equilibrated at 40C in a K-free Ringer (Steinbach, 1940). solution, had no further effects on 22Na efflux.

Transcription

1 J. Physiol. (1976), 259, pp With 6 text-figures Printed in Great Britain STIMULATION OF THE SODIUM PUMP BY AZIDE AND HIGH INTERNAL SODIUM: HANGES IN THE NUMBER OF PUMPING SITES AND TURNOVER RATE BY D. ERLIJ AND S. GRINSTEIN From the Departamento de Figiologia, entro de Investigacion, I.P.N., Mexico 14, D.F. Metxico (Received 9 April 1975) SUMMARY 1. The effects of 5 mm azide on [3H]ouabain uptake and 22Na efflux were determined. Both glycoside uptake and 22Na efflux were enhanced by azide. 2. Azide stimulated the Na pump in muscles whose pumping sites had been inhibited by ouabain and then transferred to a glycoside-free solution. This stimulation was observed before detecting any recovery of the initial pumping activity. 3. When both the resting and the azide-stimulated 22Na efflux had been blocked by ouabain, an additional exposure to azide, in a ouabain-free solution, had no further effects on 22Na efflux. 4. It is concluded that the increase in Na pumping caused by azide is due in part to an increase in the number of pumping sites. 5. [3H]ouabain binding was measured in muscles with different intracellular alkali cation concentrations. Variations in [Na]1 from 15 up to 5 mm did not significantly affect the amount of glycoside bound. A substantial increase in binding occurred when [Na]1 reached 7 mm. 6. It is proposed that the increase in Na extrusion that occurs during the recovery of Na loaded muscles mostly results from an increased turnover rate of the pump rather than from an increase in number of pumping sites. INTRODUTION In this paper we describe experiments performed to explore the mechanism of action of two procedures which markedly increase Na pumping in skeletal muscle, namely the exposure to Na azide (Horowicz & Gerber, 1965) and the addition of K Ringer at room temperature to muscles previously equilibrated at 4 in a K-free Ringer (Steinbach, 194). Since there is evidence (Erlij & Grinstein, 1976) indicating that the 2-2

2 34 D. ERLIJ AND S. GRINSTEIN stimulation of the Na pump can be mediated by an increase in the number of pumping sites in the muscle membrane, we have directed our attention to determine whether the agents studied in this investigation act by increasing the number of pumping sites or by enhancing the turnover rate of the enzyme. Our results show that both the addition of azide and extreme changes in intracellular ion composition increase the number of available pumping sites. However, we also found that in addition to the increase in number of pumping sites, azide and Na loading raise markedly the turnover rate of the pump; indeed the stimulation of Na efflux in loaded muscles is almost entirely due to an increase in the turnover rate of the pump. Some of these results have already been published in preliminary form (Grinstein & Erlij, 1974). METHODS The methods used in this investigation were mostly those described in the previous paper (Erlij & Grinstein, 1976). Sodium loaded muscles were prepared by soaking for 24 hr at 2-6 in a K-free Ringer. Up to four pairs of muscles were kept in 5 ml of vigorously stirred solution that was changed every 12 hr. Muscles for analysis were directly weighed or transferred to the recovery solutions. Muscles used for [3H]ouabain binding determinations were transferred either to the recovery solutions or to the [3H]ouabain solution to carry on the uptake procedure described in the previous paper. All the muscles in which [3H]ouabain uptake was measured were equilibrated for 1 min in 2-5 mm-k containing Ringer at room temperature before being transferred to the radioactive solution. This equilibration was carried out to perform all the binding experiments under uniform conditions. [3H]ouabain solutions always contained 2-5 mm-k. E8timation of intracellular Na and K The muscles were blotted with an ashless filter paper (Whatman No. 5) and then weighed in tared polypropylene test-tubes. After weighing, the muscles were dried overnight in an oven at 1 and weighed again. The dried muscles were dissolved in concentrated nitric acid and after dilution, Na and K were determined by flame photometry using the appropriate blank solutions. To calculate the intracellular Na and K concentrations the extracellular space was assumed to be 12-5% of the weight for both freshly dissected and soaked muscles (Desmedt, 1953). Resting potentials of muscle fibres were measured as described by Adrian & Slayman (1966). Potassium-free Ringer was prepared by omitting Kl from normal Ringer. 5 mm-k Ringer was prepared by adding 2-5 mm-k to normal Ringer. 8 mm-k solution contained (in m-molefl.) K2SO 4, Na2SO4 4, al2 2, Na2HPO4 2-5, NaH2PO, -85. Na azide was obtained from Eastman Organic hemicals.

3 AZIDE AND THE SODIUM PUMP 35 RESULTS Effects of azide on Na fluxes and [3H]ouabain binding Fig. 1 shows one experiment in which we measured the effects of 5 mm azide and ouabain in a pair of frog sartorii. z 12 5 U -1 Ringer NaN3 5mM NaN3+ouabain * Ouabain 1 166M Fig Time (min) The effects of 5 mm azide and ouabain on the rate of 22Na efflux in paired sartorius muscles. In agreement with Horowicz & Gerber (1965) we found that azide stimulates the efflux of Na from frog skeletal muscle. In ten muscles the average maximum increase in Na efflux was times the resting level. The stimulation had a variable time course; in some muscles it was sustained for several washout periods while in others it reached a maximum and started to decline rapidly. As shown by Horowicz & Gerber (1965) the addition of ouabain to muscles treated with azide reduces efflux to the level observed when ouabain is given to control muscles. The experiment in Fig. 1 also shows that azide caused a small and transient increase in Na efflux when given to a muscle immersed in ouabain solutions. Incubation in 5 mm-nan3 produced an increase in ouabain binding from to d.p.m./mg wet wt. Unmasking of pump sites in muscles with resting sites blocked by ouabain The increase in ouabain binding caused by azide suggests that it might increase the number of pumping sites available on the muscle membrane.

4 36 D. ERLIJ AND S. GRINSTEIN To test this possibility further we performed experiments, as those carried out withinsulin (see Erlij & Grinstein, 1976), in which allthe resting pumping sites in a musclewereblocked by ouabain and then the muscle was transferred to an inhibitor-free solution. Fig. 2 shows one out of four such experiments..8 -L GI W } NaN3 5mM a NaN3 5 xio_ ouabain mie In RRinger axde t S hmouabain.s-7- E - -6 z U..8 RTinger(i Time (min) Fig. 2. After a control period, ouabain (5 x 1-4 m) was added to both muscles. This was followed by a 5 mini washout in Ringer and then 5 mm NaN3 was added to the test muscle (). The paired control muscle remained in Ringer. Last, 5 x 1-4 m ouabain was added to both muscles. After a control period the muscles were transferred to 5 x 1-6 M ouabain containing solutions. This concentration abolished the ouabain sensitive component of Na efflux. The muscles were then washed for 6 min in ouabain-free Ringer solution. This treatment did not remove ouabain from specific sites, since there was no recovery of the pump. When NaN3 was added to one muscle a large increase of Na efflux was observed. The increase was equal to times (n = 4) the resting level. This stimulation of Na efflux was inhibited when ouabain (5 x 1O6 M) was added to the washout solutions. Experiments like the one in Fig. 2 give strong support to the notion that azide enhances Na pumping by increasing the number of active Na-K- ATPase sites in the muscle membrane provided it can be shown that NaN3 does not unstabilize the ouabain-enzyme complex, thus reverting the inhibition of the Na pump.

5 AZIDE AND THE SODIUM PUMP 37 The experiment of Fig. 3 was designed to explore this possibility. In this experiment we treated the test muscle with 5 mm azide. This substance caused its usual stimulating effects. After 5 min of efflux into NaN3 solutions the muscle was transferred to a Ringer solution containing ouabain (1 x 1-6 M). The efflux was markedly reduced to a level equal to -2 times the resting level; then the muscle was washed with ouabain-free 3 I I I.E_ VI,I 2 z U,o "_ t (min) Fig. 3. The effects of two successive additions of 5 mm-nan. on the rate of 2Na efflux. After the first addition of azide, the test muscle (@) was exposed to 5 x 1- M ouabain while the paired control muscle () remained in Ringer. Then both muscles were transferred to Ringer first and again to 5 m -NaN3 solutions. Ringer for 5 min and NaN3 was added for a second time. A second addition of azide did not cause a stimulation of Na efflux. The control muscle was exposed first to azide, then washed in Ringer and finally exposed for a second time to NaN3. A second addition of azide always increased the sodium efflux when ouabain had not been added after the first stimulation with azide. Do azide and insulin unma8k the same site? The experiments of azide described thus far indicate that, as insulin, this substance unmasks latent pump sites in the frog muscle membrane. We decided to find whether the sites unmasked by the two agents are the same.

6 38 D. ERLIJ AND S. GRINSTEIN Fig. 4 shows one experiment carried out to explore this question. The Na pump of the experimental muscles first received insulin to unmask all the hormone dependent pumping sites. Ouabain was then added to block both the resting and the hormone dependent sites. Following a period in which ouabain was washed from the extracellular spaces, azide was added to the efflux solutions. The companion muscle served as control to test the Ri nger Insulin Insulin+.*25 mu./ml. * uabain 1 NaN3 5mM n e 1uabain Ringer1 g _E -15 z * Time (min) Fig. 4. The effects of preincubation with insulin 25 mu./ml., on the azide-mediated stimulation of the pump. Administration of insulin to the test muscle (*) was followed by addition of solution with both insulin and 5 x 1- M ouabain. A 5 min washout period followed, and finally 5 mm azide were added. The control muscle was exposed to 5 x 16 M ouabain, and then washed in Ringer for 1 min. This muscle also received azide in the final step. effects of azide on a muscle whose resting pumps only had been blocked by ouabain. In the experimental muscle azide caused either a very small stimulation or no effects. In three muscles the stimulation was at least 6 times smaller than in the control muscles whose pumps had not been exposed by insulin. This means that after the sites exposed by insulin had been blocked by ouabain only about 17 % of the total pumping sites available for activation by azide in the control conditions can be exposed. Fig. 5 shows the converse experiment. In this case the muscle was exposed first to azide and then, after blocking with ouabain all the sites exposed by azide and washing the glycoside out of the extracellular

7 AZIDE AND THE SODIUM PUMP 39 space, the muscle was treated with insulin. Insulin stimulation was reduced to -2 times the response found in three paired muscles that were not pretreated with azide. Again this finding indicates that about 8 % of the sites available for activation by insulin in the control muscle were blocked when the test muscle was exposed to ouabain in the presence of azide. I I Insulin 25 mu./mi. E tv 3 ze 2 I._ U 'U Time (mi)n Fig. 5. The effects of preincubation with 5 mm azide on the insulin-mediated stimulation of 22Na efflux. The test muscle () was first stimulated by 5 mm azide and then inhibited by 5 x 1-6 M ouabain. This was followed by a 5 min wash in Ringer and finally insulin, 25 mu./ml., was added. The paired control (S) was subjected for 8 min to 5 x 1-6 M ouabain and then washed in inhibitor-free Ringer for the next 5 min. Insulin, 25 mu.iml., was added in the final step. Azide effects on Na pump and membrane potential Horowicz & Gerber (1965) described a parallel relationship between the increase in Na pump activity and the change in resting potential produced by either NaN3 or K. This parallelism suggests that the stimulatory effect of NaN3 on the pump may be mediated by depolarization. Since it would be of interest that the pumping activity could be controlled solely by the membrane potential, we decided to carry on experiments that would test this possibility further. With this aim we measured the effects of azide in muscles that had been depolarized in high K solutions. We reasoned that

8 4 D. ERLIJ AND S. GRINSTEIN the depolarizing effects of azide would be greatly reduced or absent in muscles whose resting potential was depressed by K. Indeed we found that the resting potential in five muscles immersed in normal Ringer had an average value of 92*3 + *7 mv (n = 51) and after immersing them in the 8 mm-k solution the resting potential dropped to mv (n = 45). 8mM -K+S mm -NaN3 * 2-5 mm -K+5 mm -NaN3 _E z N N U U Time (min) Fig. 6. The effects of 5 mli azide on the efflux of 22Na into solutions with different K concentrations. When 5 mm azide was added to this 8 mm-k solution, the resting potential was of mv (n = 53). The resting potential of these muscles recovered to mv (n = 38) when they were washed with fresh 2-5 Kl Ringer. Finally the addition of 5 mm-nan3 to these muscles reduced the resting potential to mv (n = 41) in agreement with previous findings (Horowicz, aputo & Robeson, 1962). Fig. 6 illustrated an experiment in which a sartorius muscle was exposed to a solution containing 8 mm-k. The Na efflux increased rapidly and reached a level of about 3 times the resting value. This increase was not sustained. About 1 hr after the rise in [K]O the rate constant for Na efflux approached the level observed during the control period. After this period the test muscle was exposed to 5 mm-nan3 which had its usual stimulatory effect. Although the azide induced stimulation in depolarized muscles was 2

9 AZIDE AND THE SODIUM PUMP 41 smaller than in control muscles, it still reached a level of 2 1 times the initial level, i.e., the increase was -78 times the average increase in control muscles. In other experiments we found that the azide-induced increase in Na efflux in depolarized muscles was always blocked by ouabain (14 M). The reversibility of the azide effect Fig. 3 shows, in agreement with the findings of Horowicz & Gerber (1965) that upon removal of azide the rate of Na efflux returns to a rate close to its value before the application of azide. The new rate was sometimes clearly below the original resting efflux and other times remained above it. Fig. 3 also shows that when azide was added for a second time to the solutions, Na efflux was increased again. We therefore tested whether the reversal of the effect of azide on Na flux is accompanied by changes in [3H]ouabain binding. For this purpose we measured binding in muscles that had been immersed in azide solutions for 5 min, washed in azide-free solutions for different periods of time and then exposed to [3H]ouabain. We found that the [3H]ouabain uptake of muscles that had been incubated with azide and washed with azide-free solutions for periods as long as 3 hr remained at the same high level as the uptake determined in azide containing solutions. Effects of intracelhdlar Na on ouabain binding Since the rate of pumping can be markedly increased in Na loaded muscles (Adrian & Slayman, 1966) we decided to study [3H]ouabain binding in muscles with different intracellular alkali cation concentrations. TABLE 1. The effect of variations in alkali cation concentrations on [3H]ouabain binding [3H]ouabain [Na]1 [K], binding (mm) (mm) (d.p.m./mg) Freshly dissected Soaked for 24 hr in 5 m -K at ± 67 Recovery for 1-5 hr in 5 mm-k at 19* ±354 Soaked for 6 hr in O-K at ± 5*2 3523± 738 Recovery for 1-4hr in 2-5 mm-k at 19* ± ±168 Soaked for 24 hr in ± 5*6 -K at * The muscles had been soaked for 24 hr in -K Ringer before transferring them to the recovery solutions.

10 42 D. ERLIJ AND S. GRINSTEIN Table 1 shows the values for ouabain binding at different intracellular Na and K concentrations. The concentrations were determined analytically. The extreme values of Na and K were obtained by using on the one hand freshly dissected muscles and on the other hand muscles that had been incubated for 24 hr at 4 in K-free Ringer. The intermediate values of [Na]1 were obtained by either soaking for a shorter period or by loading for 24 hr and allowing the muscles to extrude Na in K-containing solutions at 2 for different periods of time. The values found in muscles incubated for 24 hr in 5 mm-k Ringer are included as a measurement of the effects of prolonged soaking on [3H]ouabain binding. The table shows that variations in [Na]1 from 15 up to about 5 mm do not significantly affect the amount of [3H]ouabain bound by the muscles. However, when [Na]1 reached about 7 mm, a clearcut increase in binding was observed. DISUSSION The main conclusion of this investigation is that there are two mechanisms by which the rate of Na pumping in frog skeletal muscle can be controlled: an increase in the number of pumping sites and changes in the turnover rate of the enzyme. Effects of azide The first question to be considered is the mechanism of the stimulation of the Na pump by NaN3. Our evidence indicates that one of the mechanisms of azide stimulation is an increase in the number of Na pumping sites. This conclusion is based on the experiments in which azide stimulated a ouabain sensitive Na efflux in muscles whose resting pumps had been blocked by ouabain and then transferred to inhibitor-free solutions (Fig. 2), complemented by the effects of azide on ouabain binding. The possibility that the stimulation of Na efflux by NaN3 after ouabain inhibition results from a dissociation of the ouabain-enzyme complex is ruled out by experiments like that illustrated in Fig. 3. This experiment also indicates that there is a limited number of pumping sites available for opening with NaN3. The experiments in Figs. 4 and 5 show that the sites available for opening by either azide or insulin arise mostly from the same pool. Once the sites activated by one agent are blocked, the stimulation of Na efflux produced by the other agent is only about 2 % of that observed in the control experiment. Although most of the sites uncovered by insulin and azide arise from the same pool, the two agents do not share completely the same mechanism of action as the following findings indicate.

11 AZIDE AND THE SODIUM PUMP 43 (a) Stimulation by insulin is sustained while the effect of azide turns off spontaneously. (b) After all the sites exposed by azide have been blocked with ouabain a second addition of azide is without effect on the Na efflux while insulin still produces a small stimulation. The converse is also true. In addition to the increase in the number of pumping sites, it is necessary to postulate another mechanism by which azide increases the rate of Na pumping. This requirement arises from the following findings. (a) The effects of azide on Na efflux are rapidly reversed when it is washed from the bathing fluid. (b) Efflux can be stimulated for a second time if azide is reintroduced in the bathing solution. (c) [3H]ouabain uptake remains high in muscles that have been washed in azide-free solutions for up to 3 hr. It follows that after removal of NaN3 from the bathing fluid the number of ouabain binding sites remains increased although their activity is reduced by the omission of azide. Since the experiments or Figs. 4-6 show that there is a limited pool of sites for opening by either azide or insulin, the second stimulation of Na efflux caused by azide in experiments like that of Fig. 5 must be due to an increase in pumping rate rather than to an increase in the number of sites. Another point about the mechanism of azide stimulation of the Na pump is indicated by the results in muscles depolarized by high-potassium solutions. Since under these conditions azide stimulated the Na pump without modifying the resting potential we must conclude that the stimulatory action of azide on the pump cannot be explained solely by the changes it produces on membrane potential. Effects of changes in intracellular alkali cation concentration One of the points to emerge from the experiments in which the effect of varying intracellular Na and K were measured on [3H]ouabain binding is that large deviations in the cell content of these cations have to be produced before an effect on [H3]ouabain binding is detected. Since increases in Na concentration below those that cause an increase in [3H]ouabain binding produce large stimulation in the rate of Na extrusion of muscle (ross, Keynes & Rybova, 1965), it is clear that the turnover rate of the Na-K pump in skeletal muscle can be markedly increased without modifying the number of Na pumping sites. Furthermore, Adrian & Slayman (1966) estimated a maximum Na pumping rate of 27 molel/ g.hr that is 75 times larger than that of 3.57,umole/g.hr found for the resting pumping sites in our experiments. When we consider 16,um-2 as the number of Na pumping sites (Erlij & Grinstein, 1976) estimated on the basis of ouabain binding measurements and assume three Na ions pumped by each molecule of ATP hydrolysed, we arrive at a turnover rate of

12 44 D. ERLIJ AND S. GRINSTEIN 2 min- for resting muscles. This value is below the lowest value listed by Baker & Willis (1972) in their survey of pump turnover numbers in different cell types. However, when we consider that during Na loading the rate of Na extrusion can be increased up to 75-fold we find that the maximum turnover can reach levels of 15, min-'. This value is in the upper range of values quoted in the survey of Baker & Willis (1972). Increases in cytoplasmic Na also enhance pumping rate in other cell types. In the squid giant axon, Na pumping is directly proportional to the Na concentration in the cytoplasm (Hodgkin & Keynes, 1956; Brinley & Mullins, 1968; Baker, Blaustein, Hodgkin & Steinhardt, 1969). In red blood cells there is a sigmoid relationship between intracellular Na concentration and Na pumping (Garay & Garrahan, 1973). Jorgensen (1974) has pointed out that at physiological ion concentrations the rate of turnover of ATP per site in the intact cell membranes should be somewhere below 25 % of the rate at the optimum Na/K ratio in the test-tube. Provided the assumptions involved in our calculations are not in great error, we can conclude that the turnover of the Na-K pump in resting skeletal muscle is less than 3 % of the maximum turnover capacity of the ATPase. These low turnover rates of the sodium pump under physiological conditions allow for the very large changes observed when ion concentrations are modified. The findings of this and the previous paper indicate that there are at least two separate mechanisms by which the activity of the Na pump in skeletal muscle can be regulated: (a) changes in the number of active sites, (b) modification in the turnover rate of the enzyme. REFERENES ADRIAN, R. H. & SLAYmAw,. L. (1966). Membrane potential and conductance during transport of sodium, potassium and rubidium, in frog muscle. J. Phy8iol. 184, BARR, P. F., BLAusTEIN, M. P., HODGKIN, A. L. & STEINH T, E. A. (1969). The influence of calcium on sodium efflux in squid axons. J. Phygiol. 2, BAYR, P. F. & WILms, J. S. (1972). Binding of the cardiac glycoside ouabain to intact cells. J. Phy8iol. 224, BmWDy, F. J. & MULLINS, L. J. (1968). Sodium fluxes in internally dialysed squid axons. J. gen. Phy8iol. 52, Ross, S. B., KzYNEs, R. D. & RYBoVA, R. (1965). The coupling of sodium efflux and potassium influx in frog muscle. J. Physiol. 181, DESXLDT, J. E. (1953). Electrical activity and intracellular sodium concentration in frog muscle. J. Physiol. 121, ERLIJ, D. & GRINsTEIN, S. (1976). The number of sodiumionpumping sites in skeletal muscle and its modification by insulin. J. Phy8sol. 259, Giuy, R. P. & GARRAN, P. J. (1973). The interaction of sodium and potassium with the sodium pump in red cells. J. Phy8iol. 231,

13 AZIDE AND THE SODIUM PUMP 45 GwINsTEmN, S. & ERLiJ, D. (1974). hanges in the number of sodium pumping sites as a mechanism for regulation of the sodium pump in frog muscle. The Physiologist 17, 233. HODGKIN, A. L. &EKEYNEs, R. D. (1956). Experiments on the injection of substances into squid giant axons by means of a micro-syringe. J. Physiol. 131, HOROWIZ, P., APUTO,. & ROBESON, S. A. (1962). Rapid depolarization of frog muscle membranes by NaN3. Fedn Proc. 21, 151, HoRowicz, P. & GERBER,. J. (1965). Effects of sodium azide on sodium fluxes in frog striated muscle. J. gen. Phyeiot. 48, JORGENSEN, P. L. (1974). Isolation and characterization of the components of the sodium pump. Q. Rev. Biophya. 7, STEINBAH, H. B. (194). Sodium and potassium in frog muscle. J. biol. hem. 133,