Absence of effect of aldosterone on sodium efflux catalyzed by the human erythrocyte Na+, K+-ATPase in vitro

The effect of physiological and pharmacological concentrations of aldosterone on Na+ efflux catalyzed by the human erythrocyte Na+,K+-ATPase in vitro were studied. Aldosterone had no significant effect on ouabain-sensitive Na+ efflux from fresh erythrocytes. In addition, aldosterone did not alter Na+ transport activity of stimulated Na+,K+-ATPase of Na+ loaded erythrocytes. Finally, Na+ efflux from Na+ loaded erythrocytes was not changed by preincubation of the cells with aldosterone. It is concluded that aldosterone in vitro does not modify pump activity of the human erythrocyte Na+, K+-ATPase.     

The effect of membrane-bound calcium on the activity of adenosine triphosphatase from erythrocytes and erythrocyte permeability for monovalent cations]

The activities of Ca2+, Mg2+-ATPase and Na+, K+-ATPase and the permeability of reconstituted human erythrocytes for Na and K ions were measured, using Ca2+-EGTA, Ca2+ATP and Ca2+-sodium citrate buffers. It was found that the increase in the Ca2+/chelate ratio caused stimulation of Ca2+, Mg2+- and Na+, K+-Atpases and an increase in the rate constants of ouabain--dependent 42K+ influx and 22Na+ efflux from the erythrocytes. The use of the Ca2+-sodium citrate system as a calcium buffer did not change the parameters of the functional state of erythrocyte membranes. The data obtained are discussed in terms of a possible role of calcium ions, which are bound to the inner surface of the erythrocyte membrane, in the regulation of the systems of active and passive transport of cations.     

Transport of monovalent cations in erythrocytes after low temperature preservation and treatment with cryoprotective agents

The freezing-thawing of erythrocytes under protection of 1,2-propane diol and glycerol was studied for its effect on the intracellular content of K+,Na+ and state of their passive and active transport. The low-temperature conservation of cells under the effect of penetrating cryoprotectors does not change essentially the content of univalent cations. A significant increase in the Na+ transport is observed with application of the cryoprotective medium on the base of 1,2-propane diol.     

Effect of gradients of monovalent cations on active transport of Ca2+ in the sarcoplasmic reticulum and proteoliposomes

Artificially generated K+ gradient from the sarcoplasmic reticulum vesicles enhances the ATP-dependent Ca2+ transport. The effect is not specific for K+, and is observed when K+ is replaced by Na+ or choline. Dissipation of the K+, Na+, choline gradient does not influence the ATP-dependent Ca2+ transport in proteoliposomes from asolectin and purified Ca2+-ATPase. The K gradient in the presence of valinomycin stimulates the ATP-dependent Ca2+ transport in proteoliposomes.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.     

Kinetic properties of Na+, K+ activated, Mg2+ -dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis

The comparative analysis of the kinetic properties of ouabain-sensitive Na+, K+ -ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+ -ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+ -ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+ -ATPase affinity to ATP. However, Mg2+ -binding center of Na+, K+ -ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+ -ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+ -ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.     

Ion metabolism in malaria-infected erythrocytes

Malaria parasites of the genus Plasmodium spend much of their asexual life cycle inside the erythrocytes of their vertebrate hosts. Parasites presumably have to exploit metabolic and transport mechanisms to adapt themselves to the host erythrocyte's physicochemical environment. This review surveys the metabolism and transport of Ca2+, alkali cations, and H+ in malaria-infected erythrocytes. The Ca2+ content of Plasmodium-infected erythrocytes increases as the parasite matures. An increase in the influx of extracellular Ca2+ into infected erythrocytes is evident at later stages of parasite development. In infected erythrocytes, Ca2+ is almost exclusively localized in the parasite compartment and changes but little in the cytosol of the host cell. The importance of Ca2+ in supporting the growth of intraerythrocytic parasites and the invasion of erythrocytes by the merozoite has been assessed by depletion of extracellular Ca2+ with chelators, or by disturbance of the metabolism and transport of Ca2+ with a variety of Ca2+ modulators. Membranes of malaria-infected erythrocytes change their permeability to alkali cations. Hence, levels of K+ decrease and levels of Na+ increase in the cytosol of infected erythrocytes. Intraerythrocytic parasites maintain a high K+, low Na+ state, suggesting a mechanism for transporting K+ inward and Na+ outward against concentration gradients of the alkali cations across the parasite plasma membrane and/or the parasitophorous vacuole membrane (PVM). Concomitantly, P. falciparum can grow in Na(+)-enriched human erythrocytes. Experimental evidence suggests that Plasmodium possesses in its plasma membrane a proton pump which is very sensitive to orthovanadate, carbonylcyanide m-chlorophenylhydrazone, a protonophore, and dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, but is only slightly sensitive to inhibitors of bacterial and mitochondrial respiration, such as antimycin A, CN-, or N3-, and ouabain, a Na+, K(+)-ATPase inhibitor. By operating this proton pump, parasites extrude H+ and thus generate an electrochemical gradient of protons (an internal negative membrane potential and a concentration gradient of protons) across the parasite plasma membrane. The electrochemical gradient apparently drives inward movement of Ca2+ and, possibly, glucose from the cytosol of infected erythrocytes. Little is known about the transport properties of the PVM. Recent sequence studies suggest that Plasmodium contains a cation-transporting ATPase which exhibits a high homology to the Ca2(+)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Volume-dependent regulation of ion carriers in human and rat erythrocytes: role of cytoskeleton and protein phosphorylation

This study examines the effect of heat-induced cytoskeleton transitions and phosphoprotein phosphatase inhibitors on the activity of shrinkage-induced Na+, K+, 2Cl- cotransport and Na+/H+ exchange in rat erythrocytes and swelling-induced K+, Cl- cotransport in human and rat blood cells. Preincubation of human and rat erythrocytes at 49 degrees C drastically activated K+, Cl- cotransport and completely (rat) or partly (human) abolished its volume-dependent regulation. The same procedure did not affect basal activity of Na+, K+, 2Cl- cotransport but completely abolished its activation by shrinkage thus suggesting the involvement of a thermosensitive element of cytoskeleton network in the volume-dependent regulation of cotransporters. Both the shrinkage- and electrochemical proton gradient-induced Na+/H+ exchange was inhibited by the heat treatment to the same extent (50-70%), thus indicating the different signaling pathways involved in the activation of Na+, K+, 2Cl- cotransport and Na+/H+ exchange by cell shrinkage. This suggestion is in accordance with data on the different kinetics of volume-dependent activation and inactivation of these carriers as well as on their sensitivity to medium osmolality. Both swelling- and heat-induced increments of K+, Cl- cotransport activity were diminished by inhibitors of phosphoprotein phosphatases (okadaic acid and calyculin). In rat erythrocytes these compounds potentiate shrinkage-induced Na+/H+ exchange. On the contrary, neither basal nor shrinkage-induced Na+, K+, 2Cl- cotransport was affected by these compounds. Our results indicate a key role of cytoskeleton network in volume-dependent activation of K+, Cl- and Na+, K+, 2Cl- cotransport and the involvement of protein phosphorylation-dephosphorylation cycle in regulation of the activity of K+, Cl- cotransport and Na+/H+ exchange.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).     

Affinity chromatography for human Na+, K+-ATPase inhibitors in plasma and urine

Semi-purified dog kidney Na+,K+-ATPase cross-linked with ovalbumin was used in batch-wise affinity chromatography for the detection of endogenous Na+,K+-ATPase inhibitor in human plasma and urine. Ammonium acetate 1 M washed off the endogenous inhibitor from the immobilized enzyme. The inhibitory activity of the eluate from hypertensive plasma and urine was significantly higher (p less than 0.0025, n = 5 and p less than 0.005, n = 6 respectively) than that of normotensive. This latter was correlated with the ability of plasma from the same subjects to compete with ouabain binding to erythrocytes. Plasma and urine extracts inhibited the activity of Na+, K+-ATPase in a dose-dependent manner as ouabain does and were shown to contain 3 or 4 active compounds by high pressure liquid chromatography. The activity of some of these compounds was lost after peptidase treatment. These data support the heterogeneity of endogenous inhibitors of Na+,K+-ATPase activity in plasma and urine.     

Volume regulatory activity of the Ehrlich ascites tumor cell and its relationship to ion transport

The volume regulatory response of the Ehrlich ascites tumor was studied in KCl-depleted, Na+-enriched cells. Subsequent incubation in K+-containing NaCl medium results in the reaccumulation of K+, Cl-, water and the extrusion of Na+. The establishment of the physiological steady state is due primarily to the activity of 2 transport systems. One is the Na/K pump (KM for K+o = 3.5 mM; Jmax = 30.1 mEq/kg dry min), which in these experiments was coupled 1K+/1 Na+. The second is the Cl--dependent (Na+ + K+) cotransport system (KM for K+o = 6.8 mM; Jmax = 20.8 mEq/kg dry min) which mediates, in addition to net ion uptake in the ratio of 1K+:1Na+:2Cl-, the exchange of K+i for K+o. The net passive driving force on the cotransport system is initially inwardly directed but does not decrease to zero at the steady state. This raises the possibility of the involvement of an additional source of energy. Although cell volume increases concomitant with net ion uptake, this change does not appear to be a major factor regulating the activity of the cotransport system.     

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.     

Microsomal (Na- +K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors.

A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.     

Effect of membrane potential and internal pH on active sodium-potassium transport and on ATP content in high-potassium sheep erythrocytes.

Ouabain-sensitive Na+ and K+ fluxes and ATP content were determined in high potassium sheep erythrocytes at different values of membrane potential and internal pH. Membrane potential was adjusted by suspending erythrocytes in media containing different concentrations of MgCl2 and sucrose. Concomitantly either the external pH was changed sufficiently to maintain a constant internal pH or the external pH was kept constant with a resultant change of internal pH. The erythrocytes were preincubated before the flux experiment started in a medium which produced increased ATP content in order to avoid substrate limitation of the pump. It was found that an increased cellular pH reduced the rates of active transport of Na+ and K+ without significantly altering the ratio of pumped Na+/K+. This reduction was not due to limitation in the supply of ATP although ATP content decreased when internal pH increased. Changes of membrane potential in the range between -10 and +60 mV at constant internal pH did not affect the rates of active transport of Na+ or K+.     

Vanadium-induced Cl(-)-secretion in rabbit descending colon is mediated by prostaglandins.

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl- secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E2 from the tissue. Inhibitors of phospholipase A2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E2 and Cl- secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl- secretion. Although vanadate is known to inhibit Na+,K(+)-ATPase activity, no inhibition of active Na+ transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na+,K(+)-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl- secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E2.     

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.     

The energy transduction mechanism is different among P-type ion-transporting ATPases. Acetyl phosphate causes uncoupling between hydrolysis and ion transport in H+,K(+)-ATPase.

H+,K(+)-ATPase, Na+,K(+)-ATPase, and Ca(2+)-ATPase belong to the P-type ATPase group. Their molecular mechanisms of energy transduction have been thought to be similar until now. Ca(2+)-ATPase and Na+,K(+)-ATPase are phosphorylated from both ATP and acetyl phosphate (ACP) and dephosphorylated, resulting in active ion transport. However, we found that H+,K(+)-ATPase did not transport proton nor K+ when ACP was used as a substrate, resulting in uncoupling between energy and ion transport. ACP bound competitively to the ATP-binding site of H+,K(+)-ATPase. The hydrolysis of ACP by H+,K(+)-ATPase was stimulated by cytosolic K+, the half-maximal stimulating K+ concentration (K0.5) being 2.5 mM, whereas the hydrolysis of ATP was stimulated by luminal K+, the K0.5 being 0.2 mM. Furthermore, during the phosphorylation from ACP in the absence of K+, the fluorescence intensity of H+,K(+)-ATPase labeled with fluorescein isothiocyanate increased, but those of Na+,K(+)-ATPase and Ca(2+)-ATPase decreased. These results indicate that phosphorylated intermediates of H+,K(+)-ATPase formed from ACP are not rich in energy and that there is a striking difference(s) in the mechanism of energy transduction between H+,K(+)-ATPase and other cation-transporting ATPases.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.