ofguinea-pig atria that is observed with low doses ofouabainand which is assumed to be due to Na-K pump stimulation.The concentration of acetylstrophanthidin just able to produce an increase in aiN

lay between 10- and 1o-7 M (i.e. like strophanthidin). The 'threshold concentrations'for actodigin and dihydro-ouabain were also near to 10-7 M. A cardioactive steroidconcentration was defined as below threshold in our experiments if no sign of a rise inaiN. was observed within 20 min of its addition.A maximal effect on the rate of a a rise was produced by approximately

10-3 m- dihydro-ouabain (see also Fig. 1, Deitmer & Ellis, 1978b), or actodigin. Thismaximum rate appeared to be the same as could be achieved with strophanthidin oracetylstrophanthidin (10-5 M and above).

DISCUSSION

Our results confirm that inhibition of the Na-K pump causes a rise of aNa in cardiacPurkinje fibres. The maximum rate of rise of a~a is probably the best indication of thedegree ofthe pump inhibition in our experiments. The results suggest that the removalofK0 produces only a partial inhibition of the Na-K pump compared with that causedby high concentrations of cardioactive steroids. The restoration ofKo initiated a rapidrecovery of aia as would be expected from an immediate activation of the Na-Kpump. The removal of any of the cardioactive steroids used caused a somewhatdelayed and much slower recovery of a'Na. This was probably due to the rather slowdissociation of these compounds from their inhibitory binding sites. However, incontrast to ouabain, the effects of even high doses of strophanthidin, acetylstrophan-thidin, actodigin and dihydro-ouabain were completely reversible within less than2 hr.

Recovery of aN.a from K-free solutionsThe rate of recovery of a1Na from K-free solutions was dependent both on the

concentration ofK added, and also upon the level that akNa had reached. This is com-patible with the idea that a.a recovery was due to an activation of the Na-K pumpand was thereby dependent upon both [K]o and aia. The results in Fig. 3 suggeststhat the [K]0 required to half maximally activate the Na-K pump is near to 10 mM.This value however, might be inaccurate for a number of reasons. (1) Purkinje fibreshave deep intercellular clefts, i.e. restricted spaces with only limited access to thebulk solution (Mobley & Page, 1972). The activity of the Na pump would tend toreduce the K concentration in these spaces (Cohen, Daut & Noble, 1976). If, e.g. in1 mM-K, the percentage reduction of the K concentration in these spaces (comparedto the bulk solution, [K]0) is more than at higher [K]0, then this could result in over-estimation of the Km value. This effect may be partially compensated for however,by a relatively larger accumulation of K due to leakage from the cells in low [K]o.(2) The value of the Km obtained in the present experiments would have been

254

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Na

influenced by changes in the passive Na influx. The membrane potential was approxi-mately 30 mV lower in 1 mM-K solution than in 6 mm-K. Thus the driving force forinward movement of Na was reduced. However, the depolarization in low K solutionhas been suggested to be due to a decreased K and perhaps an increased Na-permeability (Carmeliet, 1961; Gadsby & Cranefield, 1977). Such an increase in Napermeability would offset the effect of a reduction in driving force for Na therebyreducing the resultant change in net passive Na influx. In experiments designed tomeasure this influx (by the addition of 10-5 M-strophanthidin) there appeared to besome change in the rate of net entry in different [K]. (between 1 and 12 mM), whichindicates that ourKmvalue of 10 mm might be underestimated by up to 23 %. Anotherfactor that must be considered is the possibility that changes in a' a might not accu-rately reflect changes in the total Na content of the cell. If changes in the akN aresignificantly buffered by an alteration of the ratio of bound to free Na ions in thecell then this could have influenced the results obtained. However, it would beanticipated that any such change in this ratio would produce a more complicatedtime course of the decline of a'a than the single exponential relationship observed(e.g. Figs. 1 and 2).

Despite the difficulties in estimating the Km value, its value in Purkinje fibresseems likely to be higher than the 15 mm reported for atrial muscle (Glitsch, Grabow-ski & Thielen, 1978).The rate of aka recovery from K-free solution appeared to be linearly related to the

at a (e.g. Fig. 2). A linear relationship between the rate of active Na efflux and internalNa has also been reported for squid axons (Hodgkin & Keynes, 1956; Brinley & Mul-lins, 1968) and for snail neurones (Thomas, 1972) but a more complicated relationshiphas been suggested in skeletal muscle (Keynes & Swan, 1959; Brinley, 1968).The apparently linear relationship between the maximum rate of a' decrease and

the ak level shown in Fig. 5 may indicate that the Km for activation of this processby internal Na is very high (i.e. 30 mm or greater). It seems likely, however, that therelationship shown is not solely an indication of the activation of Na efflux via theNa-K pump mechanism. Recent experiments (Deitmer & Ellis, 1978a) suggest thatwhen the a'a is increased, the level becomes increasingly dependent on the [Ca]0,consistent with an exchange process where Na efflux is linked to Ca influx (see alsoReuter & Seitz, 1968; Baker, Blaustein, Hodgkin & Steinhardt, 1969). At veryhigh aa levels, the normal [Ca]o (2 mM) appears to produce a decrease of a'a via sucha Na/Ca exchange (Deitmer & Ellis, 1978a). The decrease of a'a measured in thepresent experiments at the highest ata levels would therefore be due to Na-K pump-ing and Na/Ca exchange. Thus, any saturation of the internal Na-K pumping sitesby Na might be masked by an increasing component of Na/Ca exchange which doesnot appear to show any signs of saturation even at these very high a' . levels (Deitmer& Ellis, 1978a, Fig. 11). The decrease in a'aproduced by Na/Ca exchange, however,has a much slower time course than that caused by the Na-K pump. The measuredrate of a'a decrease would therefore depend only to a very small extent on Na/Caexchange (at 2 mM-Ca).The rate of aka recovery may also be affected by other factors, e.g. changes in the

intracellularK would be expected to influence this rate. If, when aiNa is raised to highlevels, [K]1 decreases, then the Na-K pump would be anticipated to be even more

255

J. W. DEITMER AND D. ELLISactive on re-addition of K0. Experiments using intracellular K+-sensitive micro-electrodes, however, showed no, or very little change of internal K in K-free solutionswithin the first 30 min (Deitmer & Ellis, unpublished observations). Glitsch et al.(1976) estimated that activation of Na efflux, by internal Na from pre-cooledguinea-pig auricles had a Km of about 22 mm (equivalent to 17 mM-akNa). The Kmfor internal Na activation of pumping in intact erythrocytes was 24 mm (equivalent to18 mM-al), when changes of al were produced by using K as the substitute cation(Post, Merrit, Kinsolving & Albright, 1960).

The effects of cardioactive steroids

Strophanthidin, acetylstrophanthidin and dihydro-ouabain have been fairlywidely used in the past because they produce rapidly reversible effects. Actodigin(AY 22,241) is a semisynthetic cardiac glycoside (Deghenghi, 1970) that has beenreported to be even more rapidly reversible than dihydro-ouabain on the dog heartlung preparation (Mendez, Pastelin & Kabela, 1974) and much more rapidly reversiblethan acetylstrophanthidin on the electrical activity of Purkinje fibres (Pastelin &Mendez, 1972). Its reversibility on isolated cardiac Na-K ATPase has also beenrecently reported (Zavec & Dutta, 1977; Dutta, Zavec, Marks, Rhee, Brar, Richards& Bhat, 1974). In the present experiments actodigin has been shown to be similar todihydro-ouabain in its ability to produce an increase in ai~a (Figs. 9 and 10). The dose-dependence of actodigin and dihydro-ouabain indicates that approximately tenfoldhigher concentrations of these two cardiac glycosides are required to produce a givenrate of increase in aia than strophanthidin or acetylstrophanthidin (Figs. 9, 10,Table 1). The reversibility of the aia change on removal of any of the cardioactivesteroids depended on their concentration and the time for which they had beenapplied. In other words the time course of the recovery appeared to depend on theextent to which the ata had increased during the period of exposure to the cardio-active steroid.

After prolonged exposures to high cardioactive steroid concentrations the akNaappears to level off at values much lower than those anticipated if the a'Na was

going to reach the level predicted from its electrochemical equilibrium (e.g. Fig. 7).This 'plateau' level depended on the cardioactive steroid concentration and appearedclosely related to the maximum rate of aia increase measured in the first 10-15 min

of exposure to the steroid. The term 'plateau' is used to indicate the level the aikareaches after 1-2 hr in the presence of the cardioactive steroid, when the aia hasalmost completely stopped increasing. The aka often continues to increase over longerperiods (5-10 hr) but the rate of increase is very slow. This 'plateau' was shown notto be due to re-stimulation of Na-K pump activity. The removal of KO under theseconditions produced no further increase and sometimes even a decrease of a'Na(Deitmer & Ellis, 1978c). Similarly, the addition of much higher cardioactive steroidconcentrations only had a minor effect on the rate of a'a increase (Fig. 7). One factorthat does influence the'plateau' level, however, is the [Ca]0 (Deitmer & Ellis, 1978 a),consistent with the presence of a Na/Ca exchange mechanism. If Ca0 is replaced byanother divalent cation or Tris in this 'plateau' level, the aka increases at a rapid rate(see e.g. Fig. 7, Deitmer & Ellis, 1978a). This rate often is slightly slower than on theinitial addition of strophanthidin, but only by an amount equivalent to the reduction

256

CARDIAC Na-K PUMP ACTIVITY AND INTERNAL Nain the Na electrochemical gradient. This therefore suggests that the tendency for theata to reach a relatively low 'plateau' level upon complete inhibition of the Na-Kpump is mainly due to an exchange of internal Na for external Ca. Recent experi-ments, where the intracellular pH of Purkinje fibres was measured under similarconditions, suggest that movements of H+ ions may also be involved in maintainingthis a' ' plateau' (J. W. Deitmer & D. Ellis, in preparation).We have assumed that the maximum rate of ata increase measured from experi-

ments like that shown in Fig. 8 is a reasonable estimate of the Na-K pump inhibitionby the various cardioactive steroid concentrations. This type of analysis would beinaccurate if the binding of the cardioactive steroid was very slow as the maximumrate of aka increase might then also partly depend on its rate of binding. This isunlikely to be the case however, as the maximum rate of a'a increase was attainedwithin the first 2-8 min after addition of the cardioactive steroids. The binding of[3H]ouabain to cardiac muscle has been reported to have a time constant of approxi-mately 5 min at 30 0C even at the low concentration of 2 x 10-7 M (Kuschinsky,Lullmann & Zweiten, 1968). This binding probably includes specific binding to pumpsites as well as a slower non-specific tissue binding (Baker & Willis, 1970). The rate ofspecific binding would therefore presumably be faster than 5 min, and faster still atour experimental temperature of 35 'C. Erdmann & Hasse (1975) found that thebinding of ouabain to Na-K-ATPase prepared from cardiac tissue follows secondorder kinetics. Their results suggest that the process has a pseudo-first order rateconstant of 4 6 x 10-3 sec-1 (time constant 218 sec) at an ouabain concentration of10-7 M at 37 0C. Furthermore, the binding ofstrophanthidintoisolatedNa-KATPasehas been found to be appreciably faster than that of ouabain (Yoda, Yoda & Sarrif,1973). It would also be anticipated that the dose-response curves in our experiments(Figs. 8 and 10) would be spread over a larger concentration range if the maximumrate of aka increase was substantially affected by the rate of cardioactive steroidbinding. It seems likely therefore, that the majority of binding in our experimentsoccurs within the first 2-8 min of exposure to the cardioactive steroid even at thelowest concentrations used.The rate of a a increase produced by maximally effective cardioactive steroid

concentrations probably reflects the rate of the net passive Na influx into the cells.This was calculated to be 2-8 p-moles/cm2 sec from the maximum rate of a¶N. rise thatcould be achieved on addition of the steroids. Under normal conditions, where the aNais kept stable at a low level, the Na-K pump would therefore have to pump at a rate of2*8 p-mole/cm2 sec. The fastest rate of a a decrease during recovery from K-freesolutions in the present experiments (Fig. 5) was up to 10 times faster than this value.In guinea-pig atria the normal rate of active Na efflux has been reported to beapproximately 8 p-mole/cm2 sec and could be increased up to a maximum of 3-4times this normal level following conditions of Na loading (Glitsch et al. 1976). Thissuggests a similar maximum rate of active Na extrusion in Purkinje fibres and atrialmuscle.

It is apparent from Fig. 8 that very low strophanthidin concentrations (10-8-10-7 M) can actually decrease the at a. A transient decrease can be observed at higherconcentrations before the aNa begins to increase. Low concentrations (near to 108 M)of ouabain can cause a similar decrease in a'a (Ellis, 1977b), and also produce an

9 PHY 284

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J. W. DEITMER AND D. ELLIS

opposite effect to high ouabain concentrations on the reversal potential of the iK,current in Purkinje fibres (Cohen et al. 1976). These and even lower ouabain concen-trations have been reported to have a positive inotropic effect on Purkinje fibres(Blood, 1975; Blood & Noble, 1977). Thus, it seems unlikely that the positive ino-tropic effect at these low ouabain concentrations is produced by Na-K pump inhi-bition and subsequent rise of aka. Higher ouabain concentrations do produce largerpositive inotropic effects (Blood, 1977) but these are normally irreversible as con-tractures or after-contractions develop. It seems likely that if the ai , is raised to highlevels by high cardioactive steroid concentrations then this could result in these toxicand, with ouabain, irreversible effects.We suggest that the use of either strophanthidin or acetylstrophanthidin as a tool

to produce a maximum rate of akNa increase, and therefore presumably a maximuminhibition of the Na-K pump, is preferable to the use of the other cardioactive steroidstested in the present study. Strophanthidin and acetylstrophanthidin have theadvantage of (1) being maximally effective at much lower concentrations thanactodigin and dihydro-ouabain, and (2) being readily reversible, in contrast to ouabain.

We are very grateful to Dr R. C. Thomas for his interest and encouragement throughout thiswork. We also thank him and Drs V. L. Lew and D. J. Miller for their comments on an earlyversion of the manuscript. The financial support by the Medical Research Council (grant to R. C.Thomas) and the Deutsche Forschungsgemeinschaft (grant to J.W.D., De 231/1, 2) is gratefullyacknowledged.

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