J. Physiol. (1975), 246, pp. 397-420

397

With 9 text-figure8 Printed in Great Britain

THE INTERACTION OF LITHIUM IONS WITH THE SODIUM-POTASSIUM PUMP IN FROG SKELETAL MUSCLE

BY LUIS BEAUGIR* From the Department of Biophysics, University of Maryland, School of Medicine, Baltimore, Maryland 21201, U.S.A.

(Received 12 August 1974) SUMMARY

1. The effects of external Li on Na and K efflux as well as those on K influx were studied in high Na muscles from Rana pipiens. 2. In the absence of external Ba, substitution of K-free Li for K-free Mg resulted in an increase of both Na and K efflux. The addition of ouabain produced an inhibition of Na efflux and at the same time a marked increase in the efflux of K. 3. K permeability was greatly reduced by adding 2 mM-Ba to the incubation solutions. Under these conditions, Li gave rise to a ouabain sensitive Na efflux which was 57 % of that in the absence of Ba. On the other hand, the efflux of K was only slightly increased and was not affected further by ouabain. 4. The activation curves of Na efflux against the stimulating cation concentration in Na-free Mg-Ba Ringer followed a more or less hyperbolic function for both K and Li. While half-maximal activation was attained at higher concentrations of Li than of K, the maximal efflux in Li was smaller than in K. 5. The extra Na efflux produced by K was increased when Li was added to the media. This increment was not a simple additive effect and was independent of the Li concentration. In addition, at some concentrations Li increased the ouabain-sensitive K influx, whereas at others it reduced it. 6. Reversible changes in membrane permeability to monovalent cations were accomplished by incubating the muscles in the presence of Nystatin, 50 /tg/ml. When internal K was reduced to values around 1-2 jtmole/g (using Li as a replacement), thus minimizing the possibility of K leaking out of the cells, both K0 and Lio were able'to promote a ouabain-sensitive extra efflux of Na. 7. The residual Na efflux in (K + Na)-free solutions was not affected by * Member of the Consejo Nacional de Investigaciones Cientificas y Argentina.

Tecnicas

of

LUIS BEA UG.9 398 the removal of Ca from the media in either Mg or Li solutions, both in the absence and the presence of Ba. On the other hand, the values for the residual efflux were higher in Mg (0-00228 min-') than in Li (0.00135 min-'). 8. These results fully support the notion that Li ions have a K-like activating action on the Na pump in muscles. In addition, they suggest that some other kind of interaction may exist between Li and the Na-K pump. INTRODUCTION

In fresh frog muscles with normal Na content, the replacement of external Na with Li produced a noticeable reduction in the efflux of Na (Keynes & Swan, 1959). This effect, also seen in choline, was taken as experimental evidence that some of the Na translocation through muscle cell membranes was due to an exchange diffusion mechanism (Ussing, 1949). On the other hand, in frog sartorius with elevated internal Na, the transfer from a K-containing sodium Ringer to a K-containing Li Ringer always produced an increase in Na efflux (Keynes, 1966; Beauge & Sjodin, 1968; Keynes & Steinhardt, 1968). If ouabain was present in the media, however, the increase was replaced by a marked reduction in the efflux (see the same references). Similar results were obtained if instead of Li, either Mg or Ca was used as a Na replacement (Horowicz, Taylor & Waggoner, 1970). If K was removed from the bathing solutions, only Li was able to produce an increase in Na efflux, whereas both Mg and Ca always brought about a reduction in Na efflux (Beauge & Ortiz, 1972). A reduction similar to that in Mg or Ca was also seen in Li Ringer, provided ouabain was added to the media. The foregoing, together with the fact that Li was able to promote net Na extrusion against an electrochemical gradient (even under conditions where the changes in free energy favouring Li entry were lower than those opposing Na exit), strongly support the notion that the Li stimulation of Na efflux is due to a direct activating effect on the Na-K pump through a K-like action. However, the idea that all extra Na efflux which occurs in Na-free Li Ringer can be attributed to a direct Li activating effect is challenged by the characteristics of both the Na pump and K fluxes in frog muscle as recently described. On one hand, there is persuasive evidence (Sjodin & Beauge, 1973) that muscles can recapture large amounts of the K leaking out of the cells in Na- and K-free conditions through the activation of the Na-K pump. On the other hand, it has been shown recently in fresh muscles (R. A. Sjodin & S. C. Wu, unpublished) that the magnitude of K efflux in K-free solutions strongly depends on the principal cation in the media. Thus the lowest values for K efflux were obtained in Mg; in Li Ringer they rose about threefold. For amultifibre system such as the frog sartorius, this could

399 Li IONS AND Na PUMP lead to a considerable accumulation of K close to the pump sites; as a consequence, part of the extra Na efflux could be due to a K rather than a Li activation of the Na pump. To resolve this question two approaches may be used: to reduce K leak and make it independent of the cationic composition of the media, or to have muscle cells with little or no K content. Both approaches were applied in the present work with an emphasis on the first one. To reduce K efflux, advantage was taken of the observation by R. A. Sjodin & S. C. Wu (unpublished) and Sjodin & Ortiz (1974) that at concentrations of a few millimoles per litre Ba considerably reduced passive K fluxes, making them insensitive to the ionic composition of the media, while it did not appreciably affect the functioning of the Na-K pump. METHODS Sartorius muscles of Rana pipiens were carefully dissected and mounted in a wire frame at about 120 % their resting length.

Solution All solutions were prepared with de-ionized water and reagent grade chemicals. The composition was as follows (mM): (a) K-free Na Ringer: NaCl, 120; CaCl2, 2; Tris-Cl (pH at 200 C = 7.4), 1; (b) K-free Li Ringer: LiCl, 120; CaCl2, 2; Tris-Cl, 1; (c) K-free Mg Ringer: MgCl2, 86-6; CaCl2, 2; Tris-Cl, 1. The total osmolarity was 231 m-osmole. When K was present, the principal cation was reduced to keep the total osmolarity constant. Different Li concentrations were obtained by adding appropriate amounts of Mg and Li Ringer. Ba was added as BaCl2 at a concentration of 2 mm. Ouabain (Sigma Co.) was added as solid at the moment of use. 22Na and 42K (New England Nuclear) were obtained as chloride salts of high specific activity; the pH was adjusted to about 7 0 before use.

Sodium and potassium fluxes and cation content of the muscles The experimental procedures were identical to those described in detail elsewhere (Beauge' & Ortiz, 1972; Sjodin & Beauge, 1973). K uptake was expressed in 1smole/g wet wt. hr, Na and K efflux as the rate of isotope loss in min-. Radioactivity was assayed in an automatic Packard gamma counter; when the counts were low they were reassayed in a low background gas flow Beckman counter. For the doublelabelled experiments, 1 week was allowed before the second counting of the samples. The cation content was expressed in ,umolefg wet wt. Temperature was 20 0C. Cation loading of the muscles High Na muscles were obtained as described elsewhere (Beaug6 & Ortiz, 1972) by incubating them for 24 hr at 30 C in K-free 120 mM-Na Ringer. In cases where both Na and K content were to be altered, the technique proposed by Cass & Dalmark (1973) for red blood cells was used with some modifications. The muscles were incubated for 2-3 hr at 20° C in an appropriate solution containing Nystatin at 50 #ug/ml.; this antibiotic had been previously dissolved in methanol at a concentration of 5 mg/ml. The replacement of internal K was made with Li. As this

400

LUIS BEA UG-9

procedure increases both monovalent cation and anion permeability the net gain of XCl and water had to be avoided. For this reason sulphate was used as an external anion. The cation composition of the solutions was as follows (mM): (a) low K muscles: Na 40, Li 100; (b) K control muscles: Na 40, K 100. CaSO4 concentration was 8 mm in all cases and the pH was maintained at 7-4 (200 C) with 1 mM-Tris sulphate. The total osmolarity was matched to 231 m-osmole with sucrose. Each muscle was incubated in 6 ml. solution which was changed every 15 min during the first hour, and every hour thereafter. During the last hour 22Na was added. The resealing was obtained by incubating the muscles in K-free Mg Ringer for 1 hr, changing tubes every 5 min. At the same time radioactive Na was washed out of the extracellular space, followed immediately by the Na efflux experiment. As all solutions were Na-free, using the final Na content and the specific activity, the cellular Na could be determined at any given time. Estimation of the initial K and/or Li contents was managed by analysing muscles submitted to the same treatment at the time the efflux began and comparing these values with those of the efflux muscles at the end of the experiment. The main disadvantage of this technique is that it produces (as it can be seen in Table 2), a net loss of total cations which is of course larger the longer the incubation time. Na loading periods as short as 15-20 min sufficed to get values about 20-25 #tmole/g. For total K replacement 2-3 hr were required. Changes in the membrane potential were followed in some cases, but they were not taken as criteria for a healthy preparation. The membrane depolarized immediately after the immersion in Nystatin to values around 0 mV regardless of the cationic composition of the solution. After the resealing the potential did not recover completely but it was usually above -70 mV. In some cases after 3 hr, and in all cases after 5 hr, muscles became opaque. These were disregarded. The criteria for viability were the following: (a) low rate of Na loss in K-free Mg, (b) stimulation of Na efflux by either external K or Li to values similar to those found in intact muscles, and (c) inhibition by ouabain of the K or Li stimulation. As a double check whereas in one pair the effect of external Li was investigated, in another pair K (at 2-5 mm concentration) was used. RESULTS

Na and K efflux into potassium-free Mg and Li-substituted Ringer in the presence and absence of 2 mM Ba The effect of K recapture in muscles produces a very low rate of K loss from high Na muscles incubated in K- and Na-free Mg solutions (Sjodin & Beaug6, 1973). When the Na-K pump is inhibited by 10-4 ouabain the rate of K loss increases about tenfold. It would then be expected that replacement of external Mg by Li would produce an increase in K efflux (R. A. Sjodin & S. C. Wu, unpublished). However, if at the same time there were an increase in K recapture, the extra K efflux would not be seen or it would be expected only in part, unless ouabain were added. To test this point, and to try to separate any possible K +Li combination effect from a true Li effect, the action on both Na and K efflux of incubating muscles in the sequence K-free Mg, K-free Li, K-free Li-ouabain was studied in the absence and presence of 2 mm-Ba in the media. The results are summarized in Fig. 1 and Table 1. In the absence of Ba, Li increased Na efflux and, as shown before (Beauge & Ortiz, 1972) this was inhibited by ouabain. At

Li IONS AND Na PUMP 0-K-Mg

I

0-K-Li

401

O-K-Li-ouabain

002r Na

Control 0-01 I-

._E

4_o 0

I

I

aI) 0 0.

12 k 0

K X 102

C

0

'U

Control

LL

0-08 _

004 I-

0

0

v}

-

0

50

-

I-

100

150

Time (min)

Fig. 1. Double isotope labelled experiment (22Na and 42K) onthe comparative effect of K-free Li and K-free Mg Ringer on Na and K efflux from high Na muscles incubated in the absence (open circles) and presence (filled circles) of 2 mM-Ba. Both muscles were dissected from the same frog and stored for 3 hr at room temperature in 5 mM-K, 120 mm-Na Ringer with 10 lc of 42K/ml. They were then transferred to 50 ml. 0-2 mM-K, 120 mM-Na Ringer with 42K of the same specific activity as before, and stored for 20 hr at 30 C. The loading with 22Na was made in 8 ml. of the same solution containing in addition 6 flc/ml. of 22Na for 3 hr at room temperature. Na and K efflux were measured at 200 C.

402 LUIS BEA UGI the same time, Li also produced an increase in the efflux of K which averaged about fivefold; when ouabain was added, the inhibition of Na efflux was accompanied by a further increase in K efflux, which now averaged elevenfold of the initial rate. In the presence of 2 mM-Ba the TABLE 1. Effect of external Li on the labelled Na and K loss

Muscles were made Na rich by cold storage and loaded with 42K and 22Na as described in the legend of Fig. 1. Na and K efflux were measured at 200 C. Values in corresponding rows are for muscles obtained from the same frog incubated in Ba-free or 2 mM-Ba containing solutions. The increment in the rate of Na loss was 0-0092 + 0-0013 min-' in Ba-free and 0-0052 + 0-0004 min-1 in Ba. The values in the last two rows correspond to two experiments of the type described in Fig. 3 Fraction of isotope lost per minute ~~~~~~~A-

f

22Na

Ba-free

Mean S.E. 2 mM-Ba

Mean S.E.

Mg 0-0032 0-0034 0-0030 0-0031 0-0035 0-0042 0-0034 + 0-0002

Li 0-0170 0-0092 0-0106 0-0110 0-0152 0-0122 0-0125 + 0-0012

0-0023 0-0023 0-0024 0-0025 0-0022 0-0028 0-0024 + 0-0002

0-0085 0-0069 0-0064 0-0072 0-0089 0-0084 0-0077 + 0-0004

42K (x 100) Liouabain 0-0015 0-0016 0-0014 0-0015 -

Mg 0-0110 0-0103 0-0074 0-0097

Li 0-0814 0-0286 0-0411 0-0398

0-0015 0-0096 + 0-00005 + 0-0008

0-0477 + 0-0115

0-0015 0-0012

0-0010 0-0012

0-0012 + 0-0001

0-0048 0-0065 0-0055 0-0055 -

Liouabain 0-1285

0-1000 0-1036 0-0934 _ 0-1064 + 0-0077

0-0090

0-0116 0-0071 0-0079 0-0105

0-0077

0-0110 0-0085

-

0-0056

0-0091

+ 0-0004

+ 0-0007

0-0093 + 0-0011

picture was quite different. While the effects on Na efflux were qualitatively the same, with Li stimulation averaging 57 % of that in the absence of Ba, the efflux of K was only slightly increased in Li, and the rate of loss was unmodified by the addition of ouabain to the media. If one assumes that the Na-K pump is slightly affected by Ba, the preceding experiments are quite illustrative. At the same time they strongly indicate a direct stimulating effect of Li on the Na pump as proposed previously (Beauge & Sjodin, 1968; Beauge & Ortiz, 1972); they also suggest that part of the

Li IONS AND Na PUMP 403 extra Na efflux in K-free, Na-free conditions is due to the accumulation of K close to the pump sites, that is to K recapture by the muscle cells. To investigate this matter further, sequences of solution change similar to those reported before with regard to their effect on Na efflux (Beauge & Ortiz, 1972), were repeated in the presence of 2 mM-Ba in the media. These experiments are described in Figs. 2 and 3. In K-free conditions the substitution of Li for Na resulted in an increase in the Na efflux, in both the absence and the presence of Ba. Although in the latter case all fluxes TABLE 2. The effect of external Li and K on Na efflux from muscles depleted of internal K The cation loading of the muscles was accomplished by a modification of the Nystatin method and it is described in detail in the text. The sodium content at zero time was calculated on the basis of the final specific activity of Na in the muscles. The estimates of the K and Li contents at zero time are approximate and were made on the basis of the cation content of muscles under similar treatment at the time efflux began, and on the basis of the final cation content of those actually used in the experiment. The same number and letter correspond to muscles dissected from the same frog. Temperature was 20° C Fraction of 22Na lost

Cation content at t = 0

(jsmole/g)

(min-1) r

Expt. 29A 29A' 31A 31A'

31B 31B' 37A

37A'

0-K-Mg 0-0072

0-0038 0-0073

0-0046

~~A

O-K-Li 0-0162 0-0148 0-0183 0-0135

Na

0-0030 0-0021

13-1 12-2

0-0035

0-0025

2-5-K-MgO-K-Mg 2-5-K-Mg ouabain 0-0065 0-0173 0.0042

0-0037

0-0205

,A

0-K-Liouabain

0-0031

0-0080

0-0240

0-0050

0-0038

0-0300

0-0026

A

28-0 21-0

K 54 5 35 1

Li 0 60 0 35

25-6 22-9 17-8 16-0

35 1 60 2

0 35 0 60

were somehow reduced, the net increment due to Li was about 70 % of that in Ba-free conditions. The experiments at the bottom of Figs. 2 and 3 are qualitatively exact reproductions of those reported by Beauge & Ortiz (1972). When passing from a K-free Na to a K-free Mg solution in the presence of 2 mM-Ba, there was a reduction in Na efflux by inhibition of the Na-Na exchange mechanism, whereas Li brought about an increase. As before, this Li-stimulating effect was reversible. As K fluxes were shown to be unmodified, this stimulation of Na efflux must have been a 14

PH Y

246

LUIS BEA UGf 404 consequence of an activation of the Na pump by Li. It is also interesting to note that in the absence of Li, Na fluxes were also lower in Ba-containing solutions (about 25-30 %). In Mg Ringer this could be interpreted as a reduction in K recapture which followed the reduction of K loss. However, 0-K-Li

0-K-Na

0

.0010r-

0

0

0

Control 0

2 mm -a

0005k

C

-E #A

.2

I

0

z

l

1/ "lo

N

l

2 mM -Ba

0 C

0

*-(U 041 310

1-

U-

0-K-Li 0-K-Na

0-005

F-

0-K-Mg

0

'If

III

I 0

t

60 Time (min)

120

Fig. 2. Effect of 2 mM-Ba on the response of the Na efflux from high Na muscles to the replacement of external sodium by Mg or Li in K-free conditions. In AA' (top) the sequence 0-K-Na-0-K-Li was studied in the absence (open circles) and presence (filled circles) of Ba. In BB' (bottom) the effect of Li (open circles) and Mg (filled circles) was studiedin Ba-containing media. Muscles with the same letter were dissected from the same frog and made Na-rich by cold storage. Temperature during efflux was 200 C.

Li IONS AND Na PUMP 405 as similar inhibitory effects were obtained in K-free Na, this could be a consequence of a real inhibition of the mechanisms) of Na efflux, Na pump included. The K concentration lodged in the interfibre space will not be the same over the entire population of fibres, but it is expected to be higher close to the inner than to the outer fibres. However, for an average effect 002 r

0-K-Li

0-K-Mg

0-K-Mg

C

E 4-

0

z

0 01 Control

0 C

0

'U U-

2 mM -Ba

0

l

l

30

60

90

Time (min)

Fig. 3. Effect of the substitution of external Li for Mg and vice versa on the Na efflux from high Na muscles in the absence (open circles) and presence (filled circles) of 2 mM-Ba. Both muscles were dissected from the same frog and made Na rich by cold storage. Efflux temperature was 200 C.

on Na efflux an average K concentration which is responsible for it can be imagined. Mullins & Noda (1963) have estimated this K accumulation to be of the order of 0-2 mm. If the reduced Na efflux seen in both Mg and Li when Ba is present is due to a prevention of K accumulation, then the addition of 0-2 mM-K to a Mg-Ba Ringer should give an efflux of Na 14-2

406 LUIS BEA UGJ2 similar to that observed in Mg Ba-free conditions. In addition, the stimulation produced by Li in both K-free and 0-2 mm-K conditions should be the same. Fig. 4 shows one of the experiments performed to test this point. As can be seen, all the expectations were borne out. The only difficulty in the interpretation of these results is the fact that (as also shown in Table 1) Na efflux in Li-ouabain was lower than in Mg without the glycoside. This finding will be treated in later sections. 0015 r Li-Ba

Mg-Ba

Li-Ba-ouabain

*.sI 0.010 0

z0 en

C40

a,, 0 005jF

LL

K-free

0

,

.

l

l

l

0

50

100

150

Time (min) Fig. 4. Typical experiment showing the effect of 0-2 mm external K on Na efflux in Mg and Li Ringer solutions containing 2 mm-Ba. Both muscles were dissected from the same frog and made Na rich by cold storage in K-free 120 mm-Na. Ouabain was at 10-4 M concentration. Temperature was 200 C.

Li effect on Na efflux in the absence of Ba in muscles depleted of internal K The results of the previous section strongly indicate that Li ions by themselves are able to stimulate the Na pump in muscles. However, to avoid the interference of K leaking out of the cells, Ba had to be used. Since some of these results as well as others already reported (Sjodin & Ortiz, 1974) also suggest an effect of Ba on the Na fluxes, it could be that the observed reduced stimulating action of Li in Ba is biased by the presence of the latter cation. To avoid both the Ba side effect and K

Li IONS AND Na PUMP 407 stimulation of the pump, it would be ideal to use K-depleted cells. However, the usual loading technique of cold storage makes it extremely difficult to accomplish a complete or nearly complete K depletion while preserving the good condition of the muscles. Thus, as an alternative the cation loading technique proposed by Cass & Dalmark (1973) for red cells was adopted with modifications (see Methods). In itself, the technique is in the first stages of development for muscles. However, in the cases where 002r

0*01

0-K-Mg

I

O-K-Li-ouabain

O-K-Li

-

a

E l

I6-

0

z

0-K-Mg

0

I

I

I

2-5-K-Mg

25 K-Mg-ouabain

)2

.o u

U-

001 F-

0

P'OO

l

1

l

30

60

90

Time (min) Fig. 5. Effect of 120 mM-Li (top figure) and 2-5 mM-K (bottom figure) on the Na efflux from control muscles (open circles) and muscles depleted of internal K (filled circles) incubated in Na-free media. The cation loading of the muscles was accomplished by a modification of the Nystatin method. Each half figure refers to paired muscles from the same frog. Temperature was 200 C. See text for details.

LUIS BEA UG.9 408 it succeeded, there is indication that it could be a useful tool for controlling intracellular cations. The results of these experiments are shown in Fig. 5 and Table 2. As far as Li is concerned the extra Na efflux stimulated over the base line in K-free Mg was the same, on the average, regardless of the K content of the muscles. Without the results with potassium activation this could have been taken as indicating that barium indeed affected largely the Na pump, reducing the Li effectiveness. But by looking at the K-activated Na efflux experiments, for comparable values of internal Na and K, one can see that the extra efflux in 2-5 mM-K was 1-6 times larger for low K cells. This would indicate that the similar extra stimulation obtained with Li was a consequence of an extra sensitive state of the Na pump to external activating cations when internal K was reduced to very low values. An increase in internal K above the normal levels has been shown to reduce the rate of ouabain-sensitive sodium extrusion (Chaplain, 1973). However in that case no figures for internal Na were given. In the present experiments, for similar values in internal Na the efflux of Na was 1-6 times more sensitive to 2-5 mm external K when K, was 1-5 ,umole/g than when it was around 50 molel. In this way these results are consistent with an inhibitory effect of internal K on the Na pump in muscle. However, as the reduction of internal K was accompanied by an introduction of Li into the cells, some stimulating effect of Li from the inside (by itself or in combination with the reduction of K) cannot be completely ruled out. Also the membrane potential must have been quite different in both groups of muscles, and this may have played an important role in the behaviour of the fluxes.

The rate of 22Na loss in K-free Mg in K-containing cells was 0-0073 + 0-0003 min- (K1 = 46 + 6-5 ,umole/g), a good deal higher than the value of 0-0034 + 0-0002 obtained in cold loaded cells (see Table 1). When the internal K was 2-3 + 0-9 /zmole in the Nystatin-treated muscles, the rate of Na loss was reduced to 0-0040 + 0-0002 min-. The difference between low Ki and control Nystatin-treated muscles could be due to the K leaking out of the cells. However, as the values for low Ki were still higher than in the cold loaded muscles, this would mean that sodium permeability remained higher than normal after resealing. This could account for the lack of complete recovery in the membrane potential in these muscles. The main conclusion which can be extracted from the foregoing is that the stimulating effect of Li ions on sodium efflux (which is ouabain sensitive) is also present when the internal K of the muscles is reduced to values so low that K recapture can be disregarded. The assertion that Li activated the Na-K pump seems inescapable. Also it seems that the presence of 2 mM-Ba in the media does not modify the Na-K exchange to an appreciable extent. For this reason, and the simplicity in the methodology, Ba was used in the following experiments.

Li IONS AND Na PUMP

409

Activation of Na efflux by external K and Li in Na-free Mg Ringer containing 2 mM-Ba Since addition of Ba to the incubation solution allowed a clear separation between the K and the Li effects on Na efflux, and as a further check of any influence of Ba on the Na-K pump, the activation curves of Na efflux as a function of the stimulating cation concentration were studied for both K and Li. The concentration of the former was followed up to 10 mm, and for the latter up to 120 mM; the total osmolarity was C

0.03 Ki

0

(A

Z/ 0

C4

0 U

-C" 0 01

/t

C

X

E U

C

0

0 I

0

I

5

K 10 I Li 120

40 80 External concentration (mM) Fig. 6. Activation curve of K (open circles) and Li (filled circles) stimulated Na efflux from high Na muscles incubated in Na-free Mg Ringer shown as a function of the concentration of the activating cation. In all cases except 110 mM-Li, one sartorius was used for K and the mate for Li activation. Muscles were Na enriched by cold storage. Each point is the mean + S.E. of mean of two to four determinations. Ba was present in all cases at 2 mm concentration. Temperature was 200 C.

matched to 231 m-osmole with MgCl2. In all cases except 110 mM-Li, one sartorius was used for K and the mate for Li. The muscles were made Na rich by cold storage as described in Methods. The results are summarized in Fig. 6 where each point is the average + S.E. of mean of two to four determinations. Both activation curves went to saturation in a more or less hyperbolic fashion. The reciprocal plots of 1/v vs. 1/s were better fitted by the K curve whereas that in Li gave more dispersion. The values obtained for the kinetic constants were Km of 3-2 mm and Vmax of 0 0300 minfor K and Km of 21 mm and Vmax of 0x0065 min' for Li. Interestingly

LUIS BEAUGJ enough Li ions not only showed a small apparent affinity for the pump site(s) but were unable to reach the same maximal activation as K. This is very clear from the Figure. This is consistent with the Li activation of nerve membrane ATPase (Skou, 1960) and has also been observed in the Cs activation of ouabain-sensitive Na efflux in squid axons (Baker, Blaustein, Keynes, Manil, Shaw & Steinhardt, 1969b) and frog muscle (L. A. Beauge & R. A. Sjodin, unpublished). 410

0015 _ A

004

B

2 mM -Li

20 mM

00C03 0010:

-Li Li-free

L~~~~~~~~~i-free

E

N

~~~~~~~0 12 K ° 0~ 00002~0-K-M

0-K-Mg

10 K-Mg

60

120 30 60 Time (mmn) Fig. 7. A, effect of 2 mM-Li (filled circles) as compared with Li-free conditions (open circles) on the activation of Na efflux by 1*2 mMi external K. B, effect of 20 mM-Li filledd circles) as compared with Li-free conditions (open circles) on the activation of Na effiuxr by 10 mM-K. The incubation solutions were Na-free Mg-substituted Ringer containing 2 mM-Ba. The pair of muscles from each Figure was dissected from the same frog and made Na-rich by cold storage. Temperature was 200 C.

A relevant question to be asked is why doesn't Li reach the sameon as K. One possibility is that, at the same time it activates the Na-K pump by some unknown mechanism, it also inhibits it. If this is the case, does it also inhibit the K activation? Part of the answer could come from the experiments nmthe first section where in Ba-free conditions Li did not prevent a pump activation by a presumably small amount of K. To analyse this problem in more detail the effect of different concentrations of external Li on the K-activated Na effiux was studied at two external K concentrations: 1-2 mw(low relative to the apparent Kin) and 10 mi (close to saturation). Na efflus was followed from both sartorii of the same frog in K-free Mg-Ba; after 90 mi both went into solutions of the same K concentration, one with and the other without Li. Fig. 7A and B shows two typical experiments and Table 3 summarizes all of them. At all K and Li con-

Li IONS AND Na PUMP 411 centrations tested, the activation of Na efflux was larger in those media containing Li. This effect did not seem to depend on the Li concentration over the range between 2 and 100 mm. In percentage, the activation was larger at 1P2 mM-K; in absolute values it was larger at 10 mM-K TABLE 3. Effect of external Li on K-stimulated Na efflux Muscles were made Na rich by cold storage in K-free 120 mm-Na Ringer for 24 hr. The radioactive Na loading was accomplished by incubating them for 3 hr at room temperature in the same solution containing 6 uc/ml. of 22Na. Prior to the efflux in K or (K +Li) media, an efflux period of 90 min was followed in K-free Mg-Ba Ringer; the average rate of Na loss in this solution was 00026 + 0.0001 min-' (n = 20). Muscles with the same number and letter were dissected from the same frog. Temperature was 20° C

Experiment

[K]0 (mM)

[Li].

34A 34A' 34B

10 10 10 10 10 10 10 10 10 10 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2 1-2

0 2 0 2 0

34B' 34C

34C' 34D 34D' 34E 34E' 35A 35A' 35B 35B' 35C

350' 35D 35Y 35E 35E'

20 0 20 0 100 0 2 0 2 0 20 0 100 0 100

Increment in (K + Li) increment 22Na loss (mm) (min-') K increment 0-0305

0-0360f 0-0339 0-0362 0-0317 0-0349 0-0328

1-18 1-107

1-10

0-0370 f 0-02921 0-0336f 0-00891 0-0107f 0-01021 0-0127

1-13

0-0094

1-38

0-0130 0-0077 0-0104f 0-0090 0-0111

1-15 1-20

1.25

1-35

1-23

(0-0039 + 0-0005 minm- against 0-0025 + 0-0003 min-' in 1-2 mm-K). From the data in Fig. 6 and on the basis of a simple additive effect, one would expect almost no change at 10 mM-K at any Li concentration. On the other hand, for 1-2 mM-K one would expect a negligible increment in 2 mm-Li and an average increment of 0-0060 min- at 100 mM-Li. None of these expectations was borne out.

LUIS BEAUGJ9

412

Effect of external Li on K influx In view of the results of the previous section a logical step was to analyse the effects of external Li on the uptake of K through the Na pump in order to correlate them with those on Na efflux. The basic experimental design consisted of two pairs of Na-enriched muscles (AA'-BB') which were incubated in Mg Ringer containing 2 mM-Ba, 42K and fixed K TABLE 4. Effect of external Li on ouabain-sensitive K uptake (,tmole/g.hr)

Muscles were made Na-rich by cold storage in K-free Na Ringer. Before the uptake began they spent 90 min in K-free Mg-Ba Ringer to make these results comparable with those in Table 3. The uptake period lasted 1 hr with points taken at 10 min intervals during the first half-hour and at 15 min intervals during the second halfhour. Values in the same row correspond to muscles obtained from the same frog. To obtain the oubain -sensitive potassium uptake the average ouabain -insensitive uptake in Li or in Li-free media was subtracted from the corresponding individual values of the total influx. The ouabain resistant K influx was the same in Li containing and Li-free solutions and had the values of 0-07 + 0-003 4Umole/g. hr (n = 10) at 1-2 mm-K and 1-48 + 0-09 /zmole/g.hr (n = 7) at 10 mm-K. Temperature was 200 C 2 mm-Li 20 mm-Li 60 mm-Li A Li-free value Value Difference Value Difference Value Difference 1-2 mm-K 7-90 8-40 7-30 4-22 5-87 6-62 6-50 7-90

8-94 9-70 7-30 -

27-3 30-0 28-1

28-4 31-0 -

+1-04 +1-30 00

-

3-22 4-51 5-80

-1-00 -1-36 - 0-82

-

3-80

-

-2-70 -2-00

5-90

_

10 mm-K

39.9 33-5

-

+1 1 +10

23-9 32-7 28-5

-

-4-2 - 7-2

-5-0

--

concentration (1-2 mm and 10 mm were both chosen). For one member of each pair (A' and B') the incubation solution also contained Li, whereas for the other (A and B) it was Li-free. The pair AA', incubated without ouabain, gave the total K uptake; the pair BB', incubated in the presence of 10-4 M ouabain, was used to estimate the ouabain-resistant influx. To calculate the ouabain-sensitive component the average values in Li and Li-free ouabain were subtracted from the corresponding individual values

413 Li IONS AND Na PUMP of the total influx. The uptake periods lasted 1 hr with points taken at 10 min intervals during the first half hour and at 15 min intervals during the second. The results of these experiments are summarized in Table 4 where only the ouabain-sensitive components of K uptake, assumed to be equivalent to the pumped K, are given. The ouabain-resistant components were very low, in agreement with Sjodin & Ortiz (1974). They amounted to 0 07 + 0 003 Iamole K/g. hr (n = 10) at 1-2 mM-K and 1-48 + 0 09 mole K/g. hr (n = 7) at 10 mM-K, and were not affected by external Li. On the ouabain-sensitive K influx the effect of external Li was a function of both its concentration and that of K. At 1 2 mM-K, 2 mm-Li increased K uptake (in two out of three experiments) by 10 % as an average. On the other hand 20 mM-Li and 60 mM-Li reduced the uptake of K 20 and 34 % respectively. When the K concentration was 10 mm, the small increment of 3-5 % shown in the data at 2 mM-Li could hardly be considered significant. When Li was present at 20 mm a reduction in the K uptake was observed which averaged 16 %, that is similar to that seen at 1 2 mM-K at the same Li concentration. Even in those cases showing a reduction, uptakes were lower than those predicted from a simple competition model. Thus, taking the apparent affinities of 3'2 mm for K and 21 mm for Li from Michaelis-Menten kinetics, the expected ratios of K uptake in Li-free to K uptake in Li media at 1-2 mM-K would have been 0 94 at Li = 2 mm, 0 59 at Li = 20 mm and 0 33 at Li = 60 mM; the actual values found were 1P09 (true activation), 0-80 and 0-66 respectively. Although the estimation of the affinity constants and the use of simple Michaelian kinetics are subject to objections, the results at 1 2 mM-K and 2 mm-Li seem to indicate a true activation of the ouabain-sensitive K uptake by external Li. In addition they show that the main K-Li interaction on the outside of the membrane occurs on the ouabain-sensitive pathway. In this regard they concur with the previously reported inhibition of the ouabainsensitive Li uptake by external K in frog muscle (Beauge & Ortiz, 1972) with no effect on the ouabain-insensitive Li influx. The possible implications of these results will be treated in the Discussion. The characteristic multicellular structure of the frog sartorius produces a slowness of diffusion in the interfibre space as compared with that in aqueous solutions (Harris & Burn, 1949; Keynes, 1954). As far as K ion is concerned this will produce an accumulation of K coming out of the cells in solutions nominally K-free (Sjodin & Beauge, 1973; this paper); in K-containing media there will be a gradient between the K concentration in the solution, [K].., and the K concentration in the vicinity of the innermost fibres, [K]0i. At K concentrations below that required for Vmax the observed Na efflux activation will be an average response of the whole muscle as a consequence of an average [K] surrounding the different fibres. The estimation of Km will not be accurate. A range of the possible true Km as well as K accumulation in the extracellular space can be obtained from the ouabain-sensitive Na efflux into (K+ Na)-free solutions assuming this is through the activation by K of the Na-K

414

LUIS BEA UGJ9

pump with Michaelian kinetics. For a Na efflux averaging 0 00112 min 1 (Beaug6 & Ortiz, 1972; Sjodin & Beauge, 1973; this paper) the upper limit of the [K] close to the innermost fibres is 0-24 mm if the Km is that obtained from the activation curve of Fig. 6. On the other hand if the Km is of the order of tenths of mm, [K]01 goes down into the micromolar range.

External cation dependence of the residual Na efflux As it can be seen in Figs. 1 and 4 and Table 1, the values for Na efflux in K-free Li-ouabain are much lower than those inK-free Mg without ouabain, both solutions containing 2 mM-Ba. The cause of this difference could be the presence of ouabain, the presence of Li, or both. Similar results can also be observed in Fig. 5 and Table 2 in those muscles where internal K was reduced to very low values; in these cases Ba was absent in the media, but the internal K leaking out of the cells could not have been enough to stimulate the pump. However, some Li leaking out could not be disregarded. If the K recapture hypothesis is correct (Sjodin & Beauge, 1973), the observed difference should be due to the Li-Mg replacement and not to ouabain. On the other hand, if by some mechanism muscle cells could have an uncoupled Na pump as described in human red cells (Lew, Hardy & Ellory, 1973), ouabain should greatly reduce Na efflux in Mg-Ba media. This being the case the possibility of an uncoupled Na pump would be supported by the fact that, in Ba, ouabain and Li produced only a slight increase in K efflux (see Table 1; L. A. Beauge, R. A. Sjodin & 0. Ortiz, unpublished). Some experiments to test this point are shown in the top and middle of Fig. 8. They show that the ouabain sensitivity of Na efflux in Mg-Ba has been greatly reduced. Actually, in some cases it seemed almost completely absent. Taking each muscle as its own control, from three experiments the average reduction seen was 0-00017 + 0-00006 min-1. This is only 15-20 % of the reduction reported in the absence of Ba by Beauge & Ortiz (1972) and Sjodin & Beauge (1973). For an internal Na of 25 ,umole/g it represents 0*255 ,umole/g . hr of ouabain-sensitive Na efflux. From the increase in K efflux produced by Li (Table 1) and by ouabain in Mg-Ba solutions (L. A. Beauge et al. unpublished), a K recapture by the pump of 0-140 to 0 175 #umole/g . hr can be estimated. Given the uncertainty due to the small changes, this seems to give support to the K recapture hypothesis (Sjodin & Beauge, 1973) and indicate that muscle cells do not have, under the experimental conditions examined in this paper, an uncoupled Na pump. Eliminating ouabain as the main reason for the reduction in Na efflux, the difference seen between Mg and Li must be caused by the main cation content in the media. This is very clear in all experiments of Fig. 8. In the presence of ouabain and Ba, Na efflux was always lower in Li than in Mg Ringer solutions. These effects of the principal cation on Na efflux were completely reversible.

Li IONS AND Na PUMP 0-K-Mg-ouabain

0-K-Mg

0003

415

0-002 0-K-Mg-ouabain | 0-K-Li-ouabain

0-K-Mg *0 001

0

0-K-Mg-ouabain

-K-Mg

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E 0

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C

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O-K-Li-ouabain | 0

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-K-Mg-ouabain | -K-Li-ouabain 100

50

150

Time (min) Fig. 8. The effect of I 04 M ouabain and the principal cation in (K+Na)free incubation solutions on the Na efflux from high Na muscles in the presence of 2 mM-Ba. Muscles were made Na rich by cold storage and loaded with 22Na as described in Methods. Before the actual efflux began, the extracellular cold and radioactive Na were washed outby 30 min incubations in a series of tubes with the same solutions as in the first tubes of the corresponding efflux sequences. In each Figure open circles correspond to one muscle and filled circles to the other muscle from the same frog. All solutions contained 2 mm-Ca. Temperature was 20° C.

~ ~ 0.

416 LUIS BEA UGcf Two important questions arise from the previous data. Is this difference between Mg and Li due to the presence of Ba in the solution? If this is the case it would more likely be considered some kind of artifact 0-003r

2 Ca-Mg 0

0 002

0 001

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The interaction of lithium ions with the sodium-potassium pump in frog skeletal muscle.

1. The effects of external Li on Na and K efflux as well as those on K influx were studied in high Na muscles from Rana pipiens. 2. In the absence of ...
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