shape. With 10 mm rubidium, when sodium was reduced from 5 mm to inflexion point was displaced to the right by ouabain.

Transcription

1 J. Phy8iol. (197), 21, pp With 17 text-figures Printed in Great Britain RUBIDIUM, SODIUM AND OUABAIN INTERACTIONS ON THE INFLUX OF RUBIDIUM IN RAT RED BLOOD CELLS BY L. A. BEAUGI* AND OLGA ORTfZ From the Instituto de Investigacion Medica, Mercedes y Martin Ferreyra, Casilla de Correo 389, Cordoba, Argentina (Received 1 December 1969) SUMMARY 1. The activation curve of rubidium influx by external rubidium in rat red cells showed an inflexion at a concentration around -2 mm. This inflexion point was displaced to the right by ouabain. 2. The removal of sodium from the external solution changed the characteristics of the activation curve of rubidium influx. At external rubidium below -5 Bm the uptake increased whereas above that concentration there was marked reduction. Thus the sodium-free effect on rubidium uptake is dependent on the external rubidium concentration. 3. With -25 mm rubidium, the relationship between increase ofrubidium influx and reduction of external sodium followed a more or less exponential function. All the increment was ouabain-sensitive. 4. With a rubidium concentration above -5 mm the reduction of the rubidium uptake, as sodium was removed, followed curves of complex shape. With 1 mm rubidium, when sodium was reduced from 5 mm to zero, there was an increase instead of a further reduction. These results suggest interactions of several effects. 5. The ouabain sensitivity of the rubidium influx in rat red cells is smaller than in other systems studied up to now. The dose-response curve was shifted to the right as the rubidium concentration increased and a plateau was obtained with rubidium only below 1 mm at 1-5 M ouabain. When plotted as a percentage of the maximal inhibition the points fell into the theoretical curve following a simple one reactant/one site reaction. 6. Ouabain inhibition seems to be a complex function of at least three variables: the concentration of the glycoside, the concentration of sodium and the concentration of rubidium. When sodium was absent, 1 /LM rubidium was able to prevent, to a great extent, the inhibition produced by 1-5 and 1 4 M ouabain. * Member of the Consejo Nacional de Investigaci6nes Cientificas y Tecnicas of Argentina.

2 52 L. A. BEAUGfl AND OLGA ORTIZ INTRODUCTION The normal mode of operation of the sodium pump in human red cells is based on the extrusion of sodium at the same time as the uptake of potassium (Glynn, 1957), with a stoichiometry of 1: 1 in net movements studies (Post, Albright & Dayani, 1967) and about 1-2 or 1-3 when unidirectional fluxes are considered. Thus one can evaluate the sodium pump mechanisms by studying either the efflux of sodium or the influx of potassium. Monovalent cations other than potassium, namely rubidium and caesium, can replace the former as external activators of the pump system in several tissues (Sjodin & Beauge, 1968; Beauge & Sjodin, 1968; Maizels, 1968). In recent work in this laboratory (Beauge & Adragna, 1969) it has been shown that in human red blood cells the uptake of rubidium is both qualitatively and quantitatively almost identical with the uptake of potassium, thus providing another method of evaluating this transport system when radioactive sodium or potassium is not available. In rat red cells Hoffman (1966) has described the activation of sodium efflux by external potassium as following an S-shaped curve. In the present work the uptake of rubidium by rat red cells and the influence of external sodium and ouabain on that uptake were studied. METHODS Blood of adult male rats was taken with heparinized syringes by heart puncture and stored in a refrigerator for 1-2 hr before use. The cells were washed three times in potassium-free sodium Ringer and twice in the incubation solution, the buffy coat being removed after the first centrifugation. Unless otherwise stated duplicate tubes were set up for each experimental condition. The temperature was 37 C and the uptake period lasted hr. Solutions. All solutions were made from 'pro-analysis' chemicals and de-ionized water, glucose (2 mg/1 ml.) being added as a solid before use. Choline chloride was purified as described by Garrahan & Glynn (1967 a). The solutions used had the following composition (nlm): (a) sodium Ringer: NaCl 15, CaCl2 1, MgCl2 1, orthophosphoric acid titrated with Tris (hydroxymethylaminomethane) to give ph 7-4 (37 C) 2. When rubidium was present the sodium concentration was 15 mm minus the rubidium concentration; (b) choline Ringer: the composition was similar to the former, sodium being replaced by equal amounts of choline; (c) mixture (sodiumcholine) Ringer: when the sodium concentration was to be reduced it was replaced by equimolar amounts of choline. The usual procedure was to mix appropriate amounts of sodium and choline Ringer. Ouabain was obtained from Sigma Co., U.S.A. Radioactive rubidium was obtained through the Comision Nacional de Energia Atomica of Argentina as rubidium chloride (86Rb) sterile solution of high specific activity. Rubidium influx. The uptake of rubidium was measured in the same way as described previously (Beauge & Adragna, 1969). As it was shown to be linear with time up to more than 1 hr with the concentrations of rubidium used, only one point at 1 hr was usually taken, in other experiments this was reduced to 3 min. The influx was

3 RUBIDIUM UPTAKE IN RAT RED CELLS 521 expressed in m-mole/(l. cells) hr. The volume of the cells was determined by analysing the content of oxyhaemoglobin of the lysate at 541 mic in a Coleman spectrophotometer, relating each time the reading to a control sample of known haematocrit from the same batch of cells. The readings of the unknown were always within 2% of the control. Counting. The activity of the samples was counted in a Beckman liquid scintillation counter without scintillator liquid (Garrahan & Glynn, 1966), counting for enough time to give a s.e. between 1 and 3% using the tritium channel. RESULTS As can be seen in Figs. 1 and 2, rubidium uptake in rat red cells, within 1 hr, at 1 or 5 mm external rubidium, follows a straight line within the resolution of the method in the different conditions in which the experiments were done. This indicates that the observed effects develop almost A E~~~~~~~~~~ Time (min) Time (min) Fig. 1 Fig. 2 Fig. 1. Influx of rubidium as a function of time in rat red cells at 1 mm rubidium. A, sodium media; A', sodium media with 1 4 M ouabain; B, choline media; B', choline media with 1- ouabain. Fig. 2. Influx of rubidium as a function of time in rat red cells at 5 mm rubidium. A, sodium media; A', sodium media with 14 M ouabain; B, choline media; B', choline media with 1 4m ouabain. instantaneously. Ouabain, as well as replacement of sodium by choline, caused a marked fall in the uptake of rubidium. The effect of a sodium-free solution was similar at both rubidium concentrations, but ouabain inhibition was greater at 1 mm than at 5 ml rubidium. These findings are described in more detail in the following sections. Influx of rubidium as a function of external rubidium concentration Fig. 3 shows one of these experiments. The uptake of rubidium reached saturation around 5 mi external rubidium and noticeable inflexion was not detected. As the activation curve of sodium efflux by external potassium in

4 522 L. A. BEAUGJE AND OLGA ORTIZ rat red cells has been described as having a strong inflexion at low potassium (Hoffman, 1966), it was decided to analyse the uptake of rubidium at concentrations up to 2 mm. Fig. 4 summarizes the results of two experiments. The shape of the uptake curve was sigmoid and not hyperbolic as in Fig. 3, with the inflexion point at about X2 mm rubidium. Ouabain produced a displacement of the inflexion point of the sigmoid to the right and the extent of the inhibition decreased as external rubidium rose. 6 ~~~~~~~~~~~6 '.- 2 -u2 EI o E x E ~ Fig 3 E P Fig 4 ~~~~~~~~~ I I red cel.etra 3 oimcnetainws1m 6 9 uiim 1 2 External Rb concn. (mm) External Rb concn. (mm) Fig. 3 Fig. 4 Fig. 3. Activation curve of rubidium influx by external rubidiumin rat red cells. External sodium concentration was 15 m~ rubidium. o control; ~* 1-4 m ouabain. Fig. 4. Activation curve of rubidium influx by low external rubidium and the effect of ouabain in rat red cells. O control; Q 16 M ouabain; * * 14 M ouabain. The effect of external sodium on rubidium influx That the decrease of rubidium uptake, after replacing sodium by choline, was not due to the poisoning effect of choline is shown in Fig. 5, where the influence of choline in human and rat red cells was studied. Both types of cells were stored in potassium-free and 1 mm potassium sodium Ringer 5 hr before use. It can be seen that, whereas the total uptake of rubidium decreased in rat cells, there was an increase in the human ones. The ouabain-sensitive fraction of the influx was also different in both cells in the presence of choline. The response was not affected by pre-incubation in media with (A) or without (B) potassium. Fig. 6 is the mean of five experiments on the activation curve of rubidium uptake by external rubidium with and without external sodium. In sodium Ringer there was an inflexion below 5 mm rubidium and a tendency to saturation, with a maximal uptake of about 6- m-mole/(1. cells) hr and a Km of 1L5 mm. In sodium-free media, whereas the uptake was noticeably increased below *5 mm rubidium, above 5 mm it was reduced. This curve has a hyperbolic shape with saturation around 3*5 m-mole/(l. cells) hr and a Km of -35 mm.

5 RUBIDIUM UPTAKE IN RAT RED CELLS 523 The function of the sodium sensitivity and the sodium dependence of the total rubidium uptake at constant rubidium is shown in Figs. 7, 8 and 9. At -25 mii rubidium external rubidium uptake increased in a more or less exponential fashion as the external sodium concentration was reduced (Fig. 7), and this increase started as early as 12 mu sodium. On the other Rat Human 3 2 I E 1 V '5 E Cx 3 I- V r_ x C. 2 I. I Na Ch Na Ch Fig. 5. Sodium-free effect and ouabain inhibition of rubidium influx in rat and human red cells at 1 m rubidium. The cells were pre-incubated for 5 hr at room temperature in potassium-free (A) and 1 rnm potassium (B) Ringer. The values represent the mean of two experiments. The ouabain concentration was 1-4 M. Hatched column represent the ouabainresistant uptake. hand, with 5 mm rubidium the uptake was reduced, and the relationship between influx and external sodium concentration followed an inverted S curve with a plateau between 3 and 1 mu sodium; below 3 m[ sodium (Fig. 8) this relationship followed a sigmoid curve, whereas above 13 mm it was exponential. When this relationship was studied at two concentrations of external rubidium (Fig. 9), different behaviour was observed both at the beginning and at the end of the curves. In fact at low sodium and

6 External Rb concn. (mm) Fig. 6. The effect of external sodium on the rubidium influx activation curve. sodium media; [] ] choline media. Each point represents the average of five different experiments + S.E. of the mean c -a -i U,- U 3 EE _ E - 2 :1E x -C C _. "",r,-5 mm-rb._.1 I I I usmi External Na concn. (mm) Fig. 7. The effect of external sodium on the rubidium influx in rat red cells and its dependence on the rubidium concentration. All media were kept isotonic with choline. L-@L 5 mm rubidium; t 25 my rubidium. 524 L. A. BEAUUGC AND OLUA ORTIZ 6 Sodium U C4, x..

7 RUBIDIUM UPTAKE IN RAT RED CELLS mm rubidium the sigmoid part of the curve had a steep rise beginning at 2 mm sodium, whereas with 1 mm rubidium the rise was smooth and did not start before the sodium concentration reached 1 mm; another remarkable point is that with 1 mm rubidium there was also an initial reduction in the uptake in very low sodium media as compared with the value in sodium-free media. At high sodium, whereas the 1 mm rubidium curve resembles the previous one at 5 mm rubidium, with 1 mm rubidium the uptake was similar to the value reached at a sodium concentration of 1 mm..c T"8 _: z~~~~~~ 5 5-b ~~~~~~~~ Ē, x.4 - x C r U. 6 S E E4 I.. X _ 1 mm-rb o -so _ 1 mm-rb o.m 6~~~~~~~~~ " External Na concn.((mm) Fig ISO External Na concn. (mm) Fig. 9 Fig. 8. Sodium dependence of rubidium influx in rat red cells at 5 mm rubidium. Choline was used as replacement for sodium. Fig. 9. Sodium dependence of rubidium influx in rat red cells at 1 mm (lower curve) and 1 mm external rubidium. Choline was used as replacement for sodium. Ouabain inhibition of rubidium influx A dose-response curve for ouabain inhibition is shown in Fig. 1. The points are means of two experiments and the lines were calculated by considering the interaction ouabain/site of action as a single one reactantone site reaction (see Mitchell, 1953); they correspond to the equation K K [I] [a 1-X x where [I] is the concentration of the inhibitor, X the percentage of inhibition and K the reciprocal of the association constant which was chosen arbitrarily to give the best fit. The value of the constants were 1-5 x 15M and 1-35 x 1-4M for 1 and 5 mm external rubidium respectively. In previous sections it was shown that when sodium was replaced by choline the ouabain-resistant rubidium influx was largely increased. Fig. 11

8 L. A. BEAUGJ9 AND OLGA ORTIZ 526 describes the sodium dependence of the ouabain inhibition at *25 mm rubidium at different concentrations of the glycoside. In all cases there was an increase of the ouabain-resistant uptake with removal of sodium, but, whereas at 1- M ouabain it started as early as 12 mm sodium, at 1-3 M there was no change before 6 mm sodium. This behaviour could be a consequence solely of the reduction of sodium, or because removal of 1 1 mm-rb 5 mm-rb -o.e 5_ E Ouabain concn. (mole/i.) log. scale Fig. 1. Dose-response curve for ouabain inhibition of rubidium influx in rat red cells plotted as % maximal inhibition. The points are the experimental ones. The lines going through them are the theoretical ones for the equation K = [I] (1-X)/X. For details see text. sodium made rubidium a better antagonist of ouabain. These possibilities were checked in the experiments shown in Fig. 12 where the inhibition by 1-3 M ouabain was studied at lower concentrations of rubidium. Ouabain inhibition was less sensitive to the removal of sodium as rubidium was reduced, and at -1 mm rubidium it was not affected before sodium reached 5 mm. As cross experiments, Figs. 13 and 14 describe the same phenomena but for a change of concentration of ouabain at -1 mx rubidium. They show that, if the concentration of rubidium is reduced

9 RUBIDIUM UPTAKE IN RAT RED CELLS 527 enough, at 1-3 M ouabain the resistant influx does not change as external sodium is removed, which indicates that the increment in the total uptake observed when choline replaced sodium is ouabain-sensitive. Fig. 15 summarizes in a dose-response plot the results at -25 mm rubidium E.3 Control E N ~~~~~~~~~~E tions oubain x mm-rb ws 1mtMouabainO c o *.. 1 mm-ba ~~~11 mm-rb External Na concn. (mm) External Na concn. (mm) *- *14Mubin-3 M ouabain. Fig. 1 1 Fig. 12 Fig. 11. The effect of varying the external sodium and ouabain concentrations on the rubidium influx in rat red cells at -25 mma rubidium. Tonicity was maintained with choline. o control; o 1-5 M ouabain; ---@1h4mouabain;@-----@1-3Mouabain. Fig. 12. The effect of varying external sodium and rubidiuim concentration on the inhibition of rubidium influx by 1-3 M ouabain at low rubidium. The replacement of sodium was made with choline. * -25 mm rubidium; *--* 1mmrubidium; *- * -5 m rubidium; * mm rubidium. The sodium dependence of ouabain inhibition at 5 mm external rubidium is shown in Fig. 16. At 15 M ouabain there was no difference from the unpoisoned fluxes; at higher concentrations of the glycoside the resistant influx began to increase as sodium was reduced, similar to that observed with -25 mm rubidium, but then it fell following changes in total uptake. DISCUSSION Sodium-free effect on rubidium influx The results of the present work provide clear evidence about the variability of the sodium-free effect on the influx of rubidium in rat red cells. The removal of external sodium not only changes the shape of the

10 L. A. BEAUGJ9 AND OLGA ORTIZ 528 activation curve, from S-shapedto hyperbolic, reducing the apparent Ki,, but also reduces the maximal uptake. Then, the sodium-free effect will depend on the concentration of rubidium chosen, resulting in an increase in the uptake below 5 mm rubidium and a reduction above that concentration. The mechanism of this dual response is not clear. One possible explanation could be that changes in external sodium and rubidium modify the cells' sodium concentration, and, if the rate of pumping varies with a power -4.4-,C, 3 E3 E ternal NControl(me) External Na control contol; - * 15sM l ouabain clo4m ouabain o 1-4Mouabain 1-3m ouabain Fi.1.Teefc fvyn h xxnlsdu and-s ouabain * ouabain cnet ti n 3h ubdu 6 9Contrlu 12 nraredl 15 a-1 m uiim 5 1 External Na concn. (mm) External Na concn. (mm) Fig. 13 Fig. 154 Fig. 13. The effect of varying the external sodium and ouabain concentrations on the rubidium influx in rat red cells at t1m rubidium. g 1-5m ouabain; *---* 1-4m ouabain; i 3 m ouabain. Solutions were made isotonic with choline. Fig. 14. The sodium and ouabain effect on the inf xof rubidium in rat red cells at very low rubidiumr (1 mm) and sodium concentrations. oqa o control;d q t* 1-5wtouabain;o ---* 1pai ouabain; m ouabain. Solutions were made isotonic, with choline. close to 2 of such a concentration, minimum changes in the internal sodium could largely modify the uptake of rubidium. The experiments of Fig. 5, where the effect was the same in cells stored in a potassium-free medium as in 1 mm potassium media, and the short time intervals taken, make this hypothesis not very probable. On the other hand, the graphs of rubidium influx versus external sodium concentration are highly complicated and suggest a complex type of interaction, possibly at the external side of the membrane. In human red cells the uptake of rubidium has been identified both qualitatively and quantitatively with that of potassium, and of course with

11 RUBIDIUM UPTAKE IN RAT RED CELLS 529 the active extrusion of sodium (Maizels, 1968; Beauge6 & Adragna, 1969). A question which immediately arises is whether the type of activationinhibition described in the present work could be responsible for the contradictory results on the sodium-free effect in those cells. Thus Sachs & Welt (1967), Garrahan & Glynn (1967b) and Beauge & Adragna (1969) 5 - ~~~~Control x C.E. _ C.t C so 5 AA; Y x~~~~~~ -- I-4.C U J2, a E x C1.olI i/ I_ 1-3M ouabain, % '' Ouabain concn.(mole/l.) log. scale Fig. 15 f I I I External Na concn. (mm) Fig. 16 Fig. 15. Sodium effect on the dose-response curve for ouabain inhibition of rubidium influx at -25 mm rubidium. * * 15 mm sodium; * * 12 M sodium; * A 9 mm sodium; V v 6 m sodium; 3 mm sodium; x x sodium-free. Fig. 16. The effect of varying external sodium and ouabain concentrations on the influx of rubidium in rat red cells at 5 mm rubidium. The replacement of sodium was made with choline. O control; *- * 1- M ouabain; *---* 1-Mouabain; - --* 1-3M ouabain. found increase or no change both in the sodium efflux and potassium or rubidium uptake when external sodium was removed, whereas Hoffman (1966) and Lubowitz & Whittam (1969) reported reduction. Differences in the experimental procedure could make this phenomena appear in some cases and remain hidden in others. Ouabain inhibition of rubidium influx It seems that the inhibitory effect of ouabain on the uptake of rubidium is not simple, but involves specific interactions with at least both rubidium and sodium ions. The exquisite sensitivity of the system is evidenced by the fact that 1 /AM rubidium can prevent about 8 % of the inhibition by

12 53 L. A. BEAUGE AND OLGA ORTIZ 1- M ouabain if external sodium is removed. The reduction of the poisoned fluxes at low sodium in Fig. 16 seems to be merely a consequence of the reduction of the total uptake in those conditions. That the same type of interactions are involved in high as well as in low external rubidium is supported by the dose-effect plots for ouabain inhibition in Figs. 15 and 17. The characteristics of both groups of curves are the same; the different '1 C C 5 o -c.c X W. -_ Ouabain concn. (mole/i.) log. scale Fig. 17. Sodium effect on the dose-response curve for ouabain inhibition of rubidium influx at 5 mm rubidium in rat red cells. * * 15 mm sodium; * * 12 mm sodium; A A 9 mm sodium; V V 6 ma sodium; * 3 mim sodium; x x sodium-free. sensitivity to external sodium can be accounted for by the twentyfold difference in rubidium concentration. Thus, the residual uptake in 5 mm rubidium choline with 1-3 M ouabain cannot be considered the true ouabain-resistant uptake, but as having a large ouabain-sensitive component which cannot be detected. In this case there is no possibility of reducing the external rubidium in order to prevent antagonism with ouabain, because it would go into concentrations where the sodium dependence of the total uptake is not detected; on the other hand the concentration of the glycoside cannot be increased further above 1-3 M. Competition seems too simple a model to explain all the above results,

13 RUBIDIUM UPTAKE IN RAT RED CELLS 531 and other types of interactions need to be postulated. As was suggested by Hoffman (1966) an allosteric effect could exist. This would be supported by the following facts: (a) the activation curves of rubidium influx are S-shaped and the glycoside produces its enhancement, similar to that which was shown in crab nerve by Baker & Connelly (1966); (b) the inhibition obtained with cardiac glycosides never reaches 1 %, as is the case with allosteric inhibitors in known enzyme systems, because the inhibitor does not act on the active centre but on other sites. The same finding has been reported for human red cells (Glynn, 1957; Beauge & Adragna, 1969) and for sodium efflux in squid giant axons (Brinley & Mullins, 1967; Baker, Blaustein, Keynes, Manil, Shaw & Steinhardt, 1969); (c) sometimes allosteric inhibitors show competitive behaviour because the allosteric centre is very close to the active one. The interpretation of these results depends in great extent on whether or not the effect of ouabain develops at once. The experiments of Figs. 1 and 2 as well as those from unpublished work, which were done at a lower ouabain concentration (1-5 M) show that this is the case under our experimental conditions. This work was supported by Grant No. 3667/69 from the Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina. We wish to thank Dr I. M. Glynn for the critical reading of the manuscript. REFERENCES BAKER, P. F. & CoNNELLY, C. M. (1966). Some properties of the external activation site of the sodium pump in crab nerve. J. Physiol. 185, BAKER, P. F., BLAIus~Tn, M. P., KEYNES, R. D., MANiL, J., SHAW, T. I. & STIN- HARDT, R. A. (1969). The ouabain-sensitive fluxes of sodium and potassium in squid giant axons. J. Phyniol. 2, BEAUGIl, L. A. & ADRAGNA, N. C. (1969). Efectos de ouabain y sodio sobre el influjo de rubidio en globulos rojos humanos. V Reuni6n Nacional S.A.I.B., p. 9. BEAUGA, L. A. & SJODiN, R. A. (1968). The dual effect of lithium ions on sodium efflux in skeletal muscle. J. yen. Physiol. 52, BRINLEY, F. J. & MuLLEs, L. J. (1967). Sodium extrusion by internally dialyzed squid axons. J. gen. Physiol. 5, 233. GARRAHAN, P. J. & GLYNN, I. M. (1966). Measurement of 24Na and 42K with a liquid scintillation counting system without added scintillator. J. Physiol. 186, 54 P. GARRAHAN, P. J. & GLYNN, I. M. (1967a). The behaviour of the sodium pump in red cells in the absence of external potassium. J. Physiol. 192, GARRAHAN, P. J. & GLYNN, I. M. (1967b). The sensitivity of the sodium pump to external sodium. J. Physiol. 192, GLYNN, I. M. (1957). The ionic permeability of the red cell membrane. Prog. Biophys. biophys. Chem. 8, HOFFMAN, J. F. (1966). The red cell membrane and the transport of sodium and potassium. Am. J. Med. 41, LuBowrrz, H. & WVIrTArM, R. (1969). Ion movement in human red cells independent of the sodium pump. J. Physiol. 22,

14 532 L. A. BEAUGJ9 AND OLGA O-STIZ MAIZELS, M. (1968). Effect of sodium content on sodium efflux from human red cells suspended in sodium-free media containing potassium, rubidium, caesium or lithium chloride. J. Physiol. 195, MITCHELL, P. (1953). Transport of phosphate across the surface of Micrococcus pyogenes: nature of the cell 'inorganic' phosphate. J. gen. Microbiol. 9, POST, R. L., ALBRIGHT, C. D. & DAYANI, K. (1967). Resolution of pump and leak components of sodium and potassium transport in human erythrocyte. J. gen. Physiol. 5, SACHS, J. R. & WELT, L. C. (1967). The concentration dependence of active potassium transport in the human red blood cells. J. din. Invest. 46, SJODIN, R. A. & BEAUGE, L. A. (1968). Coupling and selectivity of sodium and potassium transport in squid giant axons. J. yen. Physiol. 51, 1528.