Inhibition of Na,K-ATPase activity reduces Babesia gibsoni infection of canine erythrocytes with inherited high K, low Na concentrations

Babesia gibsoni multiplies well in canine red blood cells (RBCs) containing high concentrations of potassium (HK), reduced glutathione, and free amino acids as a result of an inherited high Na,K-ATPase activity, i.e., HK RBCs. To determine the role of Na,K-ATPase in the multiplication of B. gibsoni, the effect of ouabain on the proliferation of the parasites in HK RBCs was investigated. To determine the direct effect of ouabain on the parasites, the proliferation of the parasites in normal canine RBCs containing low potassium (LK) and high sodium concentrations, i.e., LK RBCs, which completely lack Na,K-ATPase activity, was observed. Ouabain at 0.1 mM significantly suppressed the multiplication of B. gibsoni in HK RBCs in vitro, whereas it had no effect on the parasites in LK RBCs. The results suggest that the multiplication of B. gibsoni in HK RBCs depends mainly on the presence of Na,K-ATPase in the cells. Therefore, the effects of ouabain on the intracellular cation and free amino acid composition of the HK RBCs were examined. In HK RBCs incubated with ouabain, a marked decrease in the concentration of potassium and an increase in sodium were observed, together with a decrease in the number of parasitized cells. These results suggest that the intracellular cation composition maintained by Na,K-ATPase might be advantageous to the parasites. Moreover, the concentrations of some free amino acids, i.e., asparagine, aspartate, glutamate, glutamine, glycine, and histidine, were markedly decreased in HK RBCs incubated with ouabain. Decreased concentrations of the free amino acids induced by inhibition of Na,K-ATPase seemed to affect the multiplication of B. gibsoni in HK RBCs. Based on these results, it is clear that the high Na,K-ATPase activity in HK RBCs contributes to the proliferation of B. gibsoni by maintaining high potassium and low sodium concentrations, as well as high concentrations of some free amino acids in the cells.     

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.     

Changes in the activity of acetylcholinesterase and Na,K-ATPase in human erythrocytes irradiated with X-rays

The response of human erythrocytes to X-rays in the dose range from 40 Gy to 600 Gy was determined on the basis of changes in the activities of AChE and ATPase. The Na,K-ATPase activity increased above the control value at doses below 200 Gy, while at the doses higher than 200 Gy, it decreased, reaching 96% of the control value at a dose of 600 Gy. In the range of doses up to 200 Gy, the AChE activity, expressed as Vmax, did not change. At higher doses, it fell drastically, reaching 33% of the control value at a dose of 600 Gy. Simultaneously, the enzyme substrate affinity decreased at 200 Gy, and then started to increase at lower values of Vmax. The obtained results suggest that under appropriate conditions, low doses of radiation may have the opposite effects to high doses.     

(Na,K)-ATPase and Ouabain binding in reticulocytes from dogs with high K and low K erythrocytes and their changes during maturation

The present study demonstrated that dog reticulocytes had considerable amounts of (Na,K)-ATPase, but lost it rapidly during maturation into erythrocytes. Furthermore, reticulocytes from dogs possessing erythrocytes characterized with high (Na,K)-ATPase activity and high K, low Na concentrations (HK dogs; Maede, Y., Inaba, M., and Taniguchi, N. (1983) Blood 61,493-499) had more ouabain binding sites than cells from normal dogs (LK dogs). Our results were as follows: i) The maximal binding capacities (Bmax) for ouabain binding at equilibrium were approximately 0 and 1,500 binding sites/cell in LK and HK dog erythrocytes, respectively. ii) Reticulocytes from LK dogs possess approximately 5,700 ouabain binding sites/cell. iii) The Bmax value for ouabain in HK reticulocytes was about 10,000 sites/cell, being 2-fold that in LK reticulocytes. iv) Ouabain-sensitive fluxes of 24Na and 42K in each type of reticulocyte were compatible with the number of ouabain binding sites on the cells. v) Ouabain binding capacity, as well as (Na,K)-ATPase activity, in the reticulocytes from LK dogs fell rapidly to nearly zero during the maturation into erythrocytes. vi) Although reticulocytes from HK dogs also showed a similar regression of (Na,K)-ATPase during maturation, they retained a certain number of ouabain binding sites even after maturation, resulting in the high activity of (Na,K)-ATPase in HK erythrocyte membrane.     

Effect of albumin on the Na, K-ATPase activity in the rat erythrocytes during immobilization stress

Immobilisation stress (IMS) led to a 42% decrease in erythrocyte Na, K-ATPase activity in rats. Pre-treatment of the "stressed" erythrocytes with human serum albumin (HSA) and 1-day exposition of the HSA prior to the IMS led to stabilising of enzyme activity at the control level. Absence of inhibiting effect of non-protein supernatants of the blood plasma of stressed rats on enzyme activity of normal erythrocytes was shown in presence of the HSA both in vitro and in vivo. The mechanism of the HSA protective effect on the Na,K-ATPase activity of erythrocytes in the IMS, is discussed.     

Cytoplasmic K+ effects on phosphoenzyme of Na,K-ATPase proteoliposomes and on the Na+-pump activity

Three phosphorylated reaction intermediates (EP) of Na,K-ATPase, and ADP-sensitive K+-insensitive EP (E1P), an ADP- and K+-sensitive EP (E*P), and a K+-sensitive ADP-insensitive EP (E2P), have been discovered at present. By using Na,K-ATPase proteoliposomes (PL) prepared from the electric eel enzyme, we found in this study that E*P existed even in the presence of K+ on both sides of the PL and that there was a sidedness difference in K+ sites between E*P and E2P. Cytoplasmic K+ (K+cyt) accelerated the conversion of E*P to E2P but did not dephosphorylate the E2P. Although the extracellular K+ accelerated the dephosphorylation of E2P, it did not interact with E*P directly. This K+cyt effect was also verified by the activation of Na+-pump in the Na+-K+ exchange mode. In the presence of K+cyt, both the ATP hydrolysis and Na+ uptake rates of the PL containing K+ inside vesicles increased sigmoidally with the concentrations of ATP and cytoplasmic Na+ (Na+cyt). However, in the absence of K+cyt, these Na+-pump reactions in PL containing K+ inside vesicles had only a hyperbolic curve. These results imply that the E*P to E2P conversion is one of the rate-limiting steps of the Na+-pump in the presence of a high concentration of ATP and that K+cyt may control this reaction step by enhancing the conversion rate of E*P to E2P.     

Na,K-ATPase function and cellular proliferative activity in culture

A study was made of the dependence of ATP hydrolysis intensity upon different ratios of sodium and potassium ions in plasma membrane of L cells and of cells of clone Lebr 625, sensitive and resistant to ethidium bromide, and of the distribution of cells according to cell cycle phases in dense and sparse cultures. In dense cultures, the cell growth is arrested on G1 phase, the hydrolytic activity of (Na+ + K+)-ATPase decreases, and the Na+, K+ ratio for maximum activity of (Na+ + K+)-ATPase changes. The higher proliferative activity of Lebr 625 cells in dense culture corresponds to the higher hydrolytic activity of (Na+ + K+)-ATPase.     

Human hypertensive placenta contains an increased amount of Na,K-ATPase with higher affinity for cardiac glycosides

Placentas of women suffering from pregnancy-induced hypertension (PIH) were found to contain a greater amount of Na,K-ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K-ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K-ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain-binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis-like factor is present in the body fluids to regulate Na,K-ATPase activity, the present results render its role quite plausible.     

Synaptosomal factors, regulating the activity of Na,K-ATPase and its sensitivity to neurotransmitters

The factors regulating the activity of synaptosomal Na,K-ATPase have been found in nerve endings cytosol. One of these (Mr 10,000) essentially inhibits, whereas the other one (Mr 60,000) in the presence of norepinephrine, 5-hydroxytryptamine and dopamine activates the Na,K-ATPase of synaptosomes. Regardless of their direct action on Na,K-ATPase, the effect of the activating factor increases in the presence of neurotransmitters. In cell ksap obtained by microsome precipitation the activating factor is absent.     

Na,K-ATPase in artificial lipid vesicles. Comparison of Na,K and Na-only pumping mode

Na,K-ATPase from rabbit kidney outer medulla was reconstituted in large unilamellar lipid vesicles by detergent dialysis. Vesicles prepared in the presence or absence of potassium allowed to study two different transport modes: the (physiological) Na,K-mode in buffers containing Na+ and K+ and the Na-only mode in buffers containing Na+ but no K+. The ATP hydrolysis activity was obtained by determination of the liberated inorganic phosphate, Pi, and the inward directed Na+ flux was measured by 22Na-tracer flux. Electrogenic transport properties were studied using the membrane potential sensitive fluorescence-dye oxonol VI. The ratio upsilon(Na,K)/upsilon(Na) of the turnover rates in the Na,K-mode and in the Na-only mode is 6.6 +/- 2.0 under otherwise identical conditions and nonlimiting Na+ concentrations. Strong evidence is found that the Na-only mode exhibits a stoichiometry of 3Na+cyt/2Na+ext/1ATP, i.e. the extracellular (= intravesicular) Na+ has a potassium-like effect. In the Na-only mode one high-affinity binding side for ATP (KM congruent to 50 nM) was found, in the Na,K-mode a high- and low-affinity binding side with equilibrium dissociation constants, KM, of 60 nM and 13 microM, respectively. The sensitivity against the noncompetitively inhibiting ADP (KI = 6 microM) is higher by a factor of 20 in the Na-only mode compared to the Na,K-mode. From the temperature dependence of the pumping activity in both transport modes, activation energies of 160 kJ/mol for the Na,K-mode and 110 kJ/mol for the Na-only mode were determined.     

Comparison of K-Cl cotransport expression in high and low K dog erythrocytes.

K-Cl cotransport plays a crucial role in regulatory volume decrease of erythrocytes. K-Cl cotransport activities in dog erythrocytes with an inherited high Na-K pump activity (HK) and normal erythrocytes (LK) were compared. Nitrite (NO(2)) stimulated K-Cl cotransport activity in HK cells around 14-fold at 2.4 mM, and it also increased the Km value of this cotransporter. Real-time PCR and western blot analysis revealed that K-Cl cotransporter 1 was dominant, and that the quantity of K-Cl cotransporter 1 protein was comparable between HK and LK erythrocytes. These results suggest that the difference in cotransport activity was not caused by the amount of K-Cl cotransport protein but by a difference in the regulation system, which is susceptible to oxidant.     

C-src locus determines increased rate of Na+,K+-cotransport and increased calcium content in erythrocytes of (SHR x WKY) F2 hybrids

26 male F2 hybrids between spontaneously hypertensive (SHR) and normotensive control (WKY) rats (SHRxWKY)F2 were segregated according to their c-src genotype into SS and WW homozygous groups, corresponding to SHR or WKY and WS heterozygous group. Na, K cotransport in erythrocytes in the WW group was equal to that of WKY and differs significantly from that of WS and SS groups (the rate of Na, K cotransport in latter groups was close to that of SHR). Ca content of RBC in WW group was equal to that of WKY, lower than that of WS and SS groups which in turn was significantly lower than in SHR, indicating polygenic control of the trait. Authors conclude that the c-src locus itself or some other loci inherited in conjunction with c-src determines both the increase of Na, K cotransport and calcium content in erythrocytes of SHR.     

Kinetics of lithium efflux through the (Na,K)-pump of human erythrocytes

Evidence from the kinetics of transport supports the hypothesis that cellular Li, Li_c, interacts with the internal aspect of the (Na,K)-pump as a congener of Na, Li_c and Na_c compete for the same sites on the internal aspect of the pump. Li_c promotes Na-activated K influx and Na_c promotes Li-activated K influx. Cellular K inhibits Li-activated K influx, indicating that the interaction of K_c with the internal aspect of the pump is qualitatively different from the interaction with either Na_c or Li_c. The Hill coefficients for Na-promoted and Li-promoted K influx are similar and are both greater than unity, indicating the same number of multiple intracellular sites per pump for the two cations. The stoichiometry of coupling between efflux and K influx is also similar for Na and Li, and is close to 3 Na or 3 Li to 2 K. The Li-activated K influx appears to be independent of the residual Na which remains in cells prepared in Na-free solutions.     

Catalytic activity of an isolated domain of Na,K-ATPase expressed in Escherichia coli.

Fusion proteins of glutathione-S-transferase and fragments from the large cytoplasmic domain of the sheep Na,K-ATPase alpha1-subunit were expressed in Escherichia coli. The Na,K-ATPase sequences begin at Ala345 and terminate at either Arg600 (DP600f), Thr610 (DP610f), Gly731 (DP731f), or Glu779 (DP779f). After affinity purification on glutathione-Sepharose, the fusion proteins were labeled with [alpha-32P]-2-N3-ATP, and incorporation of the radiolabel into the fusion proteins was measured by scintillation counting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kd values of 220-290 microM for 2-N3-ATP binding to the fusion proteins were obtained from the photolabeling experiments. Approximately 1 mol of 2-N3-ATP was calculated to be incorporated per mole of fusion protein after correction for photochemical incorporation efficiency. Labeling of all of the fusion proteins by 25 microM 2-N3-ATP was reduced in the presence of MgATP, Na2ATP, MgCl2, 2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and p-nitrophenylphosphate, and Ki values of 2-11 mM for Na2ATP, 0.2-5 mM for MgCl2, 0.1-5 mM for MgATP, and 20-300 microM for p-nitrophenylphosphate were calculated for these ligands. All of the fusion proteins catalyze the hydrolysis of p-nitrophenylphosphate. The reaction requires MgCl2 and is inhibited by inorganic phosphate, which is similar to the hydrolysis of p-nitrophenylphosphate by native Na,K-ATPase. Based on these observations, it appears that the soluble fragments from the large cytoplasmic domain of Na,K-ATPase expressed in bacterial cells are folded in an E2-like conformation and are likely to retain much of the native structure.     

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21‰ salinity), as well as potential routes of Na⁺ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase α-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na⁺ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.     

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).