Effect of ouabain on macromolecular synthesis during the cell cycle in mitogen-stimulated human lymphocytes

Ouabain inhibited in a concentration-dependent and completely reversible way, the synthesis of DNA, RNA and protein in phytohemagglutinin and concanavalin A-stimulated human lymphocytes without affecting the uptake of nucleosides and amino acids into the cells. On the other hand, ouabain even at very high concentrations was unable to interfere with the binding of [³H]concanavalin A. No correlation was found between the inhibition by ouabain of macromolecular synthesis and that of K⁺ transport. The inhibitor effect of ouabain on the stimulation of macromolecular synthesis could be partially reversed by higher concentrations of K⁺, due to the direct inhibition of ouabain binding. Ouabain added to the cultures at different stages of cell growth suppressed the incorporation of thymidine to various extents. Both ouabain sensitive stages fell in a period preceding the onset of mitosis and were characterized by very active thymidine incorporation. Lymphocytes were most sensitive to ouabain within the S phase. The results suggest that ouabain interferes with mitogen-triggered membrane-associated events, other than K⁺ transport, controlling mitosis at distinct phases of the cell cycle.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Effects of ouabain and isoproterenol on potassium influx in the turkey erythrocyte: Quantitative relation to ligand binding and cyclic AMP generation

Studies have been carried out in the turkey erythrocyte to examine: (1) the influence of external K⁺ concentration on both [³H]ouabain binding and the sensitivity of potassium influx to inhibition by ouabain and (2) the quantitative relation between β-adrenergic receptor site occupancy, agonist-directed cyclic AMP generation and potassium influx rate. Both [³H]ouabain binding and the ability of ouabain to inhibit potassium influx are markedly reduced at increasing external K⁺ concentrations, and at each K⁺ concentration the concentrations of ouabain required for half-maximal binding to the erythrocyte membrane and for half-maximal inhibition of potassium influx are identical. Both basal and isoproterenol-stimulated potassium influx rise with increasing external K⁺ concentrations. In contrast to basal potassium influx, which is 50–70% inhibitable by ouabain, the isoproterenol-stimulated component of potassium influx is entirely insensitive to ouabain. At all concentrations of K⁺, inhibition of basal potassium influx by ouabain is linear with ouabain binding, indicating that the rate of transport per unoccupied ouabain binding site is unaffected by simultaneous occupancy of other sites by ouabain. Similarly, the rate of isoproterenol-stimulated cyclic AMP synthesis is directly proportional to β-adrenergic receptor occupancy over the entire concentration-response relationship for isoproterenol, showing that at all levels of occupancy β-adrenergic receptor sites function independently of each other.Analysis of the relation of catecholamine-dependent potassium transport to the number of β-adrenergic receptor sites occupied indicates an extremely sensitive physiological system, in which 50%-maximal stimulation of potassium transport is achieved at less than 3% receptor occupancy, corresponding to fewer than ten occupied receptors per cell.     

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K⁺ ion levels in the culture medium. Lowering the extracellular K⁺ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K⁺ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K⁺ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K⁺ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, $\text{Na}\text{K}$ ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the $\text{Na}\text{K}$ ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K⁺ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.     

Adaptation of potassium metabolism and restoration of mitosis during prolonged treatment of mouse lymphoblasts with ouabain

The effects of ouabain on the growth of murine lymphoblasts in vitro have been studied. Exposure of cells to ouabain (0.1 mM) initially inhibited ⁸⁶Rb⁺ uptake rate, reduced the intracellular potassium concentration, and decreased population growth rates. Continued exposure to the same ouabain concentration resulted in an increase of ⁸⁶Rb⁺ uptake rate, intracellular potassium content and population growth rates to control values (adaptation). When treated cells were resuspended in medium free of ouabain after 12 to 15 hours of ouabain treatment, ⁸⁶Rb⁺ uptake rates and intracellular potassium levels exceeded those of untreated cells. Adaptation was inhibited by cycloheximide (3 μg/ml) and by actinomycin D (0.05 μg/ml). Kinetic analysis of transport suggested that while the total capacity of the Na⁺, K⁺ transport system increased, the affinity for both the cation (⁸⁶Rb⁺) and ouabain decreased.     

Characteristics of ouabain binding to isolated trout hepatocytes

Summary Na⁺, K⁺ exchanges were studied in isolated hepatocytes of the rainbow trout, Salmo gairdneri. Ouabain at 10^–4 M produced maximal inhibition (95%) of K⁺ uptake and enhanced intracellular Na⁺ accumulation, showing that active fluxes account for a very large proportion of Na⁺ and K⁺ exchanges. Inhibition of the Na–K pump by ouabain was significant at low concentrations (10^–8 M). When external K⁺ concentration was reduced from 7 mM to 0.5 mM, half maximum inhibition (IC₅₀) of K⁺ uptake was obtained at a 22-fold lower concentration of ouabain confirming that ouabain and potassium compete at the same pump site. Time-course analysis of [³H]ouabain binding indicated a two-component kinetics: one component saturable and dependent on K⁺ concentration in the medium, the other linear and independent of external K⁺. The ouabain binding site number, determined by Scatchard plots, remained constant (ca. 2.5·10⁵ per cell) and independent of the external K⁺ concentration (7, 0.5 or 0 mM), while the dissociation constant (K_D) decreased from 4.2 M to 7.3 nM when K⁺ was removed from the Hank's medium. These ouabain binding sites are characterized by an exceptionally low turnover rate (400 min^–1), as estimated from ouabain-sensitive K⁺ flux, in comparison to those described in other cell types of higher vertebrates. At each external K⁺ concentration studied, the inhibition of K⁺ uptake and ouabain binding measured as a function of ouabain concentration indicated a strict correlation between the degree of K pump inhibition and the amount of bound glycoside.     

The early effects of ouabain on potassium metabolism and rate of proliferation of mouse lymphoblasts

Murine lymphoblasts grown in suspension culture in the presence of ouabain showed a dose dependent and sequential decrease in ⁸⁶Rb⁺ (K⁺ analogue) influx, cellular potassium content, and growth rate. An increase in eosin staining and a decrease in cell number was observed after two hours in the presence of 1 mM ouabain; 1 μM ouabain was without effect on any of the parameters measured. Ouabain inhibition was rapidly and completely reversible at concentrations that were not cytotoxic.     

Inhibition of lymphocyte activation by ouabain Interference with the early activation of membrane phospholipid metabolism

Activation of lymphocytes by antigens and mitogens can effectively be prevented by ouabain, a known inhibitor of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$. Recently it was shown that lowering of intracellular levels of monovalent cations is not involved in the inhibitory effect of ouabain. $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$ was found to be closely associated with acylCoA: lysophosphatidylcholine acyltransferase in the plasma membrane of lymphocytes. Both enzymes are activated as an immediate consequence of mitogen binding. Human peripheral lymphocytes were stimulated with concanavalin A. Ouabain suppressed the induction of RNA and DNA synthesis in a concentration-dependent way. Increase of RNA synthesis was suppressed only if the glycoside were added within the first hours of activation. If ouabain was added later, incorporation of uridine remained at the rate that was reached at the time of glycoside administration, pointing to an early event where ouabain may be operative. Ouabain, in a dose-dependent manner similar to that affecting RNA and DNA synthesis, inhibited the increase in the incorporation of oleate into phospholipids in stimulated lymphocytes, whereas the turnover of phospholipid fatty acids in resting lymphocytes was unaffected. Increasing extracellular K⁺ concentrations reversed the binding of ouabain to lymphocytes. Simultaneously, the inhibition of stimulated RNA synthesis was decreased and the inhibition of oleate incorporation was reversed. These results suggest that the suppression of lymphocyte activation by ouabain is due to the inhibition of membrane phospholipid metabolism mediated by the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-ATPase}$.     

Involvement of Na+, K+-ATPase inhibition in K562 cell differentiation induced by bufalin

Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na⁺, K⁺ -ATPase prepared from K562 cells in vitro. N⁺, K⁺ -ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of ³H-bufalin and ³H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of ³H-bufalin was considerably higher than ³H-ouabain. The saturated level of ³H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of ³H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N⁺, K⁺ -ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin. © 1994 Wiley-Liss, Inc.     

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.     

Ouabain Binding and Potassium Transport in Young and Old Populations of Human Red Cells

Human red blood cells were separated according to density by centrifugation through mixtures of phthalate esters. The densest 20% of the erythrocyte population (old cells) had reduced volume and water content compared to the lightest 20% of the cells (young cells). Corpuscular hemoglobin content was unchanged. Young cells had 50% more potassium (K⁺) than old cells, but their total intracellular concentration was only slightly higher; old cells had a small increase in sodium (Na⁺) concentration. Active K⁺ transport of young cells was 37% higher than that of old cells. [³H] + Ouabain binding revealed that this difference was the result of more K⁺ pump sites on young cells, which bound 530 ouabain molecules per cell at 100% K⁺ pump inhibition, as compared to 400 for old cells; unseparated cells bound 450-500 molecules. The relative rates of ouabain binding were identical for the two cell types. Old cells exhibited a greater passive permeability to K⁺, haying a rate coefficient for ouabain-insensitive K⁺ influx 1.8 times that of young cells. There is evidence to suggest that in the face of reduced pump activity this augmented K⁺ “leak” might enhance the osmotic stability of the old cells and function to lengthen their life span.     

An altered response of virally transformed 3T3 cells to ouabain

The effect of ouabain on K⁺ transport was examined in 3T3 and virally transformed 3T3 cells. A 10 min exposure to ouabain (10⁻³ M) produced approximately 40% inhibition of the unidirectional K⁺ influx in all cell lines. In 3T3 cells the response was not significantly altered by up to 70 min exposure to the drug. In contrast, the continued exposure of transformed cells to ouabain produced a time-dependent increase in the K⁺ influx. This increased influx was shown to be accompanied by an increase in the K⁺ efflux. The results suggest that, in transformed cells, ouabain produces both an inhibition of Na⁺-K⁺ exchange and a stimulation of K⁺-K⁺ exchange. The latter was shown to be more readily reversible than the former.     

Localization of sodium pump sites in frog urinary bladder

[³H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and authoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na⁺ transport was completely reversible in toad bladder whereas, in frog bladder, [³H] ouabain was tightly bound and Na⁺ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [³H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further by adjusting the concentration of cecrtain (Na⁺?K⁺)-ATPase ligands (K⁺, ATP) to levels known to reduce ouabain binding. High K⁺ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [³H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na⁺ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significatly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.     

Death of ouabain-treated renal epithelial cells: evidence for p38 MAPK-mediated Na i + /K i + -independent signaling

Recent studies demonstrate that cytotoxic actions of ouabain and other cardiotonic steroids (CTS) on renal epithelial cells (REC) are triggered by their interaction with the Na⁺,K⁺-ATPase α-subunit but not the result of inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. This study examined the role of mitogen-activated protein kinases (MAPK) in the death of ouabain-treated REC. Exposure of C7-MDCK cells that resembled principal cells from canine kidney to 3 μM ouabain led to phosphorylation of p38 without significant impact on phosphorylation of ERK and JNK MAPK. Maximal increment of p38 phosphorylation was observed at 4 h followed by cell death at 12 h of ouabain addition. In contrast to ouabain, neither cell death nor p38 MAPK phosphorylation were affected by elevation of the [Na⁺]_i/[K⁺]_i ratio triggered by Na⁺,K⁺-ATPase inhibition in K⁺-free medium. p38 phosphorylation was noted in all other cell types exhibiting death in the presence of ouabain, such as intercalated cells from canine kidney and human colon rectal carcinoma cells. We did not observe any action of ouabain on p38 phosphorylation in ouabain-resistant smooth muscle cells from rat aorta and endothelial cells from human umbilical vein. Both p38 phosphorylation and death of ouabain-treated C7-MDCK cells were suppressed by p38 inhibitor SB 202190 but were resistant to its inactive analogue SB 202474. Our results demonstrate that death of CTS-treated REC is triggered by Na_i⁺,K_i⁺—independent activation of p38 MAPK.     

Transport ATPase of erythrocyte membrane: sensitivities of Na plus, K, plus-ATPase and Kplus-phosphatase activities to ouabain.

Cardiac glycosides are inhibitors of Na⁺,K⁺-ATPase, and K⁺-phosphatase activities of the transport enzyme. Previous studies have shown that when the sensitivities of these two activities to ouabain are compared by the addition of varying concentrations of the drug to the assay media, the K⁺-phosphatase is significantly less sensitive than Na⁺,K⁺-ATPase. This work was done to seek an explanation for this phenomenon. 3-O-Methyl-fluorescein phosphate was used as substrate for the continuous fluorimetric assay of K⁺-phosphatase obtained from human red cells. When ouabain was added to the assay medium, a time-dependent inhibition of K⁺-phosphatase was observed. The rate of inhibition was also influenced by the order of additions of K⁺ and ouabain. In view of these results, several enzyme samples exposed to ouabain for varying lengths of time were prepared, and their Na⁺,K⁺-ATPase and K⁺-phosphatase activities were then determined. A good correlation between the extent of inhibition of the two activities was obtained. These results prove that the previously observed discrepancies between the sensitivities of Na⁺,K⁺-ATPase and K⁺-phosphatase to ouabain are due to the different kinetics of drug interaction with the enzyme under the different conditions of the two assays and that once a certain level of ouabain binding to the enzyme is achieved, both activities are equally inhibited.     

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.