Sodium and potassium content and membrane transport properties in red blood cells from newborn puppies

The intracellular sodium and potassium concentrations and membrane transport properties for these ions were investigated in red blood cells from newborn puppies and adult dogs. At birth the intracellular concentrations of sodium and potassium are much higher than those found in adult dog red cells. During the first few weeks of life the intracellular concentrations of these ions gradually decrease until the adult level is reached. Changes in the membrane transport properties develop concurrently. The rate of active potassium influx, as measured by ouabain-sensitivity, and the pump to leak ratio are greater in red cells from newborn puppies than in those from adult animals. No ouabain-sensitive sodium efflux could be demonstrated in red cells from older puppies or adult dogs. When either puppy or adult dog red cells are depleted of ATP (by incubation at 37°C with no substrate), potassium permeability increases, and the permeability of the membrane to sodium decreases. The addition of adenosine reverses the effect of depletion.     

Relation between sodium-coupled amino acid and sugar transport and sodium/potassium pump activity in isolated intestinal epithelial cells

Cells isolated from the epithelium of the small intestine are used to study the relationship between amino acid or sugar-coupled sodium transport and potassium uptake through the sodium/potassium pump. Potassium influx is a saturable function of the external potassium concentration. Uptake in the presence of ouabain, a specific pump inhibitor, is greatly reduced. This remaining influx is linearly related to the concentration up to 6 mM potassium. Sugars and amino acids are actively accumulated by the intestinal cells. Their transport is accompanied by an initial extra influx of sodium. Although cells seem to regulate their internal sodium concentrations, this is not accompanied with a concomitant increase in potassium uptake through the pump. Thus L-alanine, 3-0-methyl-D-glucoside, and alpha-methyl-D-glucoside all fail to increase the rate of ouabain-sensitive potassium uptake. A very high coupling ratio of sodium efflux to potassium influx through the pump would be a likely explanation of the present results though they cannot be regarded as conclusive.     

Ouabain-dependent potassium-potassium exchange in the toad bladder

Summary Recent results from this laboratory have indicated the existence of two potassium compartments in the isolated toad bladder. Only one of these, containing less than 10% of total intracellular potassium, appears to be related to the sodium transport system, since potassium influx at the serosal border of this compartment is coupled to the sodium efflux which occurs there. Ouabain, which specifically inhibits serosal sodium exit, has no effect on potassium fluxes and compartment sizes in bladders mounted in normal (2.5mm K) Ringer's solution. However, in the presence of this inhibitor, removal of serosal potassium results in a significant decrease in the rate coefficient for potassium efflux into the serosal medium, while an increase in serosal potassium results in a significant rise in this parameter, which appears to saturate at approximately 5mm K. This sensitivity to serosal potassium is seen neither in the absence of ouabain nor when the sodium pump is inactivated by removal of sodium from the mucosal medium. Furosemide, which also inhibits the sodium transport system, both inhibits potassium transport parameters in normal Ringer's and abolishes the potassium-sensitive potassium efflux seen in the presence of ouabain. Thus, the Na–K pump appears to operate as a K–K exchanger when the sodium system is inhibited by ouabain; this K–K exchange mechanism is inhibited by furosemide. One explanation for these results is that ouabain effects an alteration in the affinities of the transport system for sodium and potassium.     

Ion transport across the epithelium of the rabbit caecum.

The isolated rabbit caecum was studied in vitro. Under our experimental conditions, the rabbit caecum secreted potassium and chloride and absorbed sodium. To characterize the transport properties of the apical and the basolateral barriers, transepithelial electrical and flux (22Na, 36Cl and 86Rb) measurements and their sensitivity to transport inhibitors (furosemide, DIDS, ouabain and barium) are presented together with intracellular measurements with double-barrelled microelectrodes of intracellular electrical potentials and ionic activities. The fluxes of sodium and chloride were insensitive to DIDS and furosemide. The secretion of potassium and the absorption of sodium were both inhibited by ouabain, indicating that they are coupled through the sodium pump. Ouabain induced a slow fall in the chloride net fluxes, suggesting that these fluxes are also driven by the sodium pump, albeit indirectly. The basolateral to apical fluxes of potassium are insensitive to barium added to the apical side, but are accelerated by the replacement of chloride by gluconate on the apical side, suggesting the presence of a K+/Cl- symport in the apical barrier.     

Potential-dependent membrane sodium pump current in snail giant neurons

The effect of the membrane potential on the pump current evoked by iontophoretic injection of sodium into the neuron and the effect of the intracellular sodium ion concentration on the potential dependence of the pump current were investigated by the voltage clamp method in isolated and semi-isolated neurons ofHelix pomatia andHelix italiana. The pump current was shown to change its direction in the presence of marked hyperpolarization of the membrane (by more than −80 to −120 mV). An increase in the intracellular sodium ion concentration following injection of excess ions into the neuron increases the potential dependence of the pump current. A possible connection between passive potassium permeability and the activity of the enzymic transport mechanism for the elimination of sodium from the cell is postulated.     

SODIUM AND CHLORIDE FLUXES IN SYNAPTOSOMES IN VITRO

Synaptosomes incubated in a physiological saline extrude sodium and take up potassium. As would be expected this process is completely blocked by metabolic inhibitors such as cyanide and iodoacetate. However, when metabolic inhibitors are replaced by ouabain (100 μM) there is an increase in the steady state intrasynaptosomal sodium and chloride content even though there is no change in the potassium content. The increases are prevented when synaptosomes are incubated with metabolic inhibitors in addition to ouabain. There is therefore a ouabain-insensitive process that transports sodium, chloride and concomitantly water into synaptosomes. It appears not to function when the supply of metabolic energy is inhibited. The diuretic furosemide (1 mM) in the presence of ouabain inhibits the entry of sodium and chloride without affecting the intrasynaptosomal potassium concentration. Ethacrynic acid (1 mM) has a somewhat similar effect but in addition appears to damage the synaptosome membrane. Kinetic measurements were made of the uptake of sodium, potassium and chloride under conditions of metabolic inhibition and the permeability constants of the membrane determined. Values of 0.068, 0.117 and 0.032 × 10^-6 (cm s^-1) were found for the permeability constants of the membrane to (respectively) sodium, potassium and chloride. Measurements of the rate of uptake in the presence of ouabain revealed an inwardly directed sodium and chloride flux of 5-20 pmol cm^-2 s^-1. Calculation of the fluxes from the steady state ion concentrations also reveals an inwardly directed sodium and chloride flux, though of lesser magnitude. The influx of water is less than would be expected to preserve osmotic equality suggesting that the translocation of sodium and chloride is the primary event. Although its function remains uncertain the flux has a considerable effect on the ion content of synaptosomes.     

Certain aspects of the mechanism of intestinal transport of sodium, potassium, calcium and magnesium in rats

Intestinal absorption of sodium, potassium, calcium and magnesium was studied in rats by the method of intestinal perfusion using ouabain as an inhibitor of sodium-potassium dependent ATPase. At the same time the activity of ATPase and phosphatase were determined in homogenates of intestinal mucosa. A significant effect on the concentration of the determined ions was demonstrated in the transport of these ions, and also an unquestionable participation of intestinal ATPase in the direction and intensity of this transport. It was found that the multidirectional effect of ouabain on the transport of cations depended on their concentration. In the case of concentrations of cations similar to those in the mean food rations it has been demonstrated that ouabain increased the absorption of sodium, potassium and calcium and inhibited the absorption of magnesium. With a threefold higher ions concentration the absorption of potassium and magnesium was inhibited, without changing the transport of sodium and calcium. The possible explanation of the mechanism of these effects is discussed.     

Flexibility of an Active Center in Sodium-Plus-Potassium Adenosine Triphosphatase

In plasma membranes of intact cells an enzymatic pump actively transports sodium ions inward and potassium ions outward. In preparations of broken membranes it appears as an adenosine triphosphatase dependent on magnesium, sodium, and potassium ions together. In this adenosine triphosphatase a phosphorylated intermediate is formed from adenosine triphosphate in the presence of sodium ions and is hydrolyzed with the addition of potassium ions. The normal intermediate was not split by adenosine diphosphate. However, selective poisoning by N-ethylmaleimide or partial inhibition by a low magnesium ion concentration yielded an intermediate split by adenosine diphosphate and insensitive to potassium ions. Pulse experiments on the native enzyme supported further a hypothesis of a sequence of phosphorylated forms, the first being made reversibly from adenosine triphosphate in the presence of sodium ion and the second being made irreversiblyfrom the first and hydrolyzed in the presence of potassium ion. The cardioactive steriod inhibitor, ouabain, appeared to combine preferentially with the second form. Phosphorylation was at the same active site according to electrophoretic patterns of proteolytic phosphorylated fragments of both reactive forms. It is concluded that there is a conformational change in the active center for phosphorylation during the normal reaction sequence. This change may be linked to one required theoretically for active translocation of ions across the cell membrane.     

Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

Characteristics of electrogenic sodium pumping in rat myometrium

Sodium-rich myometrium, obtained from the uteri of pregnant rats, rapidly hyperpolarized when 4.6–120 mM potassium was added to the bathing medium at 37°C. Hyperpolarization was due to sodium pumping since the process was markedly temperature dependent, was abolished by ouabain, and required both intracellular sodium and extracellular potassium. The observed membrane potential exceeded the calculated potassium equilibrium potential during hyperpolarization providing evidence that sodium pumping was electrogenic. Hyperpolarization was reduced in the presence of chloride. The rate of sodium pumping may influence potassium permeability since potassium apparently did not short-circuit the pump during hyperpolarization.     

The mechanism of energy-dependent ion transport in mitochondria

Summary The transport of potassium, sodium and various anions in rat-liver mitochondria was studied mainly by analysis of ion content and water compartmentation of the mitochondrial pellet. A comparison of spontaneous transport with valinomycin- or gramicidin-stimulated transport is made. The rate or extent of uptake, the internal concentrations and the concentration ratio (C_in/C_out) are calculated and compared to test existing models for ion transport in mitochondria.Several models of ion transport in mitochondria are based on a cation-pump which is directed inward. This hypothesis is rejected because of the following findings: (1) Valinomycin stimulates the rate of potassium uptake but does not increase the potassium concentration ratio that can be actively maintained in a steady state (in which there is no potassium flow). (2) Valinomycin greatly stimulates the efflux of⁴²K from mitochondria during the process of potassium accumulation. When potassium accumulation is stimulated the flux ratio, i.e. influx/efflux, decreases; in the presence of valinomycin this ratio approaches 1. (3) In the presence of gramicidin, the concentration ratios of potassium and sodium are about the same under a variety of conditions. These findings indicate that potassium and sodium transport are passive processes of relaxation towards electro-chemical equilibrium (of the potassium and sodium). In high external potassium concentrations the extent of potassium uptake is limited by the permeation of anions; of the permeating anions multivalent acids support a higher extent than monovalent acids. It was found that succinate, acetate and oxalate which are transported together with potassium are distributed in accordance with the pH and without any relation to the potassium concentration ratio. These findings are compatible with the hypothesis that an outward-directed proton pump creates an electrical potential gradient, which shifts the equilibrium state for the cations and drives sodium and potassium inward, and also creates proton gradient that is the driving force for anion transport.     

Regulation of Cell Volume by Active Cation Transport in High and Low Potassium Sheep Red Cells

A model cell which controls its cation composition and volume by the action of a K-Na exchange pump and leaks for both ions working in parallel is presented. Equations are formulated which describe the behavior of this model in terms of three membrane parameters. From these equations and the steady state concentrations of Na, K, and Cl, values for these parameters in high potassium (HK) and low potassium (LK) sheep red cells are calculated. Kinetic experiments designed to measure the membrane parameters directly in the two types of sheep red cells are also reported. The values of the parameters obtained in these experiments agreed well with those calculated from the steady state concentrations of ions and the theoretical equations. It is concluded that both HK and LK sheep red cells control their cation composition and volume in a manner consistent with the model cell. Both have a cation pump which exchanges one sodium ion from inside the cell with one potassium ion from outside the cell but the pump is working approximately four times faster in the HK cell. The characteristics of the cation leak in the two cell types are also very different since the HK cells are relatively more leaky to sodium as compared with potassium than is the case in the LK cells. Both cell types show appreciable sodium exchange diffusion but this process is more rapid in the LK than in the HK cells.     

The transport of Na+ and K+ ions through phospholipid bilayers mediated by the antibiotics salinomycin and narasin studied by 23Na- and 39K-NMR spectroscopy

Addition of the ionophoric antibiotics salinomycin or narasin to preparations of large unilamellar vesicles made from egg yolk phosphatidylcholine in sodium or potassium chloride solutions gives rise to dynamic effects in the 23Na- and 39K-NMR spectra. The dynamic spectra arise from the ionophore-mediated transport of the metal ions through the membrane. The kinetics of the transport are followed as a function of the concentrations of ionophore and the metal ion and are compatible in all cases with a model in which one ionophore molecule transports one metal ion. For both ionophores the transport of potassium ions is appreciably faster than that of sodium and in both cases the rate-limiting step for sodium transport is dissociation of the ionophore-metal complex. Assuming dissociation to be rate limiting in all four cases it is shown that the transport rate differences between the pairs of complexes of each metal arise solely from differences in the rates of formation. The stability constants for ionophore-metal complex formation in the membrane/water interface are evaluated.     

Ouabain Binding and Potassium Transport in Young and Old Populations of Human Red Cells

Human red blood cells were separated according to density by centrifugation through mixtures of phthalate esters. The densest 20% of the erythrocyte population (old cells) had reduced volume and water content compared to the lightest 20% of the cells (young cells). Corpuscular hemoglobin content was unchanged. Young cells had 50% more potassium (K⁺) than old cells, but their total intracellular concentration was only slightly higher; old cells had a small increase in sodium (Na⁺) concentration. Active K⁺ transport of young cells was 37% higher than that of old cells. [³H] + Ouabain binding revealed that this difference was the result of more K⁺ pump sites on young cells, which bound 530 ouabain molecules per cell at 100% K⁺ pump inhibition, as compared to 400 for old cells; unseparated cells bound 450-500 molecules. The relative rates of ouabain binding were identical for the two cell types. Old cells exhibited a greater passive permeability to K⁺, haying a rate coefficient for ouabain-insensitive K⁺ influx 1.8 times that of young cells. There is evidence to suggest that in the face of reduced pump activity this augmented K⁺ “leak” might enhance the osmotic stability of the old cells and function to lengthen their life span.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase-specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters.     

Development of hormonal regulation of ion transport in embryonic chick red cells

We have found that cation transport in red cells from chick embryos is stimulated by the hormone epinephrine and that this response develops as the embryonic definitive cells mature. Sodium efflux and potassium influx are significantly stimulated (50%) by epinephrine in red cells from embryos incubated ten days or longer, whereas cation fluxes in erythroid cells from 8- or 9-day embryos are stimulated little or not at all. The effect of epinephrine may be mediated by cyclic AMP as adenylate cyclase activity in membranes isolated from embryonic red cells is only slightly stimulated at nine days, but the response increases as the cells mature to a maximum of about 180%. Also the stimulation of cation transport by epinephrine is blocked by propranolol, but not by phentolamine. Although the younger cells respond poorly to epinephrine, cyclic AMP significantly stimulates transport. The enhancement of cation fluxes by epinephrine or cyclic AMP occurs even in the presence of ouabain. Since both K influx and Na efflux are enhanced by these agents, their action is most likely on some form of the “Na-K” pump which is not ouabain sensitive resulting in a significant increase in the maximum velocity of the pump. We suggest the hypothesis that there are two classes of “Na-K” pump in these embryonic cells. One pump is similar to that found in many erythrocytes including mammalian cells in that it selectively pumps potassium in and sodium out, is ouabain-sensitive, and is primarily involved in maintaining intracellular cation concentrations. The second pump is enhanced by epinephrine via cyclic AMP, is not inhibited by ouabain, and may have lower ion selectivity. This hormone sensitive pump activity is lost as the cells mature, a process which is completed when the animal is fully grown and no longer has significant numbers of embryonic cells in its circulation.     

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 MgCl₂ 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 MgCl₂. When muscles were enriched with sodium ions, potassium efflux into K-free, Mg⁺⁺-substituted Ringer solution fell to around 0.1 pmol/cm²·s and was increased 14-fold by addition of ouabain.     

Further studies on the partial double Donnan. Is isosmotic KCl solution isotonic with cells of respiratory trees of the holothurian Isostichopus badionotus Selenka?

As potassium, chloride and water traverse cell membranes, the cells of stenohaline marine invertebrates should swell if exposed to sea water mixed with an isosmotic KCl solution as they do when exposed to sea water diluted with water. To test this hypothesis respiratory tree fragments of the holothurian Isostichopus badionotus were exposed to five isosmotic media prepared by mixing artificial sodium sea water with isosmotic (611 mmol/l) KCl solution to obtain 100, 83, 71, 60 and 50% sea water, with and without 2 mmol/l ouabain. For comparison, respiratory tree fragments were incubated in sea water diluted to the same concentrations with distilled water, with and without ouabain. Cell water contents and potassium and sodium concentrations were unaffected by KCl-dilution or ouabain in isosmotic KCl-sea water mixtures. In tissues exposed to H(2)O-diluted sea water, cell water increased osmometrically and potassium, sodium and chloride concentrations decreased with dilution; ouabain caused a decrease in potasium and an increase in sodium but no effect on chloride concentrations. The isotonicity of the isosmotic KCl solution cannot be adscribed to impermeability of the cell membrane to KCl as both ions easily traverse the cell membrane. Rather, operationally immobilized extracellular sodium ions, which electrostatically hold back anions and consequently water, together with the lack of a cellward electrochemical gradient for potassium, resulting from membrane depolarization caused by high external potassium concentration, would explain the isotonicity of isosmotic KCl solution. The high external potassium concentration also antagonizes the inhibitory effect of ouabain on the Na(+)/K(+) ATPase responsible for sodium and potassium active transport.     

A study of passive potassium efflux from human red blood cells using ion-specific electrodes

Summary Using ion-specific electrodes, the potassium leakage induced by ouabain in human erythrocytes can be measured continuously and precisely near physiological conditions. Upon small additions of isotonic sucrose solution to a suspension of red cells in physiological saline the passive potassium efflux increases proportionally to the chloride ratio. The same result is obtained upon addition of hypertonic sucrose solution, suggesting that neither osmolarity nor intracellular concentrations have any influence on the passive potassium efflux. The independence of the potassium efflux and osmolarity can be verified by addition of a penetrating substance like glucose to the cell suspension. Adding water or hypertonic sodium chloride solution shows that the potassium efflux increases slightly in more concentrated salt solutions. Inasmuch as it can be interpreted as a pure ionic strength effect, this result supports the hypothesis of independence of potassium efflux and intracellular concentrations. The results of this investigation together with other studies show that the passive permeability of the human red blood cell to potassium depends uniquely on the membrane potential near physiological conditions, while it depends on parameters such as pH or concentrations for large membrane potentials. This suggests that two different mechanisms of transport might be involved: one would control the permeability under normal conditions; the other would represent a leak through the route normally used by anions and become important only under extreme conditions.