The transport of sodium into human erythrocytes in vivo

The relative Na²⁴ specific activity of red cells and plasma was measured at periods up to 30 hours following a single intravenous injection of Na²⁴ in normal healthy young adults. The average specific activity of the red cells relative to that of the plasma at 24 hours and beyond was found to average 0.83 ± 0.05 in a series of five normal individuals, significantly different from 1.0. This indicates that all the intracellular Na is not exchangeable in 24 hours, and confirms earlier in vitro results. The red cell Na concentration in man was shown to be 12.1 ± 1.1 m.eq. Na/liter red cell, as measured in a series of nineteen normal healthy young adults. A theoretical analysis of the data on exchangeable cell Na suggests that the red cell Na (5.3 m.eq. Na/liter blood) is divided into a fast compartment comprising 4.25 m.eq. Na/liter blood, and a slow compartment comprising 1.07 m.eq. Na/liter blood. If these compartments are arranged in parallel, the flux between plasma and fast compartment is 1.32 m.eq. Na/liter blood hour, and that between plasma and slow compartment is 0.016 m.eq. Na/liter blood hour. Results of experiments on two patients with congenital hemolytic jaundice suggest that the fraction of slowly exchanging Na may increase with the age of the red cell.     

Cholinergic stimulation of glucose transport in human erythrocytes

The effects of cholinergic stimulation on glucose equilibrium exchange rate have been studied in human erythrocytes. Carbamylcholine increases the V of equilibrium exchange by 20% but has no significant effect on K_m. The cholinergic effect is abolished by the muscarinic antagonist atropine or by alterations in intracellular calcium concentrations induced by the calcium ionophore A23187.     

A two-site model for sodium transport in human erythrocytes

A kinetic model has been proposed for human erythrocytes to account for the responses of sodium transport to alterations in the extracellular sodium concentration in the presence and absence of potassium. The proposed model characterizes the movement of sodium from the outer surface of the erythrocyte membrane to the inner surface in terms of a carrier which has two sites with differing affinities for sodium ions.     

Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.     

Exercise-induced stimulation of K(+) transport in human erythrocytes.

The hypothesis was tested that exercise-induced changes in plasma composition stimulate unidirectional K(+) transport (J(in)K) in human red blood cells (RBCs). Ten men performed two 30-s high-intensity leg-cycling tests separated by 4 min of rest. Antecubital venous blood was sampled before exercise and at the end of the second exercise bout. RBCs were separated from true exercise plasma, (42)K was added to plasma, and RBC K(+) transport was studied in vitro at 37 degrees C. In the second part of the study, blood from nine healthy men studied in vitro at 37 degrees C was used to test the hypothesis that exercise-simulated (ES) plasma stimulates net K(+) transport and J(in)K (measured using (86)Rb) in human RBCs. The J(in)K of resting RBCs added to true exercise plasma was 1,574 +/- 200 (SE) micromol. h(-1). l(-1) vs. 1,236 +/- 256 micromol. h(-1). l(-1) in true resting plasma at 2 min (controls). In true exercise and ES plasma, J(in)K was increased through activation of the ouabain-sensitive Na(+)-K(+) pump and the bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter. Increases in plasma osmolality and K(+), H(+), and epinephrine concentrations independently and in combination stimulated K(+) transport into human RBCs. In a third series of experiments, in which ES plasma K(+) concentration was continuously measured during the first 5 min of incubation of RBCs, a 1.6 +/- 0.3 mmol/l decrease in plasma K(+) concentration occurred during the first 2 min. It is concluded that RBCs transport K(+) at elevated rates in response to exercise-induced changes in plasma composition.     

Regulation of sodium transport in erythrocytes

The kinetic response of swine erythrocyte (Na + K)-ATPase to Na⁺ concentration was hyperbolic in low KCl (5–25 mm) but became increasingly sigmoidal (n = 2.2) as KCl was increased to 150 mm. The addition of 150 mm LiCl did not cause an increase in sigmoidicity although it decreased the apparent affinity for Na⁺. The dependence of ouabain-inhibited efflux of Na⁺ on internal Na⁺ concentration was measured in intact cells with intracellular cation concentrations altered by incubation in p-ehloromercuriphenyl sulfonate. The response to Na⁺ was sigmoidal (n = 2.2) in cells containing high K⁺ but hyperbolic in preparations in which most of the intracellular K⁺ was replaced by Li⁺, even in the presence of 150 mm external KCl. The data are consistent with a model in which internal K⁺ is an allosteric (feedback) inhibitor of Na⁺ efflux and there are three Na⁺ sites which interact cooperatively.     

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of $\text{3-O-[}{}^{14}\text{C}\text{]}$ methylglucose entry and exit, $\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{K}{}_{\mathrm{<mtext>si</mtext>}}$, were 8 and 7 mM, respectively. The equilibrium exchange constant, $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$, and the counterflow constant, $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$, were 9 and 11 mM, respectively. The activity constant for $\text{3-O-}\text{methylglucose}$ transport, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, defined as $\text{V/K}{}_{\mathrm{<mtext>m</mtext>}}$, was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, $\text{K}{}_{\mathrm{<mtext>go</mtext>}}$, was 2 mM and less than the counterflow constant, $\text{R}{}_{\mathrm{<mtext>g</mtext>}}$ (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, $\text{F}{}_{\mathrm{<mtext>g</mtext>}}$, was 5 ml/h perg.Under aerobic conditions, two of the dissociation-like constants ($\text{K}{}_{\mathrm{<mtext>si</mtext>}}$ and $\text{B}{}_{\mathrm{<mtext>s</mtext>}}$) for $\text{3-O-}\text{methylglucose}$ transport were significantly larger than those obtained in anaerobic cells, but the remaining two ($\text{K}{}_{\mathrm{<mtext>so</mtext>}}$ and $\text{R}{}_{\mathrm{<mtext>s</mtext>}}$) remained unchanged. The values, for $\text{K}{}_{\mathrm{<mtext>so</mtext>}}\text{, K}{}_{\mathrm{<mtext>si</mtext>}}\text{, B}{}_{\mathrm{<mtext>s</mtext><mtext>\; and</mtext>}}\text{R}{}_{\mathrm{<mtext>s</mtext>}}$ were 8, 26, 20 and 8 mM, respectively. The activity constant, $\text{F}{}_{\mathrm{<mtext>s</mtext>}}$, decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.     

Intracellular calcium content of human erythrocytes: Relation to sodium transport systems

Summary To study the possible role of intracellular Ca (Ca _i ) in controlling the activities of the Na⁺–K⁺ pump, the Na⁺–K⁺ cotransport and the Na⁺/Li⁺ exchange system of human erythrocytes, a method was developed to measure the amount of Ca embodied within the red cell. For complete removal of Ca associated with the outer aspect of the membrane, it proved to be essential to wash the cells in buffers containing less than 20nm Ca. Ca was extracted by HClO₄ in Teflon^® vessels boiled in acid to avoid Ca contaminations and quantitated by flameless atomic absorption. Ca _i of fresh human erythrocytes of apparently healthy donors ranged between 0.9 and 2.8 mol/liter cells. The mean value found in females was significantly higher than in males. The interindividual different Ca contents remained constant over periods of more than one year. Sixty to 90% of Ca _i could be removed by incubation of the cells with A23187 and EGTA. The activities of the Na⁺–K⁺ pump, of Na⁺–K⁺ cotransport and Na⁺/Li⁺ exchange and the mean cellular hemoglobin content fell with rising Ca _i ; the red cell Na⁺ and K⁺ contents rose with Ca _i . Ca depletion by A23187 plus EGTA as well as chelation of intracellular Ca²⁺ by quin-2 did not significantly enhance the transport rates. It is concluded that the large scatter of the values of Ca _i of normal human erythrocytes reported in the literature mainly results from a widely differing removal of Ca associated with the outer aspect of the membrane.     

Thiamin transport by human erythrocytes and ghosts

Summary Thiamin transport in human erythrocytes and resealed pink ghosts was evaluated by incubating both preparations at 37 or 20°C in the presence of [³H]-thiamin of high specific activity. The rate of uptake was consistently higher in erythrocytes than in ghosts. In both preparations, the time course of uptake was independent from the presence of Na⁺ and did not reach equilibrium after 60 min incubation. At concentrations below 0.5 m and at 37°C, thiamin was taken up predominantly by a saturable mechanism in both erythrocytes and ghosts. Apparent kinetic constants were: for erythrocytes,K _m =0.12, 0.11 and 0.10 m andJ _max=0.01, 0.02 and 0.03 pmol·l^–1 intracellular water after 3, 15, and 30 min incubation times, respectively; for ghosts,K _m =0.16 and 0.51 m andJ _max=0.01 and 0.04 pmol·l^–1 intracellular water after 15 and 30 min incubation times, respectively. At 20°C, the saturable component disappeared in both preparations. Erythrocyte thiamin transport was not influenced by the presence ofd-glucose or metabolic inhibitors. In both preparations, thiamin transport was inhibited competitively by unlabeled thiamin, pyrithiamin, amprolium and, to a lesser extent, oxythiamin, the inhibiting effect being always more marked in erythrocytes than in ghosts. Only approximately 20% of the thiamin taken up by erythrocytes was protein-(probably membrane-) bound. A similar proportion was esterified to thiamin pyrophosphate. Separate experiments using valinomycin and SCN^– showed that the transport of thiamin, which is a cation at pH 7.4, is unaffected by changes in membrane potential in both preparations.     

Glucose transport inhibition in human erythrocytes by calmodulin antagonists

Calmodulin antagonists (tryphtazin, lidocaine, dykain, palmitate) inhibit glucose transport from human erythrocytes. Glucose efflux inhibition is proportional to the concentration of antagonists in the medium and is of uncompetitive character. It is accompanied by a decrease in the maximum transport rate with the unchanged constant of dissociation in the complex: carrier-sugar. Calcium ionophores A23187 and divaleryldibenzo-18-crown-6 eliminated the inhibiting effect of pharmacological agents on glucose transport. The authors think that the glucose transport inhibition under the influence of calmodulin antagonists may be realized through the calmodulin-dependent chain inhibition under the influence of calmodulin antagonists in the carbohydrate transport system.     

Beta-adrenergic stimulation of volume-sensitive chloride transport in lamprey erythrocytes

We measured the effects of a beta-adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L(-1)) and hypotonic (192 mosm L(-1)) medium. Isoproterenol at a high concentration (10(-5) M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw(-1) h(-1) in the absence to 350 mmol kg dcw(-1) h(-1) (dcw=dry cell weight) in the presence of isoproterenol. Simultaneously, the half-time for volume recovery decreased from 27 to 9 min. Isoproterenol caused an increase in cellular cyclic AMP (cAMP) concentration. The stimulation of chloride transport in hypotonic conditions could be induced by application of the permeable cAMP analogue, 8-bromo-cyclic AMP, suggesting that the effect of beta-adrenergic stimulation on chloride transport occurs downstream of cAMP production. As isoproterenol did not affect unidirectional rubidium fluxes in hypotonic conditions, the transport pathway influenced by beta-adrenergic stimulation is most likely the swelling-activated chloride channel. Because the beta-adrenergic agonist only influenced the transport in hypotonic conditions despite the fact that cAMP concentration also increased in isotonic conditions, the activation may involve a volume-dependent conformational change in the chloride channel.     

Serum stimulation of sodium transport in human fibroblasts containing low and high levels of intracellular sodium

Summary The relationships between intracellular sodium content, sodium transport and serum effects were investigated in human fibroblasts. In the cells with low intracellular sodium (Na _iL ^/+ ;0.04 mol sodium/mg protein) serum stimulated the sodium-potassium pump as measured by ouabain-sensitive sodium efflux and rubidium influx and also exerted a transstimulation of ouabain-insensitive sodium transport resulting in net influx. In cells with high intracellular sodium (Na _iH ^/+ ;0.42 mol sodium/mg protein) all aspects of sodium transport were increased compared to Na _iL ^/+ cells. In these cells serum caused no change in sodium-potassium pump activity but significantly increased the ouabain-insensitive sodium fluxes resulting in net efflux. In Na _iL ^/+ cells, serum promoted net sodium influx through an amiloride-sensitive pathway that was undetectable in the basal state. In Na _iH ^/+ cells the serum-stimulated net efflux was amiloride sensitive but this pathway also contributed to a major portion of sodium transport in the basal state. This study demonstrated that sodium-potassium pump activity is directed by the supply of internal sodium and that serum can increase this supply by promoting net influx, and that serum-induced sodium transport can be modified by intracellular sodium content.     

Rubidium transport in X-irradiated human erythrocytes

Outflow of 86Rb, a radioactive analogue of potassium, from human erythrocytes X-irradiated in vitro was studied with the following results. (1) The 86Rb level in the supernatants of irradiated and control cell suspensions reflected mainly 86Rb outflow and much less its active re-uptake. (2) The effect of irradiation on 86Rb outflow was more pronounced at a low temperature (4 degrees C) than at 37 degrees C; the lowest dose of X-radiation exhibiting a significant effect on 86Rb outflow at 4 degrees C was 2.5 Gy. (3) K/Rb exchange did not seem to play an appreciable role in radiation-induced 86Rb outflow. (4) Calcium and its accumulation in irradiated cells was not found to be the cause of the effect of radiation on 86Rb outflow. (5) The effect of radiation on 86Rb outflow was higher in low Na medium but it was not inhibited by bumetanide. Rb/Na counter- or co-transport do not therefore seem to be involved in radiation-induced Rb+ outflow.     

Inhibition of phosphate transport in human erythrocytes by water-soluble carbodiimides

Phosphate entry into human erythrocytes is irreversibly inhibited by treatment of the cells with the water-soluble carbodiimides 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluene sulfonate (CMC) in the absence of added nucleophile. EDC is the more potent inhibitor (40% inhibition, 2 mM EDC, 5 min, 37 degrees C, 50% hematocrit, pH 6.9), while more than 20 mM CMC is required to give the same inhibition under identical conditions. EDC inhibition is temperature-dependent, being complete in 5 min at 37 degrees C, and sensitive to extracellular pH. At pH 6.9 only 50% of transport is rapidly inhibited by EDC, but at alkaline pH over 80% of transport is inhibited. Inhibition is not prevented by modification of membrane sulfhydryl groups but is decreased in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), a reversible competitive inhibitor of anion transport. EDC treatment leads to crosslinking of erythrocyte membrane proteins, but differences between the time course of this action and inhibition of transport indicate that most transport inhibition is not due to crosslinking of membrane proteins.