From the Departamento de Fisiologia, Centro de Investigacion

J. Physiol. (1976), 259, pp. 13-31 13With 10 text-figuresPrinted in Great Britain

THE NUMBER OFSODIUM ION PUMPING SITES IN SKELETAL MUSCLE

AND ITS MODIFICATION BY INSULIN

BY D. ERLIJ AND S. GRINSTEINFrom the Departamento de Fisiologia,

Centro de Investigacion, I.P.N., Mexico 14, D. F. Mexico

(Received 9 April 1975)

SUMMARY

1. [3H]ouabain binding by frog sartorius muscles shows at least twocomponents: one linked to inhibition of the pump and another not relatedto transport inhibition. This is suggested by the finding that [3H]ouabainuptake continued to increase when (a) the glycoside concentration wasincreased beyond that causing maximum transport inhibition, and (b)exposure times longer than those required to produce full inhibition wereused.

2. A number of 1600 pumping sites per /tm2 of membrane was estimatedconsidering only the cylindrical surface of the muscle.

3. Insulin stimulated the ouabain-sensitive components of 22Na effluxand 134Cs influx. It also increased [3H]ouabain binding to a level of 1P7times the total resting value. The increases in [3H]ouabain binding andin 22Na efflux followed a similar relationship with respect to insulinconcentration.

4. Insulin stimulated the Na pump in muscles whose pumping sites hadbeen inhibited by ouabain and then transferred to a glycoside-free solution.This stimulation was observed before detecting any recovery of the initialpumping activity.

5. When both the resting and the insulin-stimulated 22Na efflux hadbeen blocked by ouabain, an additional dose of insulin, in a duabain-freesolution, had no further effects on 22Na efflux.

6. The effects of insulin were unaffected by cycloheximide or by highconcentrations of butyryl derivatives of cyclic AMP and cyclic GMP.

7. We conclude that there are two pools of Na pumping sites in musclecells: one active and another inactive. Insulin unmasks the inactivepumping sites by a mechanism that is independent of protein synthesis,increases in intracellular [Na] or decreases in intracellular [K].

D. ERLIJ AND S. GRINSTEIN

INTRODUCTION

For more than 50 years it has been known (Briggs, Koechig, Doisy &Weber, 1923) that insulin administered to the intact animal causes areduction in the concentration of K in serum. It is probable that thishypokalaemia is due to the stimulation of the Na-K pump in skeletalmuscle caused by insulin (Zierler, 1959; Creese, 1968; Gourley, 1965;Moore, 1973; for an alternative view see Zierler, 1972).Very little is known about the mechanism of this increase in activity of

the Na-K pump caused by the hormone. A study of this problem mayprovide, on the one hand, insight into the mechanisms of regulation of theNa-K-ATPase. On the other hand, it could also provide clues to the wayinsulin alters the cell membrane. Indeed insulin also stimulates the move-ment across the cell membrane of a variety of substances that includesugars (Levine, Goldstein, Klein & Huddlestun, 1949; Kipnis, 1959) andamino acids (Manchester & Young, 1960; Kipnis & Noall, 1958).To explore the action of insulin on the Na-K-ATPase we have com-

bined measurements of the binding of [3H]ouabain, to estimate the numberof transport sites, with determinations of the movement of labelled alkalications to assay the transport system. As a necessary base line for themeasurements of the effects of insulin on [3H]ouabain binding we alsodetermined the general features of this binding in control muscles. Briefreports of this work have appeared elsewhere (Grinstein & Erlij, 1974;Erlij & Grinstein, 1974).

METHODS

Paired sartorii dissected from the same frog (Rana pipienw) were used to providecontrol and test muscles.

Cation fluxes. The technique used to measure 22Na efflux has been described pre-viously (Keynes & Swan, 1959; Horowicz, Taylor & Waggoner, 1970). An apparatusthat transfers the muscle from container to container was used in these experiments(Garcia, Leblanc & Erlij, 1969). 22Na efflux is expressed as the fraction of the radio-activity lost per unit time. This value is equivalent to a rate constant (see Keynes,1965). 134Cs influx was determined using the method of Sjodin (1961).

[3H]ouabasn uptake. [3H]ouabain was purchased from New England Nuclear(specific activity 11-7 c/m mole). Paired muscles were mounted in the apparatus forautomatic determination of fluxes and then equilibrated for a determined period oftime in solutions containing the radioactive glycoside at 20° C.

After the equilibration period had been completed the muscles were transferredthrough six tubes, 10 min in each, that contained inhibitor-free Ringer solution at00 C.

Fig. 1 shows one of four experiments in which, after equilibrating the muscles for1 hr in labelled solution, the efflux of [3H]ouabain into inhibitor-free Ringer solutionat 0° C was measured for 90 min. Following an initial period of rapid efflux theamount of label in the muscles remained practically constant. The shape of thecurve in Fig. 1 taken together with previous measurements of washout of isotopes

14

INSULIN AND Na+ PUMPING SITESfrom frog sartorius muscles (Johnson, 1955; Harris, 1963) suggests that the fastcomponent corresponds to efflux from the extracellular space and that more than95% of the [3H]ouabain in this space has been leached after 50 min of washing at0° C. Furthermore the size of the extracellular space calculated from the interceptof the fast component in the ordinate had an average value of 0-14 ml.fg (range0 10-0 18 ml./g) which is in agreement with previous determinations of the extra-cellular space (for references see Harris, 1963).

10,000

5,000

-02,000

1,000

200

Time (min)

Fig. 1. The washout of [3H]ouabain from frog sartorius muscle. The ordi-nate is the total amount of radioactivity in the muscle at any time. Theopen dots were calculated by subtracting the slow exponential from thetotal curve. The sum of the extrapolated intercepts do not add to the totalradioactivity. This is probably due to radioactive material adhering to thesurfaces of the muscle and to the holders which is washed in the first effluxtube.

The shape of these curves also suggests that there is little washout of ouabainbound to the muscle. This conclusion is also supported by the following observations:(a) when a muscle is transferred from a ouabain containing solution into an inhibitorfree solution at 200 C there is no detectable recovery of the activity of the Na pumpaduring the first hour (Abeles, 1969; Erlij & Elizalde, 1974); (b) lowering the tempera-ture drastically reduces the dissociation rate of the ouabain-ATPase complex(Baker & Willis, 1972).

After the washing procedure was completed the excess fluid ahdering to themuscles was removed by gently sliding them against the wall of a beaker. Themuscles were then weighed in a Mettler H 10 balance, digested in 0-2 ml. NCS andcounted in a toluene mixture on a Beckman LS 150 liquid scintillation counter.

15

16 D. ERLIJ AND S. GRINSTEINQuenching was determined by the External Standard-Channels Ratio method. Theestimates of total number of ouabain molecules bound were not affected by dilutingthe labelled glycoside up to tenfold with cold ouabain.

Ringer solution had the following composition (m-mole 1.): NaCl 115; KCl 2-5;CaC12 2-0; Na2HPO4 2-5; NaH2PO4 0-44. In the experiments in which the effects ofinsulin were measured in Cs solutions all KCl was substituted by 10 mM-CsCl.

Crystalline bovine insulin, cycloheximide, theophylline and the butyryl deriva-tives of cyclic nucleotides were purchased from Sigma.

All the values are given as the mean + 1 s.E. unless otherwise indicated.

RESULTS

Binding of [3H]ouabain and pump inhibitionFig. 2 illustrates one of the experiments carried out to explore the rela-

tionship between ouabain concentration and inhibition of Na efflux. Theexperiment also illustrates the time course of the inhibition of Na efflux

0010 I I I

C

_EW0

z

0C04j

U_

000751

00050

0-0025

50 100 150 200Time (min)

Fig. 2. The effects of different ouabain concentrations on the efflux of 22Nafrom paired sartorii.

under our experimental conditions. On one of the paired muscles the Naefflux was first inhibited by the glycoside at a concentration of 1 x 10-6 Mand then the concentration was further increased to 5 X 10-6 M. If allow-ance is made for the slow drift in sodium efflux which frequently occurs,

INSULIN AND Na+ PUMPING SITES

no further inhibition was detectable in this experiment when the concen-tration of the inhibitor was increased. The paired muscle was exposed toprogressively increasing ouabain concentrations at intervals of 1 hr untilthe concentration reached 1 x 10-6 M. In other experiments, we found thatconcentrations of ouabain as high as 10-3 M did not cause greater inhibi-tion than 10-6 M. With all the ouabain concentrations tested, inhibitionreached equilibrium about 50 min after the addition of the glycoside. Inagreement with previous findings maximum concentrations of ouabainonly inhibited Na efflux to 49 + 2 % the control level; most of the remaining

T5X10-6M

o t/I~~~~~~~~~~8

x 6-S /i 1~~~~~~~~~~~~~~~X10-6 M

4

25x1007m

50 100t (min)

Fig. 3. Time course of the uptake of [3H]ouabain at three concentrations ofouabain. The muscles were exposed for different periods to 5 x 10-6 M (-),10-6 M (0) or 2-5 x 10-7 M (@) [3H]ouabain. The incubation period was fol-lowed by a 60 min washout in glycoside-free Ringer at 00 C. Bars repre-sent + 1 s.E. of the mean.

efflux consists of an exchange diffusion process (Horowicz, 1965; Keynes& Steinhardt, 1968; Beaugse & Sjodin, 1968, Erlij & Leblanc, 1971).In Fig. 3 are summarized the experiments carried out to examine the

time course of [3H]ouabain uptake. The uptake of radioactive ouabainappears to have two components, a rapid one that is almost completedbetween 25 and 50 min and a slower one that continues after 50 min. Theslower phase appears to be steeper when high concentrations of ouabainare used. Comparison of Figs. 2 and 3 suggests that the rapid phase of up-take corresponds to the inhibition of Na efflux. The longer delay required

17


to reach full inhibition of Na efflux may be accounted for, at least in part,by the delay involved in washing out from the extracellular space the Napumped out before inhibition was completed. In Fig. 4 we have plottedboth the uptake of [3H]ouabain and the inhibition of 22Na efflux as afunction of glycoside concentration. To measure [3H]ouabain uptake atdifferent concentrations we selected exposure times of 50 min since theexperiments of Figs. 2 and 3 indicated that ouabain binding linked to Naflux inhibition would be complete after this interval.

6

EE

2 _

50

10-7 10-6 2 5 X 10-'M 5 X 10-6M[Ouabain]

Fig. 4. Binding of [3H]ouabain to frog sartorius muscles at different glyco-side concentrations (0). Notice that the abscissa is linear and starts fromzero. The muscles were exposed for 50 min to the glycoside and then washedfor 60 min in Ringer. Bars represent S.E. of mean. The percentual inhibi-tion of the ouabain-sensitive component of 22Na efflux is plotted as afunction of ouabain concentration (0). The dashed line is drawn throughthe origin and parallel to the component of binding remaining after fullinhibition of the pump.

The inhibition of 22Na efflux can be fitted by an equation of the form:i = I(I+ [K]b), where i is the fraction of the ouabain sensitive Na effluxat a given concentration I of ouabain (in mole/I.) and [K]1 has a value of2-6 x 10-7 M.The data show that ouabain binding increases very rapidly when con-

centrations of ouabain are increased from 10-8 to 10-6 M. When concentra-tions higher than 10-6 m are used, binding continues to increase lesssteeply, although these levels of ouabain do not cause further inhibition ofNa efflux.

18


The finding that ouabain binding continued to increase when we usedexposure times and concentrations larger than those required for completeinhibition of the pump, suggests that in frog muscle there are specificsites to which glycosides can bind as well as non-specific sites not linked toinhibition of transport. To explore further this possibility we used thebinding data to construct a Scatchard plot. These data, illustrated inFig. 5, clearly cannot be fitted by a single line. These findings show that

10,000 10,000

104 ~~~~XE 5,000

EE

x

E | -- 2000 4000

n \ ~~~~~~~~~~~~~d.p. m./mg

I ~ ~ ~ ~~I I2000 4000 6000 8000

d.p.m.r/mg

Fig. 5. The binding data of Fig. 4 are redrawn as a Scatchard plot. The linewas drawn by eye. The inset shows a Scatchard plot only for the specificbinding values obtained by subtracting the dashed line in Fig. 4 from thetotal binding. The line was calculated by the least-squares method.

[3H]ouabain binding in frog muscle conforms to the behaviour observed inother tissues (Baker & Willis, 1972; Brading & Widdicombe, 1974; Dun-ham & Hoffman, 1971).

If we assume (see Baker & Willis, 1972) that the increase in ouabainbinding that occurs at concentrations higher than 10-6 M represents alinear non-specific component of uptake, we can arbitrarily subtract it(dashed line in Fig. 4) from the total binding and thus obtain a maximumestimate of specific binding. The inset in Fig. 5 shows that after subtractingthe linear component of uptake, the resulting values given as a Scatchardplot fit a straight line (Y = 7002 - 2-55x) with a correlation coefficientr = - 0-93 (P < 0 01). From this line we derive a dissociation constant

19

20 D. ERLIJ AND S. GRINSTEIN

for the ouabain-enzyme complex of 4.4 x 10-7 M, which is similar to thevalue of 2-6 x 10-7 M obtained from Fig. 4.

If we consider only the specific component of glycoside binding, andassuming both that one ouabain molecule blocks one pump site, and a

surface: weight ratio of 0-415 cm2 mg-' for the frog sartorius muscle(Keynes & Swan, 1959) we arrive at a number of 1600 pumping sites per

square micrometre of membrane.

The action of insulinEffects on cation fluxesThe stimulation of Na efflux by insulin and its inhibition by ouabain

have already been illustrated (Grinstein & Erlij, 1974) and are also shown

8

7

6

5

_bo 4

E 3

2

1

0

2

0

100 200 300t (min)

Fig. 6. A, uptake of 134Cs in control (i) and insulin-treated (0) muscles. Theconcentration of insulin in theloadingsolution was 250 mu. ml.-'. B, uptakeof 134Cs in the presence of 5 x 10- M ouabain (@). The paired muscle (0)was loaded in a solution containing both ouabain (5 x 104 M) and insulin(250 mu. ml.-').

- A

o Insulin

* Control

.

0 0

0bI


in the first part of Fig. 9. In nine experiments insulin increased the Naefflux to 1-7 + 0-08 times the resting level. When we considered that [Nal1- 14 mm and that the rate constant that is sensitive to ouabain in theresting state is of 0 005 min-, we calculated that insulin increased Na+pumping from 3-57 to 8 56 /amole/g. hr.

Fig. 6 shows a typical experiment in which we measured the uptake of134Cs. We selected caesium for the influx measurements because it entersmuscle cells almost solely through the pump (Beauge & Sjodin, 1968).Since there are very large leakage pathways for K penetration in parallelwith the K pump, it is almost impossible to detect effects of ouabain onK uptake in unloaded frog skeletal muscle (Harris, 1957; Beauge & Sjodin,1968).In Fig. 6A one of the muscles was immersed in insulin-containing solu-

tions throughout the experiment, while the other was used as a control.The hormone produced a clearcut increase in 134Cs uptake. The average134Cs uptake by six control muscles was 1 16 + 0-08 ,mole/g. hr. In themuscles treated with insulin it averaged 2-08 + 0-25 Itmole/g. hr. Fig. 6Billustrates an experiment in which 131Cs uptake of both the control andinsulin treated muscles was measured in ouabain-containing solutions.The glycoside reduced 134Cs uptake in both control and insulin treatedmuscles to the same level (0.29+0-08 ,umole/g. hr), i.e. the ouabain-sensitive Cs influx was increased by insulin from 0- 87 to Il79 lzmole/g. hr.The ratio of ouabain-sensitive uptake between insulin treated and controlmuscles was 2l06. When we measured the effect of insulin on the Naefflux of muscles immersed in K-free Cs Ringer we found that the hormoneincreased the ouabain-sensitive efflux to 2-2 times its resting value.

Effects on [3H]ouabain uptakeWhen [3H]ouabain binding was compared in paired sartorii obtained

from the same frog the difference in the amount of label bound was neverlarger than 5% (n = 7 pairs). However, when muscles of different frogswere compared, a relatively large scatter was observed. The [3H]ouabainuptake in thirty-seven muscles incubated in 1 x 10-6 M ouabain averaged3930 d.p.m./mg ± 1256 (S.D.) The S.E. was 206 d.p.m./mg.Table 1 shows the effects of insulin on [3H]ouabain binding when two

concentrations of the inhibitor were used. Insulin produced a large increasein ouabain binding. The ratios of ouabain binding by insulin-treated overcontrol muscles averaged 1-7. The average difference in binding betweencontrol and insulin-treated paired muscles was significant for both concen-trations of ouabain used.

21


TABLE 1. Effects of insulin on [3H]ouabain binding. All binding figures are expressedin d.p.m/mg wet wt. Values are averages + s.E. of the mean (n = 7). The specificactivity of ouabain was 1 -7 c/m-mole. A (I - C) is the difference in binding betweeninsulin-treated and control muscles; P values were calculated using Student'spaired t test. I/C is the ratio of the binding of insulin-treated over control muscles

2-5x 10-7

1716+ 3202949 + 5991238 + 297(P < 0-01)1-71 + 0 07

1 x 10-6

4964+ 7658043 ± 10353079+ 607(P < 0-01)1*70 + 0-16

'Preincubated'1 X 10-6

1938 + 7713551 + 9211613 + 264(P < 0-001)2*63+0-53

Comparison of the effects of insulin on ouabain binding and Na pumpingThe increases in ouabain binding and cation pumping caused by insulin

suggest that both events may be linked. To explore further such a possi-bility, we determined in five experiments (see Fig. 7) the relationship be-tween insulin concentration and stimulation of 22Na efflux and comparedthe effects of these doses of insulin on the specific [3H]ouabain binding.Specific binding was calculated by subtracting the unspecific binding,estimated from Fig. 3, from the total uptake. The close relationship be-

160 -

80 [

50 100% A specific binding

I

40

50 100 150 200 250

Insulin (mu/ml.)

Fig. 7. Effects of increasing insulin concentration on the stimulation ofthe ouabain-sensitive component of Na efflux (A) and on the increase ofspecific [3H]ouabain binding at 106 M (0). In the inset, the increase inpumping is plotted against the increase in binding produced by eachinsulin concentration. The line was calculated by the least-squares method.

Ouabainconcn. (M)

ControlInsulini\ (I-C)

I/C

80k

C

-V-C:3.r_

V

C.

I-1

60k

40 I-

QLE

-1

20

0

0 200 -

Q_EMa-M

z 100-1

0'1.1 1 1

120 [

INSULIN AND Na+ PUMPING SITES 23

tween increases in binding and Na efflux is stressed by the graph in theinset of Fig. 7. The two effects have an index of correlation r = 0-98 withP < 001 and a slope of F96.

Unmasking of pump sites in mWucles with resting sites blocked with ouabainIf insulin stimulates Na pumping by increasing the number of Na pump

sites in the muscle membrane it ought to expose new sites even after thosesites active in the membrane of unstimulated muscles have been blockedor inactivated.

c 1: Insulin 250 Insulin 250 mu./ml0} Ringer °} .0 Ringer mu./m. 0 +ouabain 10-6M

001510 50 20 5ouabin fr4 inge andthei Ringerslto o 0mn h etmslreaie inRigr.I the f 1 ingerpeio 04 uabainwasaded6o ot

0~~~~~~~0LC0*010

0

0

N 0.005

Figu8.Afers. oto eid h uceswr mesdi 0

To test this prediction we have used the finding that when a muscle istransferred from a ouabain containing into an inhibitor-free solution thereis no detectable recovery of Na pumping for a relatively long period oftime (Abeles, 1969; Erlij &z Elizalde, 1974), suggesting that ouabain re-mains attached to the Na-K-ATPase. Thus it is possible to test the effectof insulin on muscles whose resting pumps are blocked with ouabain anddetermine whether the hormone will still increase Na pumping.

Fig. 8 shows one out of five experiments performed to test this predic-tion. After a control period, the muscles were transferred to solutionscontaining ouabain (5x 106M), which abolished the ouabain-sensitivecomponent of Na efflux. The muscles were then washed for 60 mmn in

D. ERLIJ AND S .GRINSTEIN

ouabain-free Ringer solution. No recovery of the Na efflux was observedduring this period. In ten similar experiments the ratio of the efflux valuemeasured after washing in ouabain-free Ringer for 140 min over thelowest value measured during the action of ouabain was 1*05 + 0 03. Wheninsulin was added to the muscle a large increase of Na efflux was observed.The increase was equal to 0-76 + 040 times (n = 5) the resting level. Thisstimulation of Na efflux was inhibited when ouabain (1 x 10-6 M) was addedto the wash-out solutions.Another group of experiments of the same design as those illustrated in

Fig. 8 was carried out to measure ouabain binding. After immersing boththe control and test muscles in 5 x 10-6 M unlabelled ouabain (40 min)they were washed 60 min in inhibitor-free Ringer. The test muscles werethen equilibrated with insulin and during the final period 1 X 10-6 M[3H]ouabain was added to the solutions. The control muscle also received[3H]ouabain in the final period. The results obtained are summarized inTable 1 in the column labelled 'preincubated'. The table shows that pre-incubation reduced [3H]ouabain binding to about 30% of the value ob-served in muscles that were exposed directly to [3H]ouabain. The bindingremaining after the muscles had received enough ouabain to block all theNa active transport probably represents binding to sites not linked to thecation transport process. This value of unspecific binding is similar to theone obtained from Fig. 3.

Table 1 also shows that the addition of insulin to muscles preincubatedwith unlabelled ouabain resulted again in a large and very highly signifi-cant (P < 0 001) increase in [3H]ouabain binding. The last two columns ofTable 1 shows that the increase in [3H]ouabain binding caused by insulinwas smaller in muscles preincubated in unlabelled ouabain than in musclesthat were not preincubated in inhibitor. In principle one would expect thatthe increase in labelling after insulin treatment should be the same in bothexperimental groups. We believe that the quantitative disagreement withthis expectation is due in great part to the scatter in ouabain bindingvalues between muscles obtained from different frogs mentioned pre-viously, since the difference in insulin-induced binding between bothgroups (columns 2 and 3 in Table 1) is not statistically significant (P >0 1).

Similar experiments were also performed for 134Cs uptake. In musclesthat had been preincubated with ouabain and then washed in inhibitor-free solution for 10 min, insulin caused an increase in 134Cs uptake of 0 55,umol/g. hr. It could be argued that insulin destabilizes the ouabain-enzyme complex. The dissociation of the complex would result in thetermination of the inhibitory effects of ouabain on the Na efflux and wouldmake available for binding those sites previously occupied by unlabelled

24

INSULIN AND Na+ PUMPING SITESinhibitor. This possibility is ruled out by the following observations. First,ouabain binding measured without previous exposure to unlabelled oua-bain was increased by insulin. The converse effect should be expected ifinsulin destabilized the ouabain-enzyme complex. Secondly, followingequilibration of paired muscles in [3H]ouabain for 50 min one of them was-washed out in insulin containing solutions (250 mu./ml.) and the otherone in Ringer. The ratio of [3H]ouabain remaining in the insulin-washedover the Ringer-washed muscles was 1-008 + 0 04 (n = 4).Another type of experiment that further excludes the possibility of a

dissociation of the ouabain-enzyme complex is shown in Fig. 9. In this

0 025 -I~} Ringer 0 Quabain o Ringer ° Ringer 0)Insulin0 Insulin 2Insulin r 0I

* +Ouabain0-020

0

0Cu

muscl(-0 ). 0hn5x14Moaanwsaddfrtenx 0mn h

0C

Rige souto fo00mn ial,20m.m.lislnwr de o

U_0-010

0 50 100 150 200 300t (min)

Fig. 9. After a control period, 250 mu. rnl.-' insulin were added to the testmuscle (@). Then 5 x 10-- m ouabain was added for the next 50 mmn. Thehormone and the glycoside were washed off by exposing the muscle toRinger solution for 50 min. Finally, 250 mu. ml.-' insulin were added for asecond time.

experiment, after a control period, the 22Na efflux of the test muscle wasfollowed in solutions containing insulin, 250 mu./ml., followed by the addi-tion of ouabain. Then the muscle was transferred to Ringer solution towash both the inhibitor and insulin from the extracellular space and finallyinsulin was added again. The hormone did not increase 22Na efflux.If insulin destabilized the ouabain-enzyme complex, the second additionof insulin should have increased 22Na efflux regardless of whether or notinsulin had been given before the addition of ouabain.

25


The mechanism of increase in pumping sites

In this section we describe experiments performed in an attempt toclarify the mechanism of the increase in number of pumping sites causedby insulin.

Cycloheximide. In several cell types it has been shown that there is a

system for controlling the number of Na pumps in their membranes whichinvolves protein synthesis (Vaughan & Cook, 1972; Boardman, Lamb &

o0Ringer

0 030 I-

C

E

0tv

z

N

0

r_

4.

'U

U-

0020 1-

0010

} +insulin 250 mu./ml.

I I

50 100 150Time (min)

200

Fig. 10. The control muscle (@) was exposed to 250 mu. ml.-' insulin after2 hr in Ringer solution. N6 monobutyryl cyclic AMP (104 M) and theo-phylline (0-5 mM) were added to the washout solution of the test muscle(0) after a 1 hr preincubation in Ringer. 1 hr later insulin 250 mu. ml.-.theophylline and the nucleotide were all present in the solution.

McCall, 1972). Therefore, it was of interest to test whether the insulininduced increase in number of pumping sites is mediated by protein syn-

thesis. Experiments like that illustrated in Fig. 9 indicate that the pool ofsites available for unmasking with insulin is limited. Further evidence insupport of this conclusion comes from experiments in which the effects ofinsulin on ouabain binding were tested in cycloheximide containing solu-tions (100 jtg/ml.). Incubation of paired muscles with cycloheximide was

started 40 min before the binding measurements and continued through-out the experiment. Ouabain binding was 3271 d.p.m./mg wet wt. in con-

26


trol muscles and 5504 d.p.m./mg in insulin treated muscles, i.e. the increasein ouabain binding caused by insulin was unaffected by cycloheximide.

Cyclic nucleotides and Na pumping. One of the hypothesis that has beenput forward to explain how the interaction of insulin with membranereceptors modifies the activity of several cell functions is that the hormonereduces the cytoplasmic level of 3',5'-cyclic AMP (Exton, Lewis, Ho,Robison & Park, 1971). It has also been suggested that the increasedlevels of 3',5'-cyclic GMP caused by the hormone could mediate its bio-logical effects (Illiano, Tell, Siegel & Cuatrecasas, 1973). One predictionof such hypothesis is that an imposed increase in the cytoplasmic levels ofcyclic AMP should prevent insulin action. In our experiments we havetested the effects of N6-monobutyryl-3',5'-cyclic AMP (10-4M) and ofN6,02-dibutyryl-3'5'-cyclic AMP (104 M) on the stimulation of the Napump caused by insulin. Neither nucleotide modified drastically the rest-ing Na efflux nor prevented the stimulation caused by insulin both in thepresence or absence of 0 5 mm theophylline (Fig. 10).

In similar experiments we tested the effects of N2-monobutyryl 3',5'-cyclic GMP (104 M) on Na efflux. Again the resting Na efflux and its stimu-lation by insulin were unaffected by this nucleotide.Due to the lack of effects of dibutyryl cyclic AMP on Na pumping we

measured whether this nucleotide was able to penetrate into the cellsunder the present experimental conditions. With this aim we equilibratedfour muscles with 10-4 M of [14C]N602-dibutyryl-cydlic AMP and esti-mated the intracellular concentration by subtracting the radioactivity inthe extracellular space (Desmedt, 1953) from the total uptake. After 1 hrthe intracellular concentration of dibutyryl cyclic AMP was 2 + 009 x10-5 M.

DISCUSSION

The simplest explanation for the findings described above is that insulinunmasks pumping sites in the muscle cell membrane. These latent siteswould be neither available for cation pumping nor ouabain binding atconcentrations that normally block completely Na transport. This conclu-sion derives from two independent types of evidence. One based on theeffects of insulin on Na fluxes; the other on the changes in [3H]ouabainbinding induced by the hormone.

Na flux experimentsThe most convincing finding is provided by the experiments in which

insulin stimulated a ouabain-sensitive Na efflux in muscles whose restingpumps had been blocked by ouabain and then transferred to a glycosidefree solution (Fig. 8). The possibility that this stimulation was the result of

27


a dissociation of the ouabain-enzyme complex caused by insulin has beenruled out (see page 25 and Fig. 9). The small and transient increase in Naefflux observed when insulin was added in a medium containing inhibitoryconcentrations of ouabain (fig. 1 in Grinstein & Erlij, 1974) could repre-sent the opening of new pumps that are being rapidly blocked by ouabainin the medium.

An alternative explanation for experiments like those shown in Fig. 8 can beproposed if the following assumptions are made: (a) intracellular Na increasedmarkedly while the pump was blocked before the addition of insulin, and (b) duringthe washout of ouabain from the extracellular space with inhibitory-free solutions,ouabain dissociates from a very small number of pumping sites (even though noincrease in pumping was detected). Thus the addition of insulin to a muscle with highintracellular Na would markedly stimulate Na efflux through a small number ofpumping sites. This explanation again is ruled out by experiments like those inFig. 9, where the muscles that had received insulin prior to the addition of ouabaindid not respond to a second addition of insulin.

[3H]auabain bindingFurther support for our conclusion is provided by the parallel increases

in [3H]ouabain binding and Na pumping caused by different doses of insu-lin (Fig. 7). This increase in [3H]ouabain binding was observed under con-ditions in which the sites for specific binding in the resting muscle werealready saturated (see Figs. 2 and 4 and page 18). Therefore, the sites madeavailable for [3H]ouabain binding by insulin add to the specific bindingsites already operating in resting muscle. Since the increase in [3H]ouabainbinding sites is linked to a stimulation of Na pumping we believe thatthese are, mostly, specific sites.An increase in [3H]ouabain uptake under the influence of insulin has

already been observed in cardiac muscle (Dutta, Rhee & Marks, 1972).However, it was interpreted as an increase in the transport of ouabain intothe cell. Since most of the ouabain uptake in the heart is confined to themicrosomal fraction (Kim, Bailey & Dressel, 1970) we believe that thefindings in cardiac muscle represent the same phenomenon described herefor skeletal muscle membranes.The number of pumping sites in the resting muscle estimated in this

investigation (1600 /m-2) is within the range of the values listed in thesurvey of different cell types made by Baker & Willis (1972). In our calcu-lations only the cylindrical surface of the muscle cells was considered. It ispossible therefore that we are over-estimating the number of sites per unitarea by a constant factor, since Krolenko (1971) found an enlargement ofthe transverse tubules when muscles were extruding Na at high rates.

28

INSULIN AND Nab PUMPING SITES

The insulin-induced pumping sitesTwo features of the insulin uncovered pumping sites deserve notice:

(a) the ratio of Na to Cs ouabain-sensitive fluxes is similar in the insulinstimulated and resting conditions; (b) insulin produced a greater relativestimulation of Na efflux than of [3H]ouabain binding (Fig. 7).The resting ouabain-sensitive Cs influx had a value of 0-87 ,amole/g.hr

while the resting ouabain-sensitive Na efflux was 3 67 ,umole/g. hr. Thesedeterminations give a ratio of 4: 1 for the Na: Cs ouabain-sensitive ex-change. Since insulin produced relative increases of similar magnitude inouabain-sensitive Na efflux and Cs influx the stoicheiometries of the restingand induced sites remain the same.The data in Fig. 7 show that insulin caused a relative stimulation of

ouabain-sensitive Na efflux that was almost twice as large as the relativeincrease in [3H]ouabain binding. This could be due to either an over-estimation of the amount of specific [3H]ouabain bound to resting musclesor to a higher rate of pumping by the insulin induced sites.

The mechanism of insulin stimulation of the Na pumpThe experiments in the final part ofthe results section were carried out to

test whether the insulin induced increase in sodium pumping could fit someofthe proposed mechanisms for insulin action. Although these experimentsdo not provide positive answers, they exclude some possibilities.The lack of effects of cycloheximide and the results of experiments of

the type shown in Fig. 9 make it highly unlikely that the stimulation ofNa pumping is due to synthesis of new pumps.The experiments with cyclic nucleotides make it unlikely that a reduc-

tion in the overall levels of intracellular cyclic AMP mediate the increase insodium pumping. It has been widely demonstrated that exogenous cyclicAMP (or its butyryl derivatives) mimic the action of agents that increasenucleotide production (Robison & Sutherland, 1971). These considerationssuggest that either the stimulation of the Na pump caused by insulin is notmediated only by a decrease in cyclic AMP or an increase in cyclic GMP,or that the changes in cyclic nucleotide levels occur in a region that is notaccessible to the exogenous butyrated derivatives.In conclusion our experiments show that there are two populations of

Na pumps in frog muscle cells: one active which readily binds cardiacglycosides, the other inactive and not available for inhibitor binding atconcentrations that normally block the active sites.The activation of the latent pool provides a mechanism to accelerate

pumping which is independent of increases in [Na]1 or decreases of [K]1since insulin reduces [Na]1 and raises [K], (Moore, 1973).

29


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