Sodium-sodium Exchange Research Articles

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala.

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.

Increased 22Na+-influx in lymphocytes from offspring of essential hypertensive patients

Lymphocytes were used as a cellular model for the in vitro measurements of 22Na+-influx during sodium pump inhibition by ouabain. The measurements were made using lymphocytes from young men at increased risk of developing essential hypertension in order to assess any changes and to analyse whether any such changes were associated to borderline hypertension and/or heredity.Four groups were evaluated: 28 normotensive and 20 borderline hypertensive offspring of hypertensives, 12 borderline hypertensives and 28 normotensives with normotensive parents.22Na+-influx was significantly increased in offspring of hypertensive parents especially in the normotensive offspring of hypertensive parents.The association between heredity and increased 22Na+-influx found by us in vitro may be caused by either an increased passive sodium-influx and/or an increased sodium-sodium exchange mechanism.

Increased 22Na+-influx in lymphocytes from offspring of essential hypertensive patients.

Lymphocytes were used as a cellular model for the in vitro measurements of 22Na+-influx during sodium pump inhibition by ouabain. The measurements were made using lymphocytes from young men at increased risk of developing essential hypertension in order to assess any changes and to analyse whether any such changes were associated to borderline hypertension and/or heredity. Four groups were evaluated: 28 normotensive and 20 borderline hypertensive offspring of hypertensives, 12 borderline hypertensives and 28 normotensives with normotensive parents. 22Na+-influx was significantly increased in offspring of hypertensive parents especially in the normotensive offspring of hypertensive parents. The association between heredity and increased 22Na+-influx found by us in vitro may be caused by either an increased passive sodium-influx and/or an increased sodium-sodium exchange mechanism.

Sodium pump-catalyzed sodium-sodium exchange associated with ATP hydrolysis.

Inside-out red cell membrane vesicles have been used to study sodium interactions with the ATP-dependent sodium pump at sites accessible to both membrane surfaces. ATP-dependent 22Na+ influx (equivalent to efflux from cells) shows sigmoid dependence on extravesicular Na+ concentration. This is observed both in the absence of intravesicular cations and in the presence of intravesicular K or Rb ions. The kinetic behavior is similar to that observed earlier with intact cells, (Garay, R. P., and Garrahan, P. J. (1973) J. Physiol. (Lond.) 231, 297-325) and is consistent with a ratio of close to three Na ions transported per molecule of ATP hydrolyzed. With vesicles having relatively high intravesicular sodium concentration, (approximately 50 mM NaCl), the sodium pump effects an ATP-dependent sodium efflux coupled to sodium influx and to strophanthidin-sensitive ATP hydrolysis. The influx:efflux stoichiometry is approximately 1:1, and the influx:ATP hydrolysis ratio is close to 3. This ATP-dependent exchange has a higher affinity for vanadate than ATP plus ADP-dependent sodium exchange. It is concluded that this sodium-sodium exchange mode resembles sodium-potassium exchange whereby intravesicular sodium, i.e. sodium at the extracellular surface, at relatively high concentration, behaves like potassium.

Effect of extracellular potassium on the transport of sodium and potassium in rat thymocytes.

1. The effect of extracellular potassium on the transport of sodium and potassium in rat thymocytes has been studied in vitro. 2. A significant increase in the rate constant for total and ouabain-sensitive sodium efflux was demonstrated at an extracellular potassium concentration of 1 mmol/l as compared with that at either 0 or 2 mmol/l. 3. At potassium concentrations below 3 mmol/l ouabain-sensitive sodium influx was observed suggesting sodium-sodium exchange catalysed by the sodium pump. 4. Both total and ouabain-insensitive potassium efflux rose with external potassium. A small ouabain-sensitive potassium efflux was observed at all levels of external potassium studied. 5. Total and ouabain-insensitive potassium influx increased with external potassium, but did not appear to saturate. Ouabain-sensitive potassium influx reached a maximum at an external potassium concentration of 2 mmol/l then decreased with increasing external potassium.

The magnesium dependence of sodium-pump-mediated sodium-potassium and sodium-sodium exchange in intact human red cells.

1. The magnesium content of human red blood cells was controlled by varying the magnesium concentration in the medium in the presence of the ionophore A23187. The new magnesium levels attained were very stable, which allowed the magnesium dependence of the sodium pump to be investigated.2. The effects of magnesium were shown to occur at the inner surface of the red cell membrane for the range of magnesium concentrations tested (10(-7) to 6 x 10(-3)m).3. At intracellular ionized magnesium concentrations below 0.8 mm the activation of ouabain-sensitive sodium-potassium exchange by internal ionized magnesium could be resolved into two or three components: (a) a small component, about 5% of the maximum flux, which is apparently independent of the ionized magnesium concentration below 2 mum, (b) a saturating component with a K((1/2)) of between 30 and 45 mum, and possibly (c) a component which increases linearly with ionized magnesium concentration and which only becomes apparent at concentrations above 0.1 mm.4. At intracellular ionized magnesium concentrations below 0.8 mm, activation of ouabain-sensitive sodium-sodium exchange by internal ionized magnesium could be resolved into two components: (a) a small component, about 6% of the maximal flux, which is apparently independent of the ionized magnesium concentration below 2 mum, and (b) a saturating component with a K((1/2)) of about 9 mum. At ionized magnesium concentrations between about 0.2 and 0.8 mm the rate of sodium-sodium exchange remained constant at the maximal level.5. The intracellular concentration of ATP decreased and the ADP concentration increased as the magnesium content of the cells was reduced from the normal level. A small increase in ATP and a small decrease in ADP was seen when the magnesium content was increased above the normal level. The variation in the ATP: ADP ratio from 2.5 at very low magnesium levels to about 6 at normal magnesium levels can account, at least in part, for the different K((1/2)) values of sodium-potassium and sodium-sodium exchange.6. When the concentration of ionized magnesium was increased above about 0.8 mm both sodium-potassium and sodium-sodium exchange were inhibited. Sodium-sodium exchange was more strongly inhibited than sodium-potassium exchange.7. The possible sites of action of magnesium in the sodium pump cycle are discussed.

The effects of vanadate on the fluxes of sodium and potassium ions through the sodium pump.

1. The effects of sodium orthovanadate on the fluxes of sodium and potassium (or rubidium) ions through the sodium pump have been investigated in intact human red cells and in resealed ghosts prepared from them. Sodium-potassium exchange, potassium-potassium exchange, pump reversal, sodium-sodium exchange and uncoupled sodium efflux have each been studied.2. When intact human red cells were incubated in high-sodium media containing vanadate in low concentrations, inhibition of potassium or rubidium influx was marked only if the potassium or rubidium concentration in the medium was sufficiently high to cause nearly maximal influx in the absence of vanadate. The absence of inhibition at lower potassium or rubidium concentrations cannot be explained by supposing that the onset of inhibition by vanadate is slower in these conditions.3. Lowering the extracellular sodium concentration, or raising the vanadate concentration, decreased the minimum concentration of extracellular potassium or rubidium at which inhibition by vanadate was detected.4. Experiments on potassium influx into intact red cells treated with the ionophore A23187 showed that magnesium ions act at intracellular sites to promote inhibition by vanadate.5. Measurements of potassium efflux from intact red cells incubated in high-sodium media, with or without potassium, showed that potassium-potassium exchange was inhibited by vanadate at low concentrations whereas reversal of the pump was not.6. Measurements of sodium efflux from intact red cells or resealed ghosts incubated in high-sodium media, with or without potassium, showed that vanadate had little or no effect on sodium-sodium exchange at concentrations at which sodium-potassium exchange was markedly reduced. Much higher concentrations of vanadate did cause partial inhibition of sodium-sodium exchange.7. Experiments to determine whether vanadate in low concentrations inhibited uncoupled sodium efflux were inconclusive, but suggested that the flux was inhibited. Measurements of the ATP hydrolysis that is thought to be associated with the uncoupled sodium efflux showed that this hydrolysis was strongly inhibited.8. The different effects of vanadate on the different fluxes are discussed, and related to the way in which vanadate is thought to act on the sodium pump.

Sodium-sodium exchange through the sodium pump: the roles of ATP and ADP.

1. We have developed a procedure for preparing resealed red cell ghosts that contain ADP but very little ATP. 2. The procedure involves (i) lysis of the cells in a very large volume of lysing solution, (ii) resuspension of the ghosts in a small volume, (iii) the incorporation into the ghosts, before they are resealed, of the adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (AP5A) and of hexokinase, and (iv) the removal of traces of ATP, formed by residual adenylate kinase activity, by the addition of glucose. 3. Measurements of sodium efflux from ghosts prepared in this way show that sodium-sodium exchange through the sodium pump does not occur in the absence of ATP even if ADP is present. 4. The beta:gamma imido analogue of ATP (AMP.PNP), which is incapable of phosphorylating sodium, potassium-ATPase, cannot replace ATP in supporting sodium-sodium exchange. 5. These findings support the hypothesis that the outward movement of sodium ions through the sodium pump is associated with the transfer of a phosphoryl group from ATP to the enzyme, and that the inward movement of sodium ions through the pump is associated with the return of a phosphoryl group from the phosphoenzyme to ADP.

Uptake of 22Na+ by cultured dog kidney cells (MDCK).

22Na+ uptake measurements were conducted on the dog kidney cell line, MDCK, to determine the mechanism of ouabain-insensitive sodium transport. The radioisotope was found to be taken up into monolayer cultures via an ATP-independent, saturable process (Km = 40 mM). The presence of sodium on the opposite side of the membrane gave rise to a transstimulation of the 22Na+ flux. Studies utilizing potassium and valinomycin suggested that the transport system was insensitive to changes in the membrane potential. Replacement of chloride in the assay buffer with other anions did not decrease the rate of 22Na+ uptake at 14 mMNa+, but bicarbonate and acetate were stimulatory. Potassium and rubidium increased the rate of 22Na+ influx (Ka = 13mM with 14 mM NaCL in the medium). Lithium (Ki = 7.5mM) and amiloride (Ki = 1.7 x 10(-5) M) were competitive and partially (or mixed) competitive inhibitors, respectively. The data are consistent with a mechanism of sodium uptake that includes a carrier(s) capable of catalyzing net sodium uptake and sodium-sodium exchange.

Effects of abnormal cation transport on deformability of desiccytes.

We have studied the deformability of subpopulations of red cells from a patient with "desiccytosis," a disorder characterized by increased membrane permeability to potassium and associated with a probable increase in sodium-sodium exchange. Cells become increasingly dehydrated after maturation because of continued potassium loss without compensatory sodium gain, and they exhibit a progressive increase in mean cell hemoglobin concentration (MCHC). This increase in MCHC causes the cells to become underformable at shear stress values which result in extensive deformation of normal cells. Reduction of MCHC to approximately normal levels by suspending the cells in hypotonic medium restores normal deformability to all but 0.1--0.2% of the cells. These results suggest that the major factor leading to premature destruction in this disorder is whole cell rigidity conferred by increased intracellular hemoglobin concentrations, rather than any associated membrane rigidity.

The effect of external potassium concentration on leucocyte cation transport in vitro.

1. Sodium and potassium transport rates in human leucocytes were measured in vitro at different external potassium concentrations. 2. At nominally zero external potassium concentrations, the ouabain-sensitive sodium efflux was reduced to less than 20% of its maximum value. There was evidence that under these conditions a ouabain-sensitive sodium-sodium exchange occurs. 3. Both total and ouabain-insensitive potassium influx increased with increasing external potassium concentration. The ouabain-sensitive potassium influx showed saturation. 4. Ouabain-insensitive potassium efflux was also stimulated by increasing the external potassium concentration, suggesting significant potassium-potassium exchange at physiological external potassium concentrations.

Effect of β-Adrenergic Catecholamines on Sodium Transport in Turkey Erythrocytes

Abstract Sodium fluxes across the plasma membrane of turkey erythrocytes are 2 to 3 times greater than corresponding fluxes in human erythrocytes. Extracellular potassium stimulates fractional sodium outflux and inhibits sodium influx in turkey erythrocytes. In contrast to human erythrocytes, turkey erythrocytes showed no evidence for sodium-sodium exchange, and at all sodium concentrations studied addition of extracellular potassium inhibited sodium influx. Isoproterenol stimulated accumulation of cyclic adenosine 3':5'-monophosphate (cyclic 3':5'-AMP) and produced a 2- to 4-fold increase in sodium fluxes in turkey erythrocytes. The potency of catecholamine analogs for stimulation of sodium transport correlated directly with their potency for activation of adenylate cyclase in the turkey erythrocyte membrane. Stimulation of sodium transport by isoproterenol is reproduced by adding cyclic 3':5'-AMP to the medium and is blocked by propranolol. The increase in cyclic 3':5'-AMP in response to isoproterenol progresses unimpaired for at least 2 hours. The increase in sodium transport, however, begins to diminish within 30 min after addition of isoproterenol, and after 3 hours of incubation sodium fluxes are only slightly greater than for the control. This phenomenon presumably reflects accumulation of an inhibitor or depletion of a necessary factor for the transport system activated by intracellular cyclic 3':5'-AMP. The rapid effect described here of catecholamines on sodium transport as well as certain results with other systems controlled by hormones through cyclic 3':5'-AMP suggest that endocrine regulation of sodium transport is of general biological significance.

Some ‘partial reactions’ of the sodium pump

The lecture describes recent work on a number of different partial reactions involving chemical transformation, and discusses the relations between these reactions and the movements of sodium and potassium ions. An account is given of recent experiments from the physiological laboratories at Yale and at Cambridge suggesting that ADP as well as ATP is required for sodium-sodium exchange, and that nucleotide is also required for potassium-potassium exchange.

Sodium movements from frog rod outer segments

Investigations into the sodium movements between isolated frog rod outer segments and their environment have produced evidence indicating both the presence of a sodium-sodium exchange, and a ouabain sensitive efflux of sodium from the rod outer segments which is inhibited by light. It is suggested that the sodium in the outer segments may be contained in at least two compartments.

The components of the sodium efflux in frog muscle.

1. In normal Ringer solution containing 2.5 mM-K only 37% of the efflux of labelled sodium from a freshly dissected frog muscle is blocked by treatment with ouabain; in sodium-loaded muscles the ouabain-sensitive fraction of the efflux increases to 75%.2. Under all conditions, the ouabain-insensitive component of the sodium efflux is markedly reduced if the sodium in the external medium is replaced by lithium; at least in sodium-loaded muscles, the ouabain-sensitive component is increased in lithium Ringer.3. Only the ouabain-sensitive component of the efflux is affected by the external potassium concentration.4. In the presence of 2.5 mM-K the sodium influx in a freshly dissected muscle is not significantly altered by ouabain, but in a K-free medium the influx and the efflux are both reduced by nearly 20%.5. The sodium efflux can therefore be regarded as consisting of (1) a sodium-potassium coupled component that is blocked by ouabain and involves a sodium-sodium exchange in the absence of external potassium, and (2) a potassium-insensitive component that is unaffected by ouabain and tends to reach saturation at relatively lower internal sodium concentrations.6. The evidence is considered for attributing component (1) to an efflux of sodium from the sarcoplasm proper, and component (2) to an efflux from the sarcoplasmic reticulum. Although such an interpretation is consistent with many of the observations, a definite identification of the possible sodium compartments in frog muscle cannot yet be made.