The Effect of Valinomycin on Potassium and Sodium Permeability of HK and LK Sheep Red Cells

A cyclic depsipeptide antibiotic, valinomycin, was found to produce increased selective permeability of the plasma membranes of HK and LK sheep red blood cells to potassium but not to sodium ions. The compound had relatively little effect on the active extrusion of sodium from HK sheep red blood cells or on the Na + K-stimulated ATPase activity of membranes derived from these cells. It is proposed that the selective cation permeability produced by this compound depends primarily on steric factors, particularly the relationship between the diameter of the ring and the effective diameter of the ion. The significance of these results for the problem of the mechanism of ionic selectivity in natural membranes is discussed.     

Changes from High Potassium (HK) to Low Potassium (LK) in Bovine Red Cells

Red cells of newborn calves contain 105–110 mmole K⁺ and 1–5 mmole Na⁺ per liter of cells. As the animals age the K⁺ content decreases to a value of 25–30 mmole/liter of cells after about 60 days. At approximately the same time, the sodium content reaches a value of 60–70 mmole/liter. The time required for half change (t_½) is 35–37 days for both Na⁺ and K⁺. The activity of (Na + K)-adenosine triphosphatase (ATPase) and the influx of K⁴² and Rb⁸⁶ into the red cells are high at birth and are reduced to 5 and 15% of their original values, respectively, in mature animals. t_½ for both is of the order of 30–35 days. The membrane Mg-ATPase activity is also high at birth and is reduced with a t_½ of 28–32 days to a final value of about 20% of its activity at birth. Separation of red cells according to their age showed that, in animals at the age of transition, newly formed red cells contain a higher K/Na ratio and a higher active transport capacity than older red cells of the same animal. It is suggested that the changes observed are a reflection of the average age of the red cell population as the animal grows.     

The Kinetics of Ouabain Inhibition and the Partition of Rubidium Influx in Human Red Blood Cells

In the development of ouabain inhibition of rubidium influx in human red blood cells a time lag can be detected which is a function of at least three variables: the concentrations of external sodium, rubidium, and ouabain. The inhibition is antagonized by rubidium and favored by sodium. Similar considerations could be applied to the binding of ouabain to membrane sites. The total influx of rubidium as a function of external rubidium concentration can be separated into two components: (a) a linear uptake not affected by external sodium or ouabain and not requiring an energy supply, and (b) a saturable component. The latter component, on the basis of the different effects of the aforementioned factors, can be divided into three fractions. The first is ouabain-sensitive, inhibited by external sodium at low rubidium, and requires an energy supply; this represents about 70–80% of the total uptake and is related to the active sodium extrusion mechanism. The second is ouabain-insensitive, activated by external sodium over the entire range of rubidium concentrations studied, and dependent on internal ATP; this represents about 15% of the total influx; it could be coupled to an active sodium extrusion or belong to a rubidium-potassium exchange. The third, which can be called residual influx, is ouabain-insensitive, unaffected by external sodium, and independent of internal ATP; this represents about 10–20% of the total influx.     

Separation of Adenosine Triphosphatase of HK and LK Sheep Red Cell Membranes by Density Gradient Centrifugation

Membrane fragments from high potassium (HK) and low potassium (LK) sheep red cells were separated by density gradient centrifugation. Three preparations were studied: (1) HK membranes sonicated for 20 minutes, (2) HK membranes sonicated for 3 minutes, and (3) LK membranes sonicated for 3 minutes. The adenosine triphosphatase (ATPase) activity in the maximally disrupted preparation (1) was not sensitive to Na + K and was recovered in relatively small but heavy (specific gravity 1.19) fragments which made up no more than 8 per cent of the total membrane. Both Na + K-sensitive (S) and Na + K-insensitive (I) ATPase activity were found in the more gently broken up preparations (2) and (3) but the ratio of S- to I-ATPase was much greater in HK than in LK membrane fragments. S-ATPase activity in preparation (2) was about 50 per cent that observed in HK membranes prior to sonication. S-ATPase activity was recovered from the density gradient in relatively large but light (specific gravity 1.10) fragments. As was the case with the maximally disrupted preparation (1), I-ATPase activity in both preparations (2) and (3) was recovered in small but heavy (specific gravity > 1.20) fragments. The possibility that sensitivity of sheep red cell membrane ATPase to Na + K depends on the association between units containing the enzyme(s) and large, light, phospholipid-containing components is discussed.     

Active Cation Transport and Ouabain Binding in High Potassium and Low Potassium Red Blood Cells of Sheep

Red cells from high K sheep contained 82 mM K/liter cells and had a pump flux of 0.86 mM K/liter cells x hr; similarly, LK cells had 16.5 mM K/liter cells and a pump flux of 0.12 mM K/liter cells x hr. Using [³H]-ouabain, the relation between the number of ouabain molecules bound per cell and the concomitant per cent inhibition of the pump was found to be approximately linear for both HK and LK cells. The number of glycoside molecules necessary for 100 % inhibition of the pump was 42 for HK cells and 7.6 for LK cells, after correction for six nonspecific binding sites for each type of cell. The ratio of ouabain molecules/cell at 100 % inhibition was 5.5, HK to LK, and the ratio of the normal K pump fluxes was 7.2, HK to LK. The similarity of these ratios suggests that an important difference between HK and LK cells, determining the difference in pump fluxes, is the number of pump sites. The turnover times (ions/site x min) are 6000 and 4800 for HK and LK cells, respectively. The results also indicate a high specificity of binding of ouabain to pump sites.     

The M-antigen in HK and LK sheep red cell membranes

Summary Red cells of all high-potassium-type (HK) sheep and of more than one half of all low-potassium-type (LK) sheep contained the M-antigen and were hemolyzed by iso-immune anti-M antiserum in presence of a guinea pig serum complement. It was characteristic for the hemolysis of HK red cells by the M-antiserum the all HK cells were ultimately hemolyzed at suboptimal antibody concentrations, provided the time of incubation at 37 °C was sufficiently long. Thus, the M-antigen appears to be expressed on all red cells of an individual HK sheep. The M-antibody was absorbed by HK red cells and their membranes with a high affinity, whereas M-negative LK red cells and their membranes did not bind the antibody. The ratio of the number of antibody units absorbed per cell or membrane to the number of antibody units required for lysis approached unity. The amount of antibody absorbed per membrane was unaffected by ouabain in the presence of ATP, Mg⁺⁺, Na⁺, and K⁺. The M-antigen activity depends on the integrity of the red cell membrane and was not detectable after lyophilization of HK membranes or in the membrane protein solubilized by n-butanol. The major M-antibody activity was found among the high molecular weight plasma proteins and may be attributed to the ₂ M globulins. Heterogeneity within the antibody fraction cannot be excluded since some hemolytic activity was detected in a chromatographic fraction containing predominantly -globulin. The relationship between the M-antigen and the Na⁺–K⁺ transport system in sheep red cell membranes is discussed.This work was presented in part at the 53rd annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J. 1969.     

Computational Analysis of Dynamic Interaction of Two Red Blood Cells in a Capillary

The dynamic interaction of two red blood cells (RBCs) in a capillary is investigated computationally by the two-fluid model, including their deformable motion and interaction. For characterization of the deformation, the RBC membrane is treated as a curved two-dimensional shell with finite thickness by the shell model, and allowed to undergo the stretching strain and bending deformation. Moreover, a Morse potential is adopted to model the intercellular interaction for the aggregation behavior, which is characterized as the weak attraction at far distance and strong repulsion at near distance. For validation of the present technique, the dynamic interaction of two RBCs in static blood plasma is simulated firstly, where the RBCs aggregate slowly until a balanced configuration is achieved between the deformation and aggregation forces. The balanced configuration is in good agreement with the results reported previously. Three important effects on the dynamic behavior of RBCs are then analyzed, and they are the initial RBC shape, RBC deformability, and the intercellular interaction strength. It is found that the RBC is less deformed into a well-known parachute shape when the initial RBC shape is larger. Similarly, if the elastic shear modulus and bending stiffness of RBC membrane increase, the RBC resistance to deformation becomes higher, such that the RBC is less deformed. The simulation results also demonstrate that the RBC deformability strongly depends on the intercellular interaction strength. The RBCs deform more easily as the intercellular interaction strength increases.     

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

HK/LK polymorphism and its genetic determination in Speke's gazelle

Concentrations of potassium (K) and sodium (Na) were determined in the erythrocytes of 28 members of an interbreeding herd of Speke's gazelle. The distribution of K concentrations and Na/K concentration ratios suggested the presence of the high-K/low-K (HK/LK) polymorphism known in erythrocytes of domestic bovids. The pedigree of the herd of gazelles is known completely, permitting examination of the inheritance of HK/LK polymorphism by overlaying the distribution of phenotypes on the pedigree. Statistical analyses clearly indicate a strong genetic component in the phenotypic variability that is entirely consistent with a single autosomal locus, two-allele mode of inheritance, with the LK allele being dominant. This is the first demonstration of HK/LK polymorphism in a wild bovid species. The evidence indicates that HK/LK polymorphism is of considerable evolutionary age, is of monophyletic origin, and is maintained by selection.     

Antibody-Induced Alterations in the Kinetic Characteristics of the Na:K Pump in Goat Red Blood Cells

The kinetic characteristics of the Na:K pump in high potassium (HK) and low potassium (LK) goat red cells were investigated after altering the intracellular cation concentrations. At low concentrations of intracellular K (K_c), increasing K_c at first stimulates the active K influx in HK cells, but at higher K_c the pump is inhibited. These results suggest that in HK cells K_c acts both at a stimulatory site at the inner aspect of the pump and by competition with intracellular Na (Na_c) at the Na translocation sites. In LK cells, K_c inhibits the active K influx and the sensitivity of LK cells to inhibition is much greater than the sensitivity of HK cells. Exposure of LK cells to an antibody (anti-L), raised in an HK sheep by injection of LK sheep cells, increased the active K influx at any given K_c. The effect of the antibody was greater at higher intracellular K concentrations, and in cells with very low concentrations of K the antibody had little effect on the pump rate. The failure of anti-L to stimulate the pump in low K_c LK cells was not due to failure of the antibody to bind to the cells. Anti-L combining at the outer surface of the cell reduces the affinity of the pump at the inner surface for K at the inhibitory sites. The maximal pump rate in LK cells at optimal Na and K concentrations is less than the maximal pump rate of HK cells under the same circumstances.     

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.     

Cryopreservation of Sheep Red Blood Cells

The protection of sheep erythrocytes at freezing temperatures was investigated using glycerol, dimethylsulfoxide (DMSO), glucose and four different types of polyvinylpyrrolidone (PVP) as cryoprotective agents. Depending on type (molecular weight) and concentration good protection was obtained with PVP, whereas glycerol, DMSO and glucose were unsatisfactory. Recovery of cells after thawing was most successful when the cells had been frozen at a concentration of 1–2 × 109 cells/ml. No cells tolerated freezing at −20 °G. Best results were obtained when the cells were frozen directly in liquid nitrogen (−196°G).     

Stretching and Relaxation of Malaria-Infected Red Blood Cells

The invasion of red blood cells (RBCs) by malaria parasites is a complex dynamic process, in which the infected RBCs gradually lose their deformability and their ability to recover their original shape is greatly reduced with the maturation of the parasites. In this work, we developed two types of cell model, one with an included parasite, and the other without an included parasite. The former is a representation of real malaria-infected RBCs, in which the parasite is treated as a rigid body. In the latter, where the parasite is absent, the membrane modulus and viscosity are elevated so as to produce the same features present in the parasite model. In both cases, the cell membrane is modeled as a viscoelastic triangular network connected by wormlike chains. We studied the transient behaviors of stretching deformation and shape relaxation of malaria-infected RBCs based on these two models and found that both models can generate results in agreement with those of previously published studies. With the parasite maturation, the shape deformation becomes smaller and smaller due to increasing cell rigidity, whereas the shape relaxation time becomes longer and longer due to the cell’s reduced ability to recover its original shape.     

Stretching and Relaxation of Malaria-Infected Red Blood Cells

The invasion of red blood cells (RBCs) by malaria parasites is a complex dynamic process, in which the infected RBCs gradually lose their deformability and their ability to recover their original shape is greatly reduced with the maturation of the parasites. In this work, we developed two types of cell model, one with an included parasite, and the other without an included parasite. The former is a representation of real malaria-infected RBCs, in which the parasite is treated as a rigid body. In the latter, where the parasite is absent, the membrane modulus and viscosity are elevated so as to produce the same features present in the parasite model. In both cases, the cell membrane is modeled as a viscoelastic triangular network connected by wormlike chains. We studied the transient behaviors of stretching deformation and shape relaxation of malaria-infected RBCs based on these two models and found that both models can generate results in agreement with those of previously published studies. With the parasite maturation, the shape deformation becomes smaller and smaller due to increasing cell rigidity, whereas the shape relaxation time becomes longer and longer due to the cell’s reduced ability to recover its original shape.     

Viscoelastic Transient of Confined Red Blood Cells

The unique ability of a red blood cell to flow through extremely small microcapillaries depends on the viscoelastic properties of its membrane. Here, we study in vitro the response time upon flow startup exhibited by red blood cells confined into microchannels. We show that the characteristic transient time depends on the imposed flow strength, and that such a dependence gives access to both the effective viscosity and the elastic modulus controlling the temporal response of red cells. A simple theoretical analysis of our experimental data, validated by numerical simulations, further allows us to compute an estimate for the two-dimensional membrane viscosity of red blood cells, ηmem2D ∼ 10⁻⁷ N⋅s⋅m⁻¹. By comparing our results with those from previous studies, we discuss and clarify the origin of the discrepancies found in the literature regarding the determination of ηmem2D, and reconcile seemingly conflicting conclusions from previous works.     

新型人工红细胞的制备

目的:接枝淀粉包裹血红蛋白制备新型人造红细胞的代替品。方法:利用油酸接枝淀粉,在超声条件下下自组装,包裹天然牛血红蛋白,并鉴定其物理化学及生物学性能。测定包封率、红外光谱分析(FTIR)、电镜观察形态学及粒径,测定P50和Hill系数。结果:人工红细胞呈圆球形,平均粒径250nm,包封率高,具有良好的携氧、释氧能力。结论:成功制备了人工纳米红细胞,为进一步临床应用提供了基础。     

The Effect of Ouabain and External Potassium on the Ion Transport of Rabbit Red Cells

Na efflux of rabbit RBC is approximately 10 mmoles/kg wet weight. hr. One-half of this consists of a ouabain-insensitive exchange diffusion component. Ouabain inhibits 2.5 mmoles/kg.hr of Na efflux. K influx is 3.0 mmoles/kg.hr; 2.2 mmoles/kg.hr are inhibited by ouabain. In contrast with human RBC, ouabain inhibition of Na efflux and K influx of rabbit RBC is easily reversible. After 2 hr, ouabain inhibition of Na efflux is completely compensated for by increased internal Na concentration and Na efflux returns to initial levels. Removal of ouabain at this stage results in stimulation of the efflux by 4.3 mmoles/kg.hr. Na influx is initially not affected by ouabain but is increased by 2.4 mmoles/kg.hr after 2 hr incubation with the drug. Removal of K from normal Ringer does not affect Na efflux and increases Na influx by 1.6 mmoles/kg.hr. Addition of ouabain to K-free Ringer inhibits Na efflux and influx to the same extent (1.6 mmoles/kg.hr). Removal of Na from K-free Ringer has an inhibitory effect on efflux similar to that of ouabain. These findings suggest that the fraction of Na efflux inhibited by removal of external K is completely replaced by a new, ouabain-sensitive exchange diffusion of Na ions.     

The Selective Staining of Red Blood Cells

A method for the selective staining of red blood cells is described. Material is fixed in 10% neutral formalin in .85% NaCl and imbedded in paraffin or celloidin. Sections 6-10 μ are stained 1-5 minutes in chromotrope 2R. Basophilic and the less strongly acidophilic elements are decolorized with 5% phosphotungstic acid in 95% ethyl alcohol. Red blood cells and other strongly acidophilic elements that may be present in the preparation retain the chromotrope 2R. A counterstain of methyl blue may be used for staining the decolorized basophilic elements. As a result, erythrocytes are stained red by the chromotrope 2R, and basophilic elements blue, by the methyl blue. Less strongly acidophilic elements, having little affinity for either primary or secondary dye, are colorless or gray.