Study of maturation of membrane transport function in red blood cells by X-ray microanalysis

Summary Red blood cells of certain species of animals, such as dogs and cats, contain low potassium and high sodium, whereas the erythropoietic stem cells giving rise to these cells are of high potassium type. This paper examines the sequence of membrane transport changes during erythropoiesis by analyzing the K, Na and Fe in single bone marrow cells, reticulocytes and mature red blood cells with X-ray microanalysis. The relationship between K/Na ratios and Fe/(K+Na) ratios were examined by X-ray microanalysis. The K/Na ratios give a measure of the membrane cation transport function. The Fe/(K+Na), which is analogous to hemoglobin concentration, gives an index of maturation stage. The relationships between K/Na and Fe/(K+Na) in the marrow cells of normal adult dog and those of a phenylhydrazine-injected dog with accelerated erythropoiesis show that the modification of cation composition occurs after the initiation of hemoglobin synthesis but before its completion. Similar relationships in the reticulocytes obtained from phenylhydrazine-injected dogs as well as from newborn dogs show a consistent decrease in K/Na with increased Hb, indicating a drastic change in cation composition during the maturation of the reticulocytes. Therefore the modification in membrane transport function must have occurred before or during the formation of reticulocytes.     

Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells.

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.     

The correlation of composition and morphology during the high to low potassium transition in single erythropoietic cells

The change from high potassium dog erythroid cells to low potassium red blood cells during erythropoiesis was investigated by X-ray microanalysis of single cells. A correlation of morphology and composition, using freeze-dried cryosectioned preparations, showed that during normal erythropoiesis in dog bone marrow the switch from high potassium to low potassium occurs during the change from early to late nucleated erythroid cells, and in synchrony with the beginning of iron accumulation. In contrast, during rapid erythropoiesis in dogs with phenylhydrazine-induced anemia, the most prominent change in cation composition as well as the accumulation of iron occurs during the reticulocyte stage in the peripheral blood. The determination of the absolute amounts of sodium and potassium per cell in stress reticulocytes of peripheral blood indicated that the changeover from high potassium to low potassium actually occurs by the loss of cellular potassium during volume reduction, with little change in the amount of cellular sodium. This suggests that maturation may involve a selective change in potassium permeability. Lastly, it was observed that not all cells followed the predominant pathway with respect to change in morphology, membrane permeability and hemoglobin synthesis. One particular subpopulation appeared to follow a sequence which expressed the complete HK to LK transition before the accumulation of any iron; this implies the possibility of completing protein synthesis in a low potassium intracellular milieu.     

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells.

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K+ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K+-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K+ genotype of the sheep and was not associated with either "high K+"- or "low K+"-type Na+ pump. No Ca2+-dependent change in K+ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K+ channel in the mammalian red cell and the relationship between the K+ channel and the Na+ pump are discussed.     

Human and dog erythrocytes: relationship between cellular ATP levels, ATP consumption and potassium concentrations.

The intracellular K+/Na+ ratio of various mammalian cell types are known to differ remarkably. Particularly noteworthy is the fact that erythrocytes of different mammalian species contain entirely different potassium and sodium concentrations. The human erythrocyte is an example of the supposedly "normal" high potassium cell, while the dog erythrocyte contains ten times more sodium than potassium ions (Table I). Furthermore, this difference is sustained despite the plasma sodium and potassium concentrations being almost identical in both species (high Na+ and low K+). In spite of these inorganic ion differences, both human and dog erythrocytes contain 33% dry material (mostly Hb) and 67% water. Conventional cell theory would couple cellular volume regulation with Na+ and K+ dependent ATPase activity which is believed to control intracellular Na+/K+ concentrations. Since the high Na+ and low K+ contents of dog erythrocytes are believed to be due to the lack of the postulated Na/K-ATPase enzyme, they must presumably have an alternative mechanism of volume regulation, otherwise current ideas of membrane ATPase activity coupled volume regulation need serious reconsideration. The object of our investigation was to explore the relationship between ATPase activity, ATP levels and the Na+/K+ concentrations in human and dog erythrocytes. Our results indicate that the intracellular ATP level in erythrocytes correspond with their K+, Na+ content. They are discussed in relation to conventional membrane transport theory and also to Ling's "association-induction hypothesis", the latter proving to be a more useful basis on which to interpret results.     

Ion transport in the heart atrial tissue of young and adult guinea-pigs and albino rats.

Rubidium shifts between the extracellular fluid and cells were studied in the isolated atria of guinea-pig and albino rat hearts. In the young of both species, rubidium transport from the incubation medium to the cells was much slower than in preparations from adult animals. That implies that the efficiency of membrane mechanisms for the transport of Na+ and K+ ions in the atrial tissue increases during postnatal life. This conclusion is further confirmed by the finding that the intracellular potassium concentration in the atrial tissue of the young of both species is lower, and the intracellular sodium concentration higher, than in adult animals. Conversely, the serum potassium concentration in the young is higher, and the serum sodium concentration lower, than in adult individuals.     

A calcium-activated potassium channel present in foetal red cells of the sheep but absent from reticulocytes and mature red cells

Red cells of adult sheep, like those of other ruminants, lack the calcium-activated potassium channel which is present in the membrane of human red cells. Since the activities of other transport systems in the sheep red cell are known to decrease during maturation of the cell or during development of the animal it was investigated whether the K⁺ channel is present in red cells from younger animals or in reticulocytes. Using the divalent cation ionophore A23187 to increase the intracellular Ca of intact cells, it was found that the K⁺-selective channel is present in foetal red cells from the foetus or newborn animal but not in reticulocytes. The presence of the channel showed no dependence on the K⁺ genotype of the sheep and was not associated with either “high K⁺”-or “low K⁺”-type Na⁺ pump. No Ca²⁺-dependent change in K⁺ permeability was found in red cells from either newborn or adult donkeys suggesting that its presence in the red cells of the foetus may not be general. The role of the K⁺ channel in the mammalian red cell and the relationship between the K⁺ channel and the Na⁺ pump are discussed.     

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.     

Osmotic activity and ionic permeability of membrane vesicles from Streptococcus faecalis and Micrococcus lysodeikticus cells]

Membrane fractions containing osmotically active vesicles with sufficiently low membrane permeability for K+, Na+ and Cl- ions typical for the intact cell membrane were isolated from the cells of the glycolyzing bacterium Streptococcus faecalis. In their osmotic properties and ionic permeability the membrane fractions of S. faecalis were found similar to those of the respiring bacterium Micrococcus lysodeikticus, which are capable of the energy-dependent potassium transport. It may be thus assumed that the S. faecalis fractions obtained may be used to study ionic transport. The removal of proton-dependent ATPase of the S. faecalis membrane preparations did not affect the permeability of membranes for K+ ions which is indicative of different mechanisms of proton and potassium translocation.     

Changes in the cation composition and active K+ transport in the red cells of fetal sheep prepartum

The red blood cells of lambs, genotypically low potassium type, undergo a transition from high potassium to low potassium cell type from parturition onwards. This involves gradual changes in cell ion content, sodium pump activity, and ouabain binding. In the present study we investigated the properties of fetal red blood cells from 30 days prepartum using the chronically cannulated pregnant ewe preparation. We demonstrate that intracellular sodium increases and potassium decreases from -30 days onwards. Sodium pump activity monitored either by tracer potassium influx or ouabain binding is markedly higher in the early fetal samples examined and declines fourfold during the final month in utero. Unlike the maternal low potassium cells the early fetal red cells are refractory in terms of sodium pump stimulation by anti-L, the antibody in fact consistently inhibiting the pump. Finally, we have investigated the volume sensitivity and development of the ouabain-insensitive potassium fluxes in these cells and found that both fetal and maternal cells show a marked chloride-dependent, volume-sensitive passive potassium flux. We conclude that the decrease in active sodium transport between fetal red cells and adult low potassium cells is achieved partly by a reduction in the density of sodium pumps per cell, and then later by the introduction into the circulation of cells with Lp-antigen-modified sodium pumps.     

An Adenosinetriphosphate-Activated Hemolytic System : III. Effect of rabbit hemolytic factor on the membrane permeability of human red cells

Lysis of human red cells in vitro by an enzyme obtained from rabbit red cell hemolysates and the inhibition of this lytic activity by human stroma have been shown to require Mg⁺⁺ and ATP, and ATP utilization has been demonstrated in both reactions. We find that sodium or potassium ions are also required for the lytic phenomenon and that they enhance the inhibition. The rate of hemolysis is not affected by the internal concentrations of these ions but depends only on the external concentration. The rate of influx and efflux of Na²²Cl and K⁴²Cl in surviving red cells is greatly enhanced both during and after treatment with rabbit hemolytic factor whereas the entry of C²⁴-sucrose, a small foreign molecule, is mediated only in the presence of hemolytic factor. Glycolysis neither protects against lysis nor enhances the activity of this system, and cardiac glycosides which are known inhibitors of active transport of ions also have no effect. It appears that lysis in this system is not brought about by increased active transport of ions into the cell but that the rabbit factor degrades or combines with some membrane component, altering permeability and resulting in increased diffusion, first of sodium and potassium ions and other small molecules, and finally of large molecules (hemoglobin) out of the cell.     

Potassium uniport and ATP synthesis in Halobacterium halobium.

Light-driven potassium ion uptake in Halobacterium halobium is mediated by bacteriorhodopsin. This uptake is charge-balanced by sodium ions and not by proton release. Light-induced shifts in concentrations of divalent cations were found to be negligible. The transient changes in extracellular pH (alkaline overshoot) can be understood by the concomitant processes of ATP synthesis, proton/sodium exchange and potassium uptake. The driving force of potassium ion uptake is the membrane potential, no ATP-dependent potassium transport process is found. Fluorescence measurements indicate a high permeability of the membrane to potassium ions compared to sodium ions. Therefore the potassium ion diffusion potential contributes to the membrane potential (about 30 mV/decade) and thereby influences the ATP level. Sudden enhancement of the diffusion potential by the potassium ionophore monactin leads to the expected transient increase in cellular ATP level. Due to the large size (up to 100-fold) of the potassium ion gradient and its high capacity (intracellular concentration up to 3 M) the potassium ion gradient can well serve the cell as a long term storage form of energy.     

Potassium permeability of Rickettsia prowazekii.

The potassium permeability of Rickettsia prowazekii was characterized by chemical measurement of the intracellular sodium and potassium pools and isotopic flux measurements with 86Rb+ as a tracer. R. prowazekii, in contrast to Escherichia coli, did not maintain a high potassium-to-sodium ratio in their cytoplasm except when the potassium-to-sodium ratio in the extracellular medium was high or when the extracellular concentrations of both cations were low (ca. 1 mM). Both influx and efflux assays with 86Rb+ demonstrated that the rickettsial membrane had limited permeability to potassium and that incorporation of valinomycin into these cells increased these fluxes at least 10-fold. The transport of potassium showed specificity and dependence on rickettsial metabolism. The increased flux of potassium which results from the incorporation of valinomycin into the rickettsial membrane was detrimental to both lysine transport and lysis of erythrocytes by the rickettsiae.     

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.     

Cation and ATP content of ferret red cells

Ferret red cells were shown to have the following properties: 1. They have a high sodium (96 mmol/l cell) and low potassium (3.9 mmol/l cell) content. 2. The majority do not appear to have an active sodium pump in their membranes. 3. Their membranes are highly permeable to rubidium indicating that they are probably also highly permeable to potassium. 4. Their magnesium (3.01 mmol/l cell) and calcium (0.01 mmol/l cell) contents are similar to those of red cells from other species. 5. Their ATP content (0.6 mmol/l cell) is similar to that of cat and dog red cells and is sufficiently high to activate known ion transport systems.     

Cationic amino acid transport by two renal epithelial cell lines: LLC-PK1 and MDCK cells

LLC-PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC-PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans-stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.     

Resolution of Pump and Leak Components of Sodium and Potassium Ion Transport in Human Erythrocytes

Further support for the pump-leak concept was obtained. Net transport was resolved into pump and leak components with the cardiac glycoside, ouabain. The specificity of ouabain as a pump inhibitor was demonstrated by its ineffectiveness when the pump was already inhibited by lack of one of the three pump substrates, sodium ion, potassium ion, or adenosine triphosphate. In the presence of ouabain the rates of passive transport of sodium and potassium ions changed almost in proportion to changes in their extracellular concentrations when one ion was exchanged for the other. In the presence of ouabain and at the extracellular concentrations which produced zero net transport, the ratio of potassium ions to sodium ions was 1.2-fold higher inside the cells than outside. This finding was attributed to a residual pump activity of less than 2% of capacity. The permeability to potassium ions was 10% greater than the permeability to sodium ions. A test was made of the independence of pump and leak. Conditions were chosen to change the rate through each pathway separately or in combination. When both pathways were active, net transport was the sum of the rates observed when each acted separately. A ratio of three sodium ions pumped outward per two potassium ions pumped inward was confirmed.     

The M-L blood group system and its influence on red cell potassium levels in sheep

Five phenotypes within the M-L blood group system of sheep were identified with three reagents: anti-L(m), anti-M and anti-M'. Factor M' only occurred in combination with M. The serological properties were studied and the potassium concentrations in the red cells of the various phenotypes were compared. Like M, the L antigen was only weakly developed on the red cells of newborn lambs, and it was only when adult low potassium levels were established that full L activity was reached. These results support the hypothesis that antigen L is acting as an inhibitor of active potassium transport.     

NMR studies of intracellular free calcium, free magnesium and sodium in the guinea pig reticulocyte and mature red cell

During the maturation process reticulocytes lose their intracellular organelles and undergo changes in membrane lipid composition and ion transport properties. While several reports indicate differences in the levels of magnesium, sodium and calcium in reticulocytes and erythrocytes, controversy remains concerning the actual magnitude and direction of ionic alterations during reticulocyte maturation. One problem with all of these studies is that the techniques used are invasive and are limited to measuring only the total cell ion content. We have used 31P, 23Na and 19F nuclear magnetic resonance (NMR) spectroscopy to compare the intracellular free ion and phosphometabolite levels in guinea pig reticulocytes and mature red blood cells. In contrast to a sharply decreased concentration of ATP in erythrocytes in comparison to reticulocytes, the intracellular free magnesium, measured using 31P-NMR, was increased by about 65% upon maturation (150 mumol/l cell water in reticulocytes in comparison to 250 mumol/l cell water in erythrocytes). Sizeable but opposite changes in intracellular sodium (5.5 mumol/ml cells in reticulocytes vs. 8.5 mumol/ml cells in erythrocytes) and intracellular free calcium (99 nM vs. 31 nM in reticulocytes and mature red cells, respectively) were also observed, suggesting that alterations in the kinetics of membrane ion transport systems, accompanying changes in phospholipid and cholesterol content, occur during the process of red cell maturation. However, in contrast to dog red blood cells, there was no evidence for the presence of a Na+/Ca2+ exchanger in guinea pig reticulocytes or erythrocytes.     

Changes in the Membrane Permeability of Frog's Sartorius Muscle Fibers in Ca-Free EDTA Solution

The changes in the membrane permeability to sodium, potassium, and chloride ions as well as the changes in the intracellular concentration of these ions were studied on frog sartorius muscles in Ca-free EDTA solution. It was found that the rate constants for potassium and chloride efflux became almost constant within 10 minutes in the absence of external calcium ions, that for potassium increasing to 1.5 to 2 times normal and that for chloride decreasing about one-half. The sodium influx in Ca-free EDTA solution, between 30 and 40 minutes, was about 4 times that in Ringer's solution. The intracellular sodium and potassium contents did not change appreciably but the intracellular chloride content had increased to about 4 times normal after 40 minutes. By applying the constant field theory to these results, it was concluded that (a) P_Cl did not change appreciably whereas P_K decreased to a level that, in the interval between 10 and 40 minutes, was about one-half normal, (b) P_Na increased until between 30 and 40 minutes it was about 8 times normal. The low value of the membrane potential between 30 and 40 minutes was explained in terms of the changes in the membrane permeability and the intracellular ion concentrations. The mechanism for membrane depolarization in this solution was briefly discussed.