Delivery of ion pumps from exogenous membrane-rich sources into mammalian red blood cells.

Using polyethylene glycol-mediated fusion of ATP-ase-enriched (native) microsomes with red blood cells, we have delivered sarcoplasmic reticulum (SR) Ca-ATPase and kidney Na,K-ATPase into the mammalian erythrocyte membrane. Experiments involving delivery of the SR Ca-ATPase into human red cells were first carried out to assess the feasibility of the fusion protocol. Whereas there was little detectable 45Ca2+ uptake into control cells in either the absence or presence of extracellular ATP, a marked time-dependent uptake of 45Ca2+ was observed in the presence of ATP in cells fused with SR Ca-ATPase. Comparison of the kinetics of uptake into microsome-fused cells versus native SR vesicles supports the conclusion of true delivery of pumps into the red cell membrane. Thus, the time to reach steady state was more than two orders of magnitude longer in the (large) cells versus the native SR vesicles. Na,K-ATPase from dog and rat kidney microsomes were fused with red cells of humans, sheep, and dogs. Using dog kidney microsomes fused with dog red cells which are practically devoid of Na,K-ATPase, functional incorporation of sodium pumps was evidenced in ouabain-sensitive Rb+ uptake and Na+ efflux energized by intracellular ATP, as well as in ATP-stimulated Na+ influx and Rb+ efflux from inside-out membrane vesicles prepared from the fusion-treated cells. From analysis of the biphasic kinetics of ouabain-sensitive Na+ efflux under conditions of limited intracellular Na+ concentration, it is concluded that the kidney pumps are incorporated into a relatively small fraction (approximately 15%) of the red cells. This system provides a uniquely useful system for studying the behavior of native sodium pumps in a compartment (red cell) of small surface/volume ratio. The newly incorporated native kidney pumps, while of the same isoform as the endogenous red cell pump, behave differently from the endogenous red cell sodium pump with respect to their very low "uncoupled" Na+/O flux activity.     

Membrane transport of potassium ions in erythrocytes of the American black bear, Ursus americanus

1. Membrane transport of K ions was investigated in red blood cells of bears by methods of measurement of unidirectional isotopic fluxes. 2. Unlike red cells of dogs, red cells of bears exhibited a significant, though small, component of ouabain-sensitive K influx. 3. Ouabain-insensitive K influx, as in other carnivore cells, was activated by swelling and inhibited by shrinkage. Swelling-induced K influx was dependent upon presence of chloride ions but was not inhibited by furosemide or bumetanide. 4. Ouabain-sensitive K influx was largest with ATP and with high concentration of Na in the cell, but it persisted in the absence of cytoplasmic Na or ATP. It was also resistant to the drug, harmaline, at a concentration that in other cells fully inhibits ouabain-sensitive K influx. 5. It was concluded that under such adverse conditions ouabain-sensitive K influx represents another mode of the Na/K pump not fully described elsewhere. 6. Also, as in low K red cells of sheep and goat, apparent absence of Na/K pump activity in carnivore red cells may represent suppression rather than elimination of activity. 7. Ouabain-insensitive K influx showed a seasonal pattern with minima occurring in early winter, earlier than for the minimum observed in Na influx. 8. Ouabain-sensitive K influx tended to be lower in the hibernation season of the bear, but the seasonal pattern was not consistent.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Lithium transport pathways in human red blood cells

In human red cells, Li is extruded against its own concentration gradient if the external medium contains Na as a dominant cation. This uphill net Li extrusion occurs in the presence of external Na but not K, Rb, Cs, choline, Mg, or Ca, is ouabain-insensitive, inhibited by phloretin, and does not require the presence of cellular ATP. Li influx into human red cells has a ouabain-sensitive and a ouabain-insensitive but phloretin-sensitive component. Ouabain-sensitive Li influx is competitively inhibited by external K and Na and probably involves the site on which the Na-K pump normally transports K into red cells. Ouabain does not inhibit Li efflux from red cells containing Li concentrations below 10 mM in the presence of high internal Na or K, whereas a ouabain-sensitive Li efflux can be measured in cells loaded to contain 140 mM Li in the presence of little or no internal Na or K. Ouabain-insensitive Li efflux is stimulated by external Na and not by K, Rb, Cs, choline, Mg, or Ca ions. Na-dependent Li efflux does not require the presence of cellular ATP and is inhibited by phloretin, furosemide, quinine, and quinidine. Experiments carried out in cells loaded in the presence of nystatin to contain either only K or only Na show that the ouabain-insensitive, phloretin-inhibited Li movements into or out of human red cells are stimulated by Na on the trans side and inhibited by Na on the cis side of the red cell membrane. The characteristics of the Na-dependent unidirectional Li fluxes and uphill Li extrusion are similar, suggesting that they are mediated by the same Na-Li countertransport system.     

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.     

Kinetics of the sodium pump in red cells of different temperature sensitivity

Ouabain-sensitive K influx into ground squirrel and guinea pig red cells was measured at 5 and 37 degrees C as a function of external K and internal Na. In both species the external K affinity increases on cooling, being three- and fivefold higher in guinea pig and ground squirrel, respectively, at 5 than at 37 degrees C. Internal Na affinity also increased on cooling, by about the same extent. The effect of internal Na on ouabain-sensitive K influx in guinea pig cells fits a cubic Michaelis-Menten-type equation, but in ground squirrel cells this was true only at high [Na]i. There was still significant ouabain-sensitive K influx at low [Na]i. Ouabain-binding experiments indicated around 800 sites/cell for guinea pig and Columbian ground squirrel erythrocytes, and 280 sites/cell for thirteen-lined ground squirrel cells. There was no significant difference in ouabain bound per cell at 37 and 5 degrees C. Calculated turnover numbers for Columbian and thirteen-lined ground squirrel and guinea pig red cell sodium pumps at 37 degrees C were about equal, being 77-100 and 100-129 s-1, respectively. At 5 degrees C red cells from ground squirrels performed significantly better, the turnover numbers being 1.0-2.3 s-1 compared with 0.42-0.47 s-1 for erythrocytes of guinea pig. The results do not accord with a hypothesis that cold-sensitive Na pumps are blocked in one predominant form.     

The use of ionophores of rapid loading of human red cells with radioactive cations for cation-pump studies.

Techniques are described for the rapid loading of intact human red cells with radioactive isotopes of alkali cations or Ca2+ by using ionophorous compounds (nigericin, gramicidin D and A 23187). Loading was rapid and efficient if the membrane potential of the cells was rendered more negative inside. After cation loading the ionophores could be bound to albumin and removed by repeated washings. The ATP and 2,3-DPG contents of the cells were practically unaltered by this treatment. Passive membrane permeability to Na+ and Ca2+ returned to normal. Loaded erythrocytes pumped out Na+ in a ouabain-sensitive and Ca2+ in a lanthanum-sensitive way. Ca2+ -loaded red cells were microspherocytes and exhibited a rapid K+ -efflux. Parallel with the extrusion of Ca2+ cells regained their biconcave shape and normal passive permeability to K+.     

Influence of Ca/Na exchange and diamide on membrane partitioning of glyceraldehyde-3-phosphate dehydrogenase in dog red cells

1. Ca/Na exchange in dog red cells is greatly stimulated by pretreatment of the cells with diamide, but only if the diamide preincubation is carried out in a Na-free, Ca-containing medium. 2. Membranes prepared from dog red cells that had been pre-exposed to diamide in solutions containing various combinations of Ca and Na were subjected to polyacrylamide gel electrophoresis and Western blotting. 3. The band representing glyceraldehyde-3-phosphate dehydrogenase was selectively increased under the very same ionic conditions that result in a diamide-induced stimulation of Ca/Na exchange. 4. Glyceraldehyde-3-phosphate dehydrogenase may participate in the modulation by diamide of Ca/Na exchange in dog red cells.     

Heterogeneity among dog red blood cells

A phthalate density-separation technique has been used to study the heterogeneity of dog red blood cells that becomes manifest when they are suspended in KCl media. It is demonstrated that the proportions of cells that separate into light and dense fractions can be varied by altering the tonicity of the KCl medium. This results from the fact that the Na and K permeabilities of each cell are continuous functions of cell volume. It was found that quinidine inhibits selectively the volume dependence of Na permeability. In the presence of this drug, the heterogeneity demonstrated by KCl incubation disappears. The notion that dog red blood cells are heterogeneous in their permeabilities to Na and K is thus upheld, but the heterogeneity is not an abruptly discontinuous one, as has been claimed. A sample of dog blood does not contain two discrete populations of red cells.     

Cellular Inhomogeneity in Dog Red Cells As Revealed by Sodium Flux

Unidirectional ²⁴Na fluxes across the dog red blood cell membrane were measured. The kinetics were incompatible with a single time constant but could be accounted for in terms of a two-series compartment cell model, with approximately 1% of cell Na in the outer compartment. Dog red blood cells are known to be inhomogeneous in their Na and K permeabilities. Theoretical analysis showed that such cellular inhomogeneity in the Na permeability coefficient might in principle account for the flux data. In order to evaluate the inhomogeneity effect, a technique based on the differential response of cells suspended in isosmolar high K buffers was devised to measure the variations in Na permeability in the cell population. A variation in the Na permeability coefficient of approximately 30% was found. This inhomogeneity is insufficient to account for the flux data.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape.

Na+ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na+ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the 31P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg2+/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

Increase of Na+ gradient-dependent L-glutamate and L-aspartate transport in high K+ dog erythrocytes associated with high activity of (Na+, K+)-ATPase

As reported previously, some dogs possess red cells characterized by low Na+, high K+ concentrations, and high activity of (Na+, K+)-ATPase, although normal dog red cells contain low K+, high Na+, and lack (Na+, K+)-ATPase. Furthermore, these red cells show increased activities of L-glutamate and L-aspartate transport, resulting in high accumulations of such amino acids in their cells. The present study demonstrated: (i) Na+ gradient-dependent L-glutamate and L-aspartate transport in the high K+ and low K+ red cells were dominated by a saturable component obeying Michaelis-Menten kinetics. Although no difference of the Km values was observed between the high K+ and low K+ cells, the Vmax values for both amino acids' transport in the high K+ cells were about three times those of low ones. (ii) L- and D-aspartate, but not D-glutamate, competitively inhibited L-glutamate transport in both types of the cells. (iii) Ouabain decreased the uptake of the amino acids in the high K+ dog red cells, whereas it was not effective on those in the low K+ cells. (iv) The ATP-treated high K+ cells [(K+]i not equal to [K+]o, [Na+]i greater than [Na+]o) showed a marked decrease of both amino acids' uptake rate, which was almost the same as that of the low K+ cells. (v) Valinomycin stimulated the amino acids' transport in both of the high K+ and the ATP-treated low K+ cells [( K+]i greater than [K+]o, [Na+]o), suggesting that the transport system of L-glutamate and L-aspartate in both types of the cells might be electrogenic. These results indicate that the increased transport activity in the high K+ dog red cells was a secondary consequence of the Na+ concentration gradient created by (Na+, K+)-ATPase.     

Seasonal changes in cation transport in red blood cells of grey squirrel (Sciurus carolinensis) in relation to thermogenesis and cellular adaptation to cold.

1. Unidirectional influx of 42K was measured in red cells of grey squirrels at seasonal intervals over two years. 2. Na/K pump-related (i.e. ouabain-sensitive) K influx at 37 degrees C was maximal in cells collected in January and was more than three times greater than cells collected in summer. Na/K pump activity, maximized by loading the cells with Na, exhibited a similar difference. 3. At 5 degrees C in fresh cells, ouabain-sensitive K influx, expressed as per cent of that at 37 degrees C, was highest in March. In Na-loaded cells it was lowest in summer. 4. Passive "leak" K influx (i.e., the residual influx remaining in presence of ouabain and bumetanide) was highest in October, and declined progressively to the summer months, when it was only 27% of that in October. 5. Cotransport (i.e., bumetanide-sensitive K influx) exhibited the same seasonal pattern as Na/K pump activity in fresh cells. 6. Net gain of Na in cells stored at 5 degrees C for three days in March was less than half of that in January or summer. 7. High transport activity in January may correlate with a requirement for increased non-shivering thermogenesis. However, red cells of grey squirrels exhibit maximum resistance to low temperature in March and at this time resemble the red cells of hibernating mammals.     

Temperature Adaptation of Active Sodium-Potassium Transport and of Passive Permeability in Erythrocytes of Ground Squirrels

Unidirectional active and passive fluxes of ⁴²K and ²⁴Na were measured in red blood cells of ground squirrels (hibernators) and guinea pigs (nonhibernators). As temperature is lowered, "active" (ouabain-sensitive) K influx and Na efflux were more greatly diminished in guinea pig cells than in those of ground squirrels. The fraction of total K influx which is ouabain sensitive in red blood cells of ground squirrels was virtually constant at all temperatures, whereas it decreased abruptly in guinea pig cells as temperature was lowered. All the passive fluxes (i.e., Na influx, K efflux, and ouabain-insensitive K influx and Na efflux) decreased logarithmically with decrease in temperature in both species, but in ground squirrels the temperature dependence (Q₁₀ 2.5–3.0) was greater than in guinea pig (Q₁₀ 1.6–1.9). Thus, red blood cells of ground squirrel are able to resist loss of K and gain of Na at low temperature both because of relatively greater Na-K transport (than in cells of nonhibernators) and because of reduced passive leakage of ions.     

Red cell sodium in DOCA-salt hypertension: a Brattleboro study.

The alteration of red cell Na+ content (Na+i), its causes and the possible relationship to the development of DOCA-salt hypertension were studied in Brattleboro rats. A pronounced hypertension developed in heterozygous (non-DI) animals that synthesize vasopressin (VP) although no substantial Na+i elevation was observed in their erythrocytes. In contrast, Na+i rose progressively in red cells of homozygous VP-deficient (DI) rats in which only marginal increase of systolic blood pressure was found after six weeks of DOCA-salt regimen. DOCA-salt treatment of non-DI rats did not cause major alterations in ouabain-resistant (OR) net Na+ uptake or ouabain-sensitive (OS) net Na+ extrusion but moderately increased furosemide-sensitive (FS) Rb+ uptake. The same treatment of DI rats doubled Na+i by an increased OR net Na+ uptake (due to a major elevation in both Na(+)-K+ cotransport and Na+ leak). Consequently, OS net Na+ extrusion was augmented in red cells of these animals. This was accompanied by an about threefold elevated FS Rb+ uptake. It can be concluded that a) the alterations of OR and/or OS Na+ or K+ transport observed in erythrocytes of Brattleboro DI rats are not essential for the development of severe DOCA-salt hypertension, b) red cell ion transport abnormalities revealed in DOCA-salt treated DI rats might be rather ascribed to cell potassium depletion, and c) increased inward Na(+)-K+ cotransport and Na+ leak causes red cell Na+i elevation that stimulates Na(+)-K+ pump activity.     

Intracellular Potassium : A Determinant of the Sodium-Potassium Pump Rate

Normal human red cells which have had their intracellular sodium (Na_c) reduced have a diminished Na-K pump rate, but only if intracellular potassium (K_c) is high. If most of the K_c is replaced by tetramethylammonium or choline, both ouabain-sensitive Na efflux and K influx are significantly increased even with Na_c below normal. Cells with reduced Na_c and high K_c have an unchanged Na efflux if external potassium (K_ext) is removed. In contrast, low-Na, low-K cells have a large ouabain-sensitive Na efflux which shows a normal response to removal of K_ext. Neither low-K nor high-K cells have an altered ouabain-sensitive K efflux. Measurement at constant low Na_c and varying K_c shows the pump Na efflux to be an inverse function of K_c. Thus, in low-Na cells, K_c appears to act as an inhibitor of the pump. Inhibition by high K_c can be seen even when Na_c is normal. The effects attributed to K_c are distinguished experimentally from other variables such as cell volume, adenosine triphosphate concentration, effects of the replacement cations, and the method used to alter intracellular cation concentrations. A role is proposed for K_c, in cooperation with Na_c, in regulating the pump rate of normal human red cells.     

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.     

The molecular chaperone alpha-crystallin incorporated into red cell ghosts protects membrane Na/K-ATPase against glycation and oxidative stress.

Alpha-crystallin, a molecular chaperone and lens structural protein protects soluble enzymes against heat-induced aggregation and inactivation by a variety of molecules. In this study we investigated the chaperone function of alpha-crystallin in a more physiological system in which alpha-crystallin was incorporated into red cell 'ghosts'. Its ability to protect the intrinsic membrane protein Na/K-ATPase from external stresses was studied. Red cell ghosts were created by lysing the red cells and removing cytoplasmic contents by size-exclusion chromatography. The resulting ghost cells retain Na/K-ATPase activity. alpha-Crystallin was incorporated in the cells on resealing and the activity of Na/K-ATPase assessed by ouabain-sensitive 86Rb uptake. Incubation with fructose, hydrogen peroxide and methylglyoxal (compounds that have been implicated in diabetes and cataract formation) were used to test inactivation of the Na/K pump. Intracellular alpha-crystallin protected against the decrease in ouabain sensitive 86Rb uptake, and therefore against inactivation induced by all external modifiers, in a dose-dependent manner.     

Affinities or Apparent Affinities of the Transport Adenosine Triphosphatase System

The interactions of potassium ions and ATP on transport ATPase activity are discussed, and the interpretation of these interactions is shown to be often ambiguous. Caldwell''s (1968) Physiological Review model is discussed with particular reference to the observed kinetics of sodium: sodium exchange in red cells. Recent experimental work on the properties of the ouabain-sensitive component of potassium efflux from red cells is described. This component of efflux occurs only if either sodium or potassium are present in the external medium, but the effects of external sodium and potassium are not additive. The relation between ouabain-sensitive potassium efflux and the external concentration of sodium (in a potassium-free medium) or of potassium (in low- and high-sodium media) are described. When starved sodium-poor red cells are poisoned with iodoacetamide, loaded with phosphate, and incubated in high-sodium potassium-free media, the ouabain-sensitive efflux of potassium appears to be accompanied by the reversal of the entire ATPase system. About two to three potassium ions leave by the ouabain-sensitive route for each molecule of ATP synthesized. If potassium is present in the external medium, no ouabain-sensitive synthesis of ATP occurs and the ouabain-sensitive efflux of potassium presumably involves the reversal of only the last part of the ATPase system.     

Evidence for bumetanide-sensitive, Na(+)-dependent, partial Na-K-Cl co-transport in red blood cells of a primitive fish

Tracer uptake studies identified the major routes for K+ transport in hagfish red cells, resolving them into ouabain-sensitive, loop diuretic-sensitive, and residual components. The K1/2 values for ouabain, bumetanide, and furosemide were 10(-5), 6 x 10(-7), and 5 x 10(-6) M, respectively. The properties of the Na-K-Cl co-transporter were investigated further by varying K+, Na+, and Cl- concentrations. The measured K1/2 values were similar to those for human red cells. Finally, the stoichiometry of Na:K:Cl uptake was determined, giving 1:1 for K+:Cl-; in contrast, no significant Na+ flux could be measured, although Na+ content must be present for measurable bumetanide-dependent K+ or Cl- flux to occur. The Na-K-Cl transport therefore shows Na(+)-dependent KCl co-transport or partial flux of the system.