Activation of Na+-K+-adenosine triphosphatase by spermine.

Spermine activated Na⁺-K⁺-ATPase when the concentrations of K⁺ and ATP were low, whereas it inhibited K⁺-dependent and ouabain-inhibitable monophosphatase. The activating effect of sperimine was not due to the substitution for K⁺ or Na⁺. Excess K⁺ inhibited Na⁺-K⁺-ATPase partially, and reduced the spermine activation. When 1 mM ATP was used, spermine at higher concentrations inhibited Na⁺-K⁺-ATPase, and did not activate at all. It is suggested that the K⁺-sites essential to Na⁺-K⁺-ATPase and the K⁺-phosphatase co-exist at different places of the enzyme.     

Identification of a third isoform of Na+, K+-ATPase activity in rat brain synaptosomes

³H-ouabain binding and ouabain-inhibitable ⁸⁶Rb⁺ (K⁺) uptake were investigated as a means to identify a third isoform of Na⁺, K⁺-ATPase in crude synaptosome preparations. The specific binding of low concentrations (10 nM and 1 uM) of ³H-ouabain, in crude synaptosome preparations, was markedly inhibited by K⁺ (0.5–5 mM). Accordingly, ⁸⁶Rb⁺ (K⁺) uptake, in the presence of 5 mM K⁺ was not sensitive to inhibition by low concentrations (10⁻¹¹–10⁻⁷ M) of ouabain. Higher concentrations (10⁻⁶–10^−2.6 M) of ouabain resulted in a biphasic inhibition of K⁺ uptake, which distinguished the activities of the presumed alpha 2 and alpha 1 isozymes of Na⁺, K⁺-ATPase. Reduction of K⁺ (1.25 mM and 0.5 mM) in the incubation, resulted in the observation of a third component of ouabain- sensitive K⁺ uptake. This Na⁺, K⁺-ATPase activity, which was defined, pharmacologically, as very sensitive (VS) to ouabain, exhibited IC₅₀s of 3.6 nM and 92 nM at 1.25 mM K⁺ and 0.5 mM K⁺, respectively. Inhibition of ouabain binding and VS-dependent K⁺ uptake, at a high, physiological cocentration (5 mM) of K⁺, suggests that VS may be an inactive isoform of brain Na⁺, K⁺-ATPase under resting conditions.     

Na+, K+-ATPase in HeLa cells after prolonged growth in low K+ or ouabain

Effects of long-term, subtotal inhibition of Na⁺-K⁺ transport, either by growth of cells in sublethal concentrations of ouabain or in low-K⁺ medium, are described for HeLa cells. After prolonged growth in 2 × 10^?8 M ouabain, the total number of ouabain molecules bound per cell increases by as much as a factor of three, mostly due to internalization of the drug. There is only about a 20% increase in ouabain-binding sites on the plasma membrane, representing amodest induction of Na⁺, K⁺-ATPase. In contrast, after long-term growth in low K⁺ there can be a twofold or greater increase in ouabain binding per cell, and in this case the additional sites are located in the plasma membrane. The increase is reversible. To assess the corresponding transport changes, we have separately estimated the contributions of increased intracellular [Na⁺] and of transport capacity (number of transport sites) to transport regulation. During both induction and reversal, short-term regulation is achieved primarily by changes in [Na⁺]_i. More slowly, long-term regulation is achieved by changes in the number of functional transporters in the plasma membrane as assessed by ouabain binding, V_max for transport, and specific phosphorylation. Parallel exposure of cryptic Na⁺, K⁺-ATPase activity with sodium dodecyl sulfate in the plasma membranes of both induced and control cells showed that the induction cannot be accounted for by an exposure of preexisting Na⁺, K⁺-ATPase in the plasma membrane. Analysis of the kinetics of reversal indicates that it may be due to a post-translational event.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Cytosolic sodium regulation in mouse cortical astrocytes and its dependence on potassium and bicarbonate

Sodium plays a major role in different astrocytic functions, including maintenance of ion homeostasis and uptake of neurotransmitters and metabolites, which are mediated by different Na⁺-coupled transporters. In the current study, the role of an electrogenic sodium-bicarbonate cotransporter (NBCe1), a sodium-potassium-chloride transporter 1 (NKCC1) and sodium-potassium ATPase (Na⁺-K⁺-ATPase) for the maintenance of [Na⁺]_i was investigated in cultured astrocytes of wild-type (WT) and of NBCe1-deficient (NBCe1-KO) mice using the Na⁺-sensitive dye, asante sodium green-2. Our results suggest that cytosolic Na⁺ was higher in the presence of CO₂/HCO₃⁻ (15 mM) than CO₂/HCO₃⁻-free, HEPES-buffered solution in WT, but not in NBCe1-KO astrocytes (12 mM). Surprisingly, there was a strong dependence of cytosolic [Na⁺] on the extracellular [HCO₃⁻] attributable to NBCe1 activity. Pharmacological blockage of NKCC1 with bumetanide led to a robust drop in cytosolic Na⁺ in both WT and NBCe1-KO astrocytes by up to 6 mM. There was a strong dependence of the cytosolic [Na⁺] on the extracellular [K⁺]. Inhibition of the Na⁺-K⁺-ATPase led to larger increase in cytosolic Na⁺, both in the absence of K⁺ as compared with the presence of ouabain and in NBCe1-KO astrocytes as compared with WT astrocytes. Our results show that cytosolic Na⁺ in mouse cortical astrocytes can vary considerably and depends greatly on the concentrations of HCO₃⁻ and K⁺, attributable to the activity of the Na⁺-K⁺-ATPase, of NBCe1 and NKCC1.     

Effects of alloxan-diabetes on the sodium-potassium adenosine triphosphatase enzyme system in dog hearts

The effects of alloxan-diabetes on the partial reaction of (Na⁺+K⁺)-ATPase, K⁺-activated para-nitrophenylphosphatase, and on ouabain binding were studied in isolated adult dog heart myocytes. The Km of K⁺-activated para-nitrophenylphosphatase for K⁺ activation was increased from 2.5 to 7.7 mM with no change in Vmax. The Scatchard plots for ouabain binding between control and diabetic animals were indistinguishable. These results indicate that in acute diabetes induced by alloxan, the number of Na⁺-K⁺ pumping sites in the heart is not altered but the affinity of the system for K⁺ is decreased. It is suggested that the decrease in K⁺ affinity of the (Na⁺+K⁺)-ATPase enzyme system is at least in part responsible for the altered K⁺ homeostasis in the diabetic state.     

Effect of external ATP on the plasma membrane permeability and (Na++K+)-ATPase activity of mouse neuroblastoma cells

1. Addition of 3.5 mM ATP to mouse neuroblastoma Neuro-2A cells results in a selective enhancement of the plasma membrane permeability for Na⁺ relative to K⁺, as measured by cation flux measurements and electro-physiological techniques. 2. Addition of 3.5 mM ATP to Neuro-2A cells results in a 70% stimulation of the rate of active K⁺ -uptake by these cells, partly because of the enhanced plasma membrane permeability for Na⁺. Under these conditions the pumping activity of the Neuro-2A (Na⁺+K⁺)-ATPase is optimally stimulated with respect to its various substrate ions. 3. External ATP significantly enhances the affinity of the Neuro-2A (Na⁺+K⁺)-ATPase for ouabain, as measured by direct [³H]ouabain-binding studies and by inhibition studies of active K⁺ uptake. In the presence of 3.5 mM ATP and the absence of external K⁺ both techniques indicate an apparent dissociation constant for ouabain of 2·10^?6 M. Neuro-2A cells contain (3.5±0.7)·10⁵ ouabain-binding sites per cell, giving rise to an optimal pumping activity of (1.7±0.4)·10^?20 mol K⁺/min per copy of (Na⁺+K⁺)-ATPase at room temperature.     

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Estimation of ouabian specifically bound to dog renal (Na+ + K+)-ATPase in vivo

It is not known whether ouabain injected into the kidney in vivo is bound exclusively to the (Na⁺ + K⁺)-ATPase and whether the reduction of sodium pumping capacity is large enough to account for the reduction in sodium reabsorption. In the present study on dogs the total amount of parenchymal ouabain was therefore estimated and the specific renal binding compared to the reduction in (Na⁺ + K⁺)-ATPase activity. Ouabain, 120 nmol/kg body weight, was injected into the renal artery in vivo reducing the (Na⁺ + K⁺)-ATPase activity by 3lmost 80%. After nephrectomy, tissue ouabain could be quantified by radioimmunoassay after heating the homogenate to 70°C for 30 min; negligible amounts were detectable without heating. No correlation between ouabain binding and tissue volume, protein content, DNA content or Mg²⁺-ATPase content could be found when comparing the following four fractions of the kidney: outer cortex, inner cortex, outer medulla and papilla. For the whole kidney, mean parenchymal tissue concentration of ouabain equalled 0.58 ± 0.03 μmol/100 g wet tissue. Only 21.3 ± 1.2% of the ouabain was confined to the outer medulla corresponding to 54 ± 4 nmol giving a tissue concentration of 1.08 ± 0.05 μmol/100 g wet tissue. The renal ouabain concentrations were highly correlated to the reduction in (Na⁺ + K⁺)-ATPase activity, giving a ratio between the reduction in hydrolysis rate and bound ouabain (turnover number) of 6105 min^?1 which is close to the value of 7180 min^?1 found by in vitro Scatchard analysis. No ouabain seems to be bound to other tissue components than the (Na⁺ + K⁺)-ATPase and the present method is therefore a simple way of measuring the number of inhibited (Na⁺ + K⁺)-ATPase molecules after in vivo injection of ouabain.     

Characterization of the stimulation of neuronal Na+, K+-ATPase activity by low concentrations of ouabain

Low concentrations (< 10^?7 M) of ouabain stimulate the activity of Na⁺, K⁺-ATPase in whole homogenates of rat brain. The magnitude of this stimulation varies from 5 to 70%. The concentrations of ouabain which induces maximal stimulation is also highly variable and ranges between 10^?9 to 10^?7 M. The ouabain stimulation disappears following 1:50 dilution and 2 h preincubation or freezing and thawing of the membranes or their treatment with deoxycholate. “Aging” of a preparation of ATPase also results in loss of its ability to be stimulated by ouabain but ouabain inhibition is preserved. No stimulation of enzyme activity by ouabain is observed in rat brain microsomal fraction. The β-adrenergic blocker propranolol does not inhibit the ouabain induced stimulation of ATPase activity. It is suggested that the stimulation of Na⁺, K⁺-ATPase activity by low concentrations of cardiac glycosides if a result of either the displacement of an endogenous ouabain-like compound from the enzyme or an indirect effect by changing membrane surrounding environment of the Na⁺, K⁺-ATPase.     

Insensitivity of lepidopteran tissues to ouabain: Physiological mechanisms for protection from cardiac glycosides

Neuronal tissues from Manduca sexta, the tobacco hornworm, Hyalophora cecropia, the silkmoth and Danaus plexippus, the Monarch Butterfly, contain Na⁺K⁺-ATPase which is sensitive to cardiac glycoside (ouabain). The K_m for K⁺ stimulation of Na⁺K⁺-ATPase in M. sexta and D. plexippus is 2.2 mM and for Na⁺ stimulation in D. plexippus, 6.0 mM. In vitro ouabain concentrations of 1.0 × 10^?5 M and 5.0 × 10^?5 M in the presence of 7.5 mM K⁺ inhibited Na⁺K⁺-ATPase activity in H. cecropia and M. sexta by 50% respectively. Na⁺K⁺-ATPase from D. plexippus was approximately 300 times less sensitive. High concentrations (10^?3 M in haemolymph) of ouabain had no effect on M. sexta in vivo. This is largely explained by haemolymph K⁺ (>; 30 mM) antagonizing the binding of ouabain to Na⁺K⁺-ATPase. As demonstrated in vitro, 30 mM K⁺ totally protects Na⁺K⁺-ATPase from inhibition by 7.5 × 10^?3 M ouabain in D. plexippus and protects the enzyme by 65% in M. sexta. At least part of the physiological burden incurred in utilization of cardiac glycoside ingestion and storage for protection from predation, however, is probably related to the toxic effects of cardiac glycosides on neuronal Na⁺K⁺-ATPase.     

K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

Effects of nucleotides and Na + on ouabain activation of the phosphatase associated with Na + , K + -ATPase

Ouabain activation of the phosphatase associated with Na⁺,K⁺-ATPase is a time-dependent process which is stimulated by ATP and other nucleotides. Further stimulation by Na⁺ is observed under certain conditions. The stimulatory effect of ATP was found to be due to an increase in the affinity of the enzyme for ouabain. The time required for maximal ouabain activation to be achieved was decreased by ATP and further decreased by ATP + Na⁺.These conditions for maximal activation by ouabain are similar to those required for maximal ouabain binding and suggest that the same ouabain site is responsible for activation of Mg²⁺-dependent phosphatase and for inhibition of Na⁺,K⁺-ATPase and K⁺-phosphatase.     

Exchange between inorganic phosphate and adenosine triphosphate in (Na+,K+)-ATPase

(Na⁺,K⁺)-ATPase is able to catalyze a continuous ATP?P_i exchange in the presence of Na⁺ and in the absence of a transmembrane ionic gradient. At pH 7.6 the Na⁺ concentration required for half-maximal activity is 85 mM and at pH 5.1 it is 340 mM. In the presence of optimal Na⁺ concentration, the rate of exchange is maximal at pH 6.0 and varies with ADP and P_i concentration in the assay medium. ATP?P_i exchange is inhibited by K⁺ and by ouabain.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

Inhibition of Na+, K+-ATPase activates swelling-induced taurine efflux in a human neuroblastoma cell line

The Na⁺ pump (Na⁺, K⁺-ATPase) has been implicated in the regulation of many cellular functions, including cell volume regulation. The effects of inhibiting Na⁺ pump activity on cell volume and taurine efflux were evaluated in the human neuroblastoma cell line CHP-100. Cell volume changes monitored with the Coulter Multisizer technique and confocal microscopy showed that neuroblastoma cells exposed to ouabain swelled by 22 ± 4% (n = 5). The rapid cell swelling was followed by regulatory volume decrease (RVD). In cells treated with ouabain, ¹⁴C-taurine efflux increased by 183 ± 11% compared with controls. However, cells exposed simultaneously to ouabain and hypoosmotic solution resulted in a ¹⁴C-taurine efflux of 207 ± 18%. Western blot and immunofluorescence microscopy with specific monoclonal antibodies for the catalytic α isoforms of Na⁺, K⁺-ATPase demonstrated high levels of the ubiquitously expressed α1 and the neuronal-specific α3. Ouabain-binding data showed that CHP-100 cells express ∼3 × 10⁵ pump units/cell. The present data indicate that efflux of taurine may be involved during volume recovery subsequent to blockade of Na⁺, K⁺-ATPase in CHP-100 cells. J. Cell. Physiol. 174:145–153, 1998. © 1998 Wiley-Liss, Inc.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

Crosstalk between Na+,K+-ATPase and a volume-regulated anion channel in membrane microdomains of human cancer cells

Low concentrations of cardiac glycosides including ouabain, digoxin, and digitoxin block cancer cell growth without affecting Na⁺,K⁺-ATPase activity, but the mechanism underlying this anti-cancer effect is not fully understood. Volume-regulated anion channel (VRAC) plays an important role in cell death signaling pathway in addition to its fundamental role in the cell volume maintenance. Here, we report cardiac glycosides-induced signaling pathway mediated by the crosstalk between Na⁺,K⁺-ATPase and VRAC in human cancer cells. Submicromolar concentrations of ouabain enhanced VRAC currents concomitantly with a deceleration of cancer cell proliferation. The effects of ouabain were abrogated by a specific inhibitor of VRAC (DCPIB) and knockdown of an essential component of VRAC (LRRC8A), and they were also attenuated by the disruption of membrane microdomains or the inhibition of NADPH oxidase. Digoxin and digitoxin also showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges, and these effects were blocked by DCPIB. In membrane microdomains of cancer cells, LRRC8A was found to be co-immunoprecipitated with Na⁺,K⁺-ATPase α1-isoform. These ouabain-induced effects were not observed in non-cancer cells. Therefore, cardiac glycosides were considered to interact with Na⁺,K⁺-ATPase to stimulate the production of reactive oxygen species, and they also apparently activated VRAC within membrane microdomains, thus producing anti-proliferative effects.