Anion-dependent cation transport in erythrocytes

A selective survey of the literature reveals at least three major anion-dependent cation transport systems, defined as Na+ + Cl-, K+ + Cl- and Na+ + K+ + Cl- respectively. In human red cells, kinetic data on the fraction of K+ and Na+ influx inhibitable by bumetanide are presented to indicate an Na+:K+ stoichiometry of 1:2. For LK sheep red cells the large Cl- -dependent K+ leak induced by swelling is shown to share many characteristics with that induced by N-ethylmaleimide (NEM) treatment. NEM has complex effects, both inhibiting and then activating Cl- -dependent K+ fluxes dependent on NEM concentration. The alloantibody anti-L can prevent the action of NEM. In human red cells NEM induces a large Cl- -dependent specific K+ flux, which shows saturation kinetics. Its anion preference is Cl- greater than Br- greater than SCN- greater than I- greater than NO3- greater than MeSO4-. This transport pathway is not inhibited by oligomycin or SITS, although phloretin and high concentrations of furosemide and bumetanide (over 0.3 mM) do inhibit. Quinine (0.5 mM) is also an inhibitor. It is concluded that at least two distinct Cl- -dependent transport pathways for K+ are inducible in mammalian red cells, although the evidence for their separation is not absolute.     

Triacylglycerol lipase in bovine erythrocytes

Acid lipase activity was found in the bovine erythrocyte ghosts, but little neutral or alkaline lipase activity was observed in the erythrocytes. The membrane-bound lipase showed a remarkable activity in the ghosts only after hemolysis. The membrane-bound lipase showed its maximum activity at pH 4.5, 38 degrees C, and it was stable below 40 degrees C. The hydrolysis rate was linear with time up to 60 min, and was proportional to the amount of enzyme up to 0.4 mg protein. The bound lipase was activated markedly by bovine serum albumin and slightly by octyl-glycoside. The lipase was remarkably inhibited by bovine serum.     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

The cation content of phospholipides from swine erythrocytes

Phospholipides from swine erythrocytes were isolated and separated into four reproducible fractions. One of the fractions seems to be pure phosphatidylserine. The others are almost certainly not single compounds, although the analytical data indicate that they represent mixtures considerably simpler than the parent mixture extracted from the cells. All four fractions contained Na⁺ and K⁺, but very little Ca²⁺. Sodium was the predominant cation in two of the fractions under all conditions although the major intracellular cation was potassium. In the other two fractions the ratio Na/K varied with the extraction procedure largely because the quantity of K⁺ seemed to depend on the solvent system used. There appear to be reasons to believe that the entire system of phospholipides binds Na⁺ preferentially. In addition, it was observed that the quantity of Na⁺ found in the lipide extracts varied when the extrusion of Na⁺ from the cells was made to vary. Both of these observations are consistent with the possibility that the phospholipides play some part in the extrusion of Na⁺ from these cells.     

The connexion between active cation transport and metabolism in erythrocytes

1. A study has been made of the dependence on the concentrations of internal Na(+) and external K(+) of lactate and phosphate production in human erythrocytes. 2. Lactate production was stimulated by Na(+) and K(+) but only when they were internal and external respectively. The stimulation was counteracted by ouabain. The production of phosphate was affected in the same way. 3. There is a quantitative correlation between these effects and those previously found for cation movements and the membrane adenosine triphosphatase. 4. It is concluded that the rate of energy production in glycolysis is partly controlled by the magnitude of active transport; the extent of this regulation is shown to vary from 25 to 75% of a basal rate that is independent of active transport. 5. The activity of the membrane adenosine triphosphatase was also compared with rates of Na(+) and K(+) transport. The latter were varied by altering the concentrations of internal Na(+) and external K(+), and by inhibiting with ouabain. 6. A threefold variation of active transport rate was accompanied by a parallel change in the membrane adenosine-triphosphatase activity. The results show a constant stoicheiometry for the number of ions moved/mol. of ATP hydrolysed, independent of the electrochemical gradient against which the ions were moved. 7. Calculations show that the amount of ATP hydrolysed would provide enough energy for the osmotic work. The results are discussed in relation to possible mechanisms for active transport.     

Radioprotection of bovine erythrocytes to haemolysis

The effect of several free-radical scavengers on the radiation-induced haemolysis of bovine erythrocytes (50 krad, 60Co, cell concentration: 0.05 v/v) was studied. A two-phase effect of the additives was found with a maximum of the protective action in the millimolar concentration range. A correlation was established between the degree of protection at a fixed concentration (5mM) and the rate constants of reactions of respective compounds with OH. A radiosensitizing effect of diethyldithiocarbamate, an inhibitor of superoxide dismutase (SOD) was not confirmed and no specific protective effect of SOD was seen.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

Decreased cation channel activity and blunted channel-dependent eryptosis in neonatal erythrocytes

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca²⁺-permeable cation channels with subsequent entry of Ca²⁺ and stimulation of Ca²⁺-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl^– removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca²⁺ activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E₂ (PGE₂) formation, cation channel activity, Ca²⁺ entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl^– in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE_2, and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca²⁺ leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes. annexin V; osmotic cell shrinkage; calcium; apoptosis     

Hypertension: cation transport and the physical properties of erythrocytes

In recent years, many reports have appeared describing altered Na+ and K+ transport in erythrocytes of individuals with essential hypertension. Collectively, the interpretation of these results has been unclear. Our studies revealed that the active ouabain-sensitive K+ influx, the furosemide-sensitive K+ influx and the residual passive K+ influx in both human and rat erythrocytes can vary considerably among individual persons or rats and that these measurements alone can not be used to distinguish normotensive from hypertensive individuals. The only consistent cation transport difference observed was an increased Na+ permeability in spontaneously hypertensive rat (SHR) erythrocytes. We have also examined certain physical properties (equilibrium density distribution and sedimentation velocity) of erythrocytes from normotensive Wistar-Kyoto (WKY) and SHR rats, since these characteristics may be altered in response to abnormalities of ion transport. It was found that the erythrocytes from geographically, environmentally, and age-matched littermates of WKY and SHR rats have identical equilibrium density distributions. It was also found that the density distribution of erythrocytes can vary among geographically dispersed colonies of the same strain of rat, and even among successive litters of the same rat colony. However, the sedimentation time required for erythrocytes to reach their equilibrium density was always shorter in the normotensive WKY samples than in the matched SHR. Utilizing a simple centrifugation method, we were able to clearly show that for any population of erythrocytes with the same upper limit of cell density, normotensive WKY cells always sediment at a faster rate than those of the hypertensive SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of active K+ transport in cation dimorphic sheep erythrocytes

Arrhenius diagrams of K⁺ pump fluxes measured between 15°C and 41°C were discontinuous in high K⁺ but not in low K⁺ sheep red cells. Exposure of low K⁺ cells to anti-L caused a bimodal temperature response of K⁺ pump flux with a transition temperature, $\text{T}\text{c}$, similar to that found in high K⁺ cells but with comparatively higher activation energies above $\text{T}\text{c}$.