Strophanthidin-sensitive transport of cesium and sodium in muscle cells.

Uptake of cesium-134 ions into muscle cells is reduced to very low values by the presence of 10(-5)M strophanthidin in the Ringer solution. Cesium ions can induce extrusion of sodium from muscle cells in which the intracellular sodium content is elevated. The cesium-induced extra efflux of sodium-22 is inhibited by the external presence of 10(-5)M strophanthidin. The coupling between inward movement of cesium and outward movement of sodium appears to be chemical in nature. The evidence suggests that cesium ions are transported into muscle cells by a system of sites or carriers that requires a source of metabolic energy for ion turnover to occur.

The ATP dependence of a ouabain-sensitive sodium efflux activated by external sodium, potassium and lithium in human red cells.

The stimulation of ouabain-sensitive Na+ efflux by external Na+, K+ and Li+ was studied in control and ATP-depleted human red cells. In the presence of 5 mM Na+, with control and depleted cells, Li+ stimulated with a lower apparent affinity than K+, and gave a smaller maximal activation than K+. The ability of Na+, K+ and Li+ to activate Na+ efflux was a function of the ATP content of the cells. Relative to K+ both Na+ and Li+ became more effective activators when the ATP was reduced to about one tenth of the control values. At this low ATP concentration Na+ was absolutely more effective than K+.

Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons.

Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na+ efflux goes through the Na+ pump. We found that (i) internal K+ had a strong inhibitory effect on Na+ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na+, (ii) a reduction in ATP levels from 3 mM to 50 microM greatly increased the apparent affinity for external K+, but reduced its effectiveness compared with other monovalent cations, as an activator of Na+ efflux, and (iii) the relative effectiveness of different K+ congeners as external activator of the Na+ efflux, though affected by the ATP concentration, was not affected by the Na+/K+ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na+ + K+)-ATPase can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.

The independence of membrane potential and potassium activation of the sodium pump in muscle.

The membrane potential (Em) of sartorius muscle fibers was made insensitive to [K+] by equilibration in a 95 mM K+, 120 mM Na+ Ringer solution. Under these conditions a potassium-activated, ouabain-sensitive sodium efflux was observed which had characteristics similar to those seen in muscles with Em sensitive to [K+]. In addition, in the presence of 10 mM K+, these muscles were able to produce a net sodium extrusion against an electrochemical gradient which was also inhibited by 10- minus 4 M oubain. This suggests that the membrane potential does not play a major role in the potassium activation of the sodium pump in muscles.

An analysis of the leakages of sodium ions into and potassium ions out of striated muscle cells.

Net sodium influx under K-free conditions was independent of the intracellular sodium ion concentration, [Na](i), and was increased by ouabain. Unidirectional sodium influx was the sum of a component independent of [Na](i) and a component that increased linearly with increasing [Na](i). Net influx of sodium ions in K-free solutions varied with the external sodium ion concentration, [Na](o), and a steady-state balance of the sodium ion fluxes occurred at [Na](o) = 40 mM. When solutions were K-free and contained 10(-4) M ouabain, net sodium influx varied linearly with [Na](o) and a steady state for the intracellular sodium was observed at [Na](o) = 13 mM. The steady state observed in the presence of ouabain was the result of a pump-leak balance as the external sodium ion concentration with which the muscle sodium would be in equilibrium, under these conditions, was 0.11 mM. The rate constant for total potassium loss to K-free Ringer solution was independent of [Na](i) but dependent on [Na](o). Replacing external NaCl with MgCl(2) brought about reductions in net potassium efflux. Ouabain was without effect on net potassium efflux in K-free Ringer solution with [Na](o) = 120 mM, but increased potassium efflux in a medium with NaCl replaced by MgCl(2). When muscles were enriched with sodium ions, potassium efflux into K-free, Mg(++)-substituted Ringer solution fell to around 0.1 pmol/cm(2).s and was increased 14-fold by addition of ouabain.

The kinetics of ouabain inhibition and the partition of rubidium influx in human red blood cells.

IN THE DEVELOPMENT OF OUABAIN INHIBITION OF RUBIDIUM INFLUX IN HUMAN RED BLOOD CELLS A TIME LAG CAN BE DETECTED WHICH IS A FUNCTION OF AT LEAST THREE VARIABLES: the concentrations of external sodium, rubidium, and ouabain. The inhibition is antagonized by rubidium and favored by sodium. Similar considerations could be applied to the binding of ouabain to membrane sites. The total influx of rubidium as a function of external rubidium concentration can be separated into two components: (a) a linear uptake not affected by external sodium or ouabain and not requiring an energy supply, and (b) a saturable component. The latter component, on the basis of the different effects of the aforementioned factors, can be divided into three fractions. The first is ouabain-sensitive, inhibited by external sodium at low rubidium, and requires an energy supply; this represents about 70-80% of the total uptake and is related to the active sodium extrusion mechanism. The second is ouabain-insensitive, activated by external sodium over the entire range of rubidium concentrations studied, and dependent on internal ATP; this represents about 15% of the total influx; it could be coupled to an active sodium extrusion or belong to a rubidium-potassium exchange. The third, which can be called residual influx, is ouabain-insensitive, unaffected by external sodium, and independent of internal ATP; this represents about 10-20% of the total influx.

Strophanthidin-sensitive components of potassium and sodium movements in skeletal muscle as influenced by the internal sodium concentration.

"Low sodium" muscles were prepared which contained around 5 mmoles/kg fiber of intracellular sodium. "High sodium" muscles containing between 15 and 30 mmoles/kg fiber of intracellular sodium were also prepared. In low sodium muscles application of 10(-5)M strophanthidin reduced potassium influx by about 5%. Potassium efflux was unaffected by strophanthidin under these conditions. In high sodium muscles, 10(-5)M strophanthidin reduced potassium influx by 45% and increased potassium efflux by 70%, on the average. In low sodium muscles sodium efflux was reduced by 25% during application of 10(-5)M strophanthidin while in high sodium muscles similarly treated, sodium efflux was reduced by about 60%. Low sodium muscles showed a large reduction in sodium efflux when sodium ions in the Ringer solution were replaced by lithium ions. The average reduction in sodium efflux was 4.5-fold. Of the amount of sodium efflux remaining in lithium. Ringer's solution, 40% could be inhibited by application of 10(-5)M strophanthidin. The total sodium efflux from low sodium muscles exposed to Ringer's solution in which lithium had been substituted for sodium ions for a period of 1 hr can be fractionated as 78% Na-for-Na interchange, 10% strophanthidin-sensitive sodium pump, and 12% residual sodium efflux. It is concluded that large strophanthidin-sensitive components of sodium and potassium flux can be expected only at elevated sodium concentrations within the muscle cells.

The dual effect of lithium ions on sodium efflux in skeletal muscle.

Sartorius muscle cells from the frog were stored in a K-free Ringer solution at 3 degrees C until their average sodium contents rose to around 23 mM/kg fiber (about 40 mM/liter fiber water). Such muscles, when placed in Ringer's solution containing 60 mM LiCl and 50 mM NaCl at 20 degrees C, extruded 9.8 mM/kg of sodium and gained an equivalent quantity of lithium in a 2 hr period. The presence of 10(-5)M strophanthidin in the 60 mM LiCl/50 mM NaCl Ringer solution prevented the net extrusion of sodium from the muscles. Lithium ions were found to enter muscles with a lowered internal sodium concentration at a rate about half that for entry into sodium-enriched muscles. When sodium-enriched muscles labeled with radioactive sodium ions were transferred from Ringer's solution to a sodium-free lithium-substituted Ringer solution, an increase in the rate of tracer sodium output was observed. When the lithium-substituted Ringer solution contained 10(-5)M strophanthidin, a large decrease in the rate of tracer sodium output was observed upon transferring labeled sodium-enriched muscles from Ringer's solution to the sodium-free medium. It is concluded that lithium ions have a direct stimulating action on the sodium pump in skeletal muscle cells and that a significantly large external sodium-dependent component of sodium efflux is present in muscles with an elevated sodium content. In the sodium-rich muscles, about 23% of the total sodium efflux was due to strophanthidin-insensitive Na-for-Na interchange, about 67% being due to strophanthidin-sensitive sodium pumping.

The influence of potassium- and sodium-free solutions on sodium efflux from squid giant axons.

The sensitivity of sodium efflux to the removal of potassium ions from the external solution and the change in sodium efflux occurring when sodium ions are also removed were observed to be related. When Tris was used to replace external sodium ions, increases in sodium efflux were always observed whether the sensitivity of sodium efflux to external potassium ions was weak or strong. Greater percentage increases in sodium efflux occurred, however, the greater the sensitivity of sodium efflux to external potassium ions. When lithium ions were used to replace external sodium ions, increases in sodium efflux occurred if the sensitivity of efflux to external potassium ions was strong whereas decreases in sodium efflux took place if the sensitivity of efflux to external potassium ions was weak. Intermediate sensitivities of efflux to external potassium resulted in no change in efflux upon substitution of lithium ions for external sodium ions. In the presence of 10(-5)M ouabain, substitution of Tris for external sodium ions always resulted in a small decrease in sodium efflux. The data can be described in terms of a model which assumes the presence of efflux stimulation sites that are about 98% selective to potassium ions and about 2% selective to sodium or lithium ions.

An analysis of the influence of membrane potential and metabolic poisoning with azide on the sodium pump in skeletal muscle.

1. Activation of the Na pump in muscle by the external K concentration, [K]O, is independent of the membrane potential (Em) as shown by experiments in which Em was either stabilized during variation of [K]O or varied by application of azide at constant or zero [K]O. 2. Application of azide to Na-enriched muscles causes a transient increase in 22Na efflux which occurs either in the presence or in the absence of external K. 3. The increased 22Na efflux induced by azide is abolished by addition of ouabain and is greatly reduced by removal of almost all of the external Na concentration, [Na]o. 4. Azide-treated muscles show a rather normal K sensitivity of 22Na efflux and [K]O induces a net Na extrusion from Na-enriched muscles in the presence of azide. 5. Azide reduces ouabain-sensitive K influx to low values thus interfering with K pump but not with the ability of K to activate the Na pump. 6. The experiments provide evidence that azide promotes a ouabainsensitive Na-Na exchange in Na-enriched muscles and that it partially uncouples the Na-K exchange normally observed.

Sodium ions, acting at high-affinity extracellular sites, inhibit sodium-ATPase activity of the sodium pump by slowing dephosphorylation.

1. It is known that extracellular Na+ ions, in low concentrations, inhibit Na+-ATPase activity in resealed red cell ghosts and that this inhibition is reversed by high concentrations of extracellular Na+. We have attempted to elucidate these actions of extracellular Na+ by investigating the dependence on Na+ concentration of (a) ATP-ADP exchange and Na+-ATPase activity both in native and in N-ethylmaleimide (NEM)-treated (Na+ + K+)-ATPase from pig kidney, and (b) the rate of hydrolysis of the phosphorylated kidney enzyme in the absence of K+ ions. 2. With the native enzyme, ATP-ADP exchange and Na+-ATPase activity showed similar responses to changes in Na+ concentration: a steep but S-shaped rise between 0 and 2.5 mM, a slight fall (exchange) or a plateau (ATPase) between 2.5 and 10 mM, and a roughly linear rise between 10 and 150 mM. With NEM-treated enzyme, the ATP-ADP exchange, which was greatly accelerated, showed no sign either of inhibition at intermediate Na+ concentrations or of the reversal of that inhibition at higher concentrations. The exchange rate increased with Na+ concentration in a smooth curve and was half-maximal at about 7 mM. 3. The effects, on ATP-ADP exchange, of changing the concentrations of ATP, ADP and Mg have also been investigated. With both native and NEM-treated enzyme, the interactions of ATP, ADP and Mg are complicated; they show that, for the reaction leading to ATP formation, either free ADP rather than MgADP is the substrate, or Mg2+ ions are inhibitory (or both). 4. Since NEM, in the conditions in which we have used it, is believed to act by inhibiting the conversion of an ADP-sensitive form of the phosphoenzyme (E1P) to an ADP-insensitive form (E2P), the absence of Na+ inhibition of ATP-ADP exchange in NEM-treated enzyme, together with the parallel effects of Na+ ions on the ATP-ADP exchange activity and on the Na+-ATPase activity of native enzyme, suggests that the inhibitory effect of external Na+ occurs after the conversion of E1P into E2P. 5. To test whether this inhibitory effect of Na+ reflected inhibition of the hydrolysis of E2P, we measured the rate of loss of incorporated 32P when enzyme, newly phosphorylated by [gamma32P]ATP, was squirted into a large volume of ice-cold solution containing 1,2-cyclohexylenedinitrilotetraacetic aicd (CDTA), unlabelled ATP and 0, 5 or 150 mM-Na+. The rate of loss of radioactivity from the membranes was least at 5 mM-Na+, about twice as great at 150 mM-Na+, and about 5 times as great at 20 microM (final) Na+. 6. An unexpected feature of the results was that the pattern of stimulation of ATP-ADP exchange in intact cells. If Na+ ions are absent externally, a different could be fitted better on the assumption that activation by internal Na+ occurs at two sites with equal affinities, than on the assumptions that activation occurs at a single site or at three sites with equal affinities.

The equilibrium between different conformations of the unphosphorylated sodium pump: effects of ATP and of potassium ions, and their relevance to potassium transport.

1. Changes in the intrinsic fluorescence of Na, K-ATPase protein have been used to monitor the interconversion of E(1) (low fluorescence) and E(2) (high fluorescence) forms of the unphosphorylated enzyme.2. In media lacking sodium and nucleotides, 1 mM-potassium was sufficient to convert practically all of the enzyme into the E(2) form. In media containing 1 mM-potassium, 1 mM-EDTA, and no sodium or magnesium, the addition of ATP, or its beta, gamma-imido or methylene analogues, converted the enzyme back into the E(1) form. The relation between nucleotide concentration and the fraction of the enzyme that was in the E(1) form could be described by a rectangular hyperbola, with a K((1/2)) of about 15 muM for ATP, 65 muM for adenylyl-imidodiphosphate (AMP-PNP) and 180 muM for adenylyl (beta, gamma-methylene)-diphosphonate (AMP-PCP). ADP also converted the enzyme back into the E(1) form, with a K((1/2)) of about 25 muM, but the relation between concentration and fraction converted was not well described by a rectangular hyperbola.3. In similar media containing 50 mM-potassium, much higher concentrations of ATP were required to convert the enzyme back into the E(1) form, and the conversion was probably incomplete.4. If we assume that ATP and potassium ions affect each other's binding solely by altering the equilibrium between E(1) and E(2) forms of the enzyme, we are able to conclude (i) that potassium ions bind to the E(1) form with a moderately low affinity, (ii) that, in the absence of nucleotides, the equilibrium between E(1)K and E(2)K is poised strongly in favour of E(2)K, (iii) that the binding of ATP to a low-affinity site alters the equilibrium constant for the interconversion of E(1)K and E(2)K by two to three orders of magnitude, so that, at saturating levels of ATP, the equilibrium is probably slightly in favour of E(1)K, and (iv) that in sodium-free, potassium-containing media, ATP will appear to bind to the enzyme more tightly than would be expected from the dissociation constant of the E(2)K. ATP complex.5. The pattern of the equilibrium constants for the various reactions between E(1), E(2), ATP and potassium is compatible with the hypothesis that the ATP-accelerated conversion of E(2)K into E(1)K, and the subsequent release of potassium ions from low-affinity inward-facing sites, are part of the normal sequence of events during potassium influx in physiological conditions.

Further evidence for a potassium-like action of lithium ions on sodium efflux in frog skeletal muscle.

1. The efflux of labelled sodium as well as net sodium and lithium changes were studied in aged high sodium sartorius muscles of the South American frog Leptodactilus ocelatus.2. In the presence of 2.5 mM potassium in the media, the replacement of external sodium with lithium or magnesium resulted in an increase in sodium efflux. The magnitude of such increase was always larger in lithium.3. With the absence of potassium in the media, the response of sodium efflux to replacement of external sodium varied with the cation used as a substitute. In lithium Ringer there was always a noticeable increase, whereas in magnesium there was always a marked reduction. The same results were observed when calcium was substituted for magnesium.4. The replacement of 60 mM external sodium with sucrose did not prevent the stimulating effect of 5 mM potassium on sodium efflux, nor the inhibitory action of 10(-4)M ouabain. This indicates that neither sucrose by itself, nor the lowering of the ionic strength, modified to an appreciable extent the function of the sodium pump.5. Net sodium extrusion took place against an electrochemical gradient in potassium-free - 50 mM sodium - mM lithium Ringer. About 75% of this efflux was ouabain sensitive.6. Muscles made both sodium and lithium rich and incubated in potassium-free - 60 mM sodium - 50 mM lithium Ringer also showed net sodium extrusion against an electrochemical gradient, which was 85% ouabain sensitive. This extrusion took place even under conditions where the changes in free energy favouring lithium entry were always lower than the changes in free energy opposing sodium going out. This indicates that a sodium-lithium exchange by a counter-transport process is unlikely.7. External potassium reduced the ouabain sensitive lithium influx in muscles incubated in lithium Ringer. The values found were 5.90 +/- 0.39 mu-mole/g.hr and 2.66 +/- 0.43 mumole/g.hr in potassium-free and 15 mM potassium respectively. At the same time potassium had no effect on the ouabain-insensitive lithium uptake.8. Muscles incubated in potassium-free-magnesium Ringer had a residual sodium efflux which could not be accounted for by passive movement. About 40% of it was abolished by 10(-4)M ouabain. This ouabain-sensitive part could be a consequence of some stimulation of the sodium pump by potassium leaking out of the cells. If this is correct it should be inhibited by external sodium and should not contribute to the total sodium efflux in potassium-free sodium media.9. Magnesium was used as the reference cation to study the sodium-stimulated sodium efflux under potassium-free conditions. The total sodium efflux amounted to 0.668 hr(-1) (rate constant) and was 71% ouabain sensitive.10. The present experiments demonstrated that lithium ions have a direct stimulating effect on sodium efflux in high sodium skeletal muscle, and strongly support the notion that this effect is produced by an activation of the sodium pump through a potassium-like action.

Transport of caesium in frog muscle.

1. The entry of caesium into sartorius muscle cells is strongly suppressed by the presence of 10(-5)M strophanthidin in Ringer solution.2. The amount by which caesium entry is reduced in the presence of strophanthidin is dependent on the intracellular sodium concentration and is greater the higher the intracellular sodium concentration.3. The magnitude of caesium influx in the absence of strophanthidin is highly dependent on the intracellular sodium concentration.4. Caesium uptake by muscles in which sodium has been largely replaced by lithium is reduced to very low values.5. Caesium can promote the extrusion of sodium from muscles with high intracellular sodium concentrations. The effects of 25 mM caesium and 5 mM potassium on sodium extrusion are roughly the same.6. External potassium inhibits the entry of caesium ions into muscle cells presumably by competing for transport sites.7. The drug strophanthidin has no effect on (134)Cs efflux provided that muscles have been loaded with tracer ions for a long period of time. Caesium efflux from the intracellular compartment appears to occur by a process not mediated by metabolism.8. The action of strophanthidin on (134)Cs efflux from muscles exposed to tracer for short times suggests that caesium ions are transported inwardly by an active process after first accumulating in a superficial reservoir.

Rubidium, sodium and ouabain interactions on the influx of rubidium in rat red blood cells.

1. The activation curve of rubidium influx by external rubidium in rat red cells showed an inflexion at a concentration around 0.2 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 0.5 mM 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 0.25 mM rubidium, the relationship between increase of rubidium 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 0.5 mM the reduction of the rubidium uptake, as sodium was removed, followed curves of complex shape. With 10 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 10(-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, 10 muM rubidium was able to prevent, to a great extent, the inhibition produced by 10(-5) and 10(-4)M ouabain.