Variant HeLa cells selected for their resistance to ouabain

The cardiac glycoside, ouabain, normally kills HeLa cells at concentrations of about 10⁻⁷ m or greater. By treating a population of HeLa cells with increasingly higher concentrations of the drug, a variant population was obtained of HeLa cells capable of growing in medium containing 10⁻⁴ M ouabain. Inhibition of volume regulation of cells subjected to hypotonic shock was used as a measure of inhibition of active transport of Na across the plasma membrane. In that way dose-response curves for the rapid effects of ouabain and other inhibitors of active Na transport were obtained with both the original, ouabain-sensitive (OS) and the variant, ouabain-resistant (OR) cells. Three other cardiac glycosides (digoxin, digitoxin and hellebrin) and two aglycones (digitoxigenin and strophanthidjn) were found to be equally as effective as ouabain in inhibiting volume regulation of the OS cells; the concentration which produced half-maximum inhibition, I(max/2), was about 6 × 10⁻⁷ M in each case. Similar inhibition of the OR population by ouabain was observed only when the concentration exceeded 10⁻⁴ m [I(max/2)∼2.5 × 10⁻⁴ m], and the other steroid compounds had no effect on the variant cells at the highest concentrations tested (∼2 × 10⁻⁵ m). OR and OS cells differed also in their sensitivities to the cardioactive erythrophleum alkaloid, coumingine; I(max/2) for OS and OR cells was 5 × 10⁻⁸ m and 6 × 10⁻⁷ M, respectively. These results, in addition to results of ouabain binding experiments and measurements of the rates of reversal of inhibition of volume regulation, suggest that a major reason for the differential sensitivities of the two phenotypes to these drugs is different affinities of their sodium pumps for inhibitors of active transport.     

Decreased ouabain binding in cystic fibrosis fibroblasts in potassium-free medium

We have previously demonstrated that cystic fibrosis (CF) cells show increased survival compared to normal cells when exposed to ouabain in medium lacking potassium. In this report, we show that the CF cells bind significantly less ouabain than the normal cells in potassium-deficient medium. Using age-matched normal and CF skin fibroblast strains, we show that (1) at ouabain concentrations <20 × 10⁻⁹ M binding for both normal and CF cells is linear with time for 1 h and reaches equilibrium after 4 h; (2) at ouabain concentrations between 2 and 20 × 10^-9 M, the initial rate of binding for CF cells is approx. 70% of the normal cells; (3) under equilibrium-binding conditions, Scatchard analysis reveals that three different CF strains have 12, 16, and 44% fewer ouabain-binding sites than their matched normal controls. In addition, studies in potassium-free medium of the inhibition of ⁸⁶Rb flux (a K⁺ analogue) into the cell by ouabain show no differences between CF and normal cells. We have also previously shown that in a low glucose, potassium-deficient medium the CF cells survived ouabain exposure no better than normal cells. In this report, equilibrium-binding studies with [³H]ouabain clearly show that CF cells bind less ouabain under these conditions than normal cells. These results indicate that ouabain resistance in CF cells is not solely a function of differences in ouabain binding. Furthermore, the differential ouabain killing may not be due to ion transport differences, but rather to as yet unknown mechanisms. CF cells thus appear to be unlike previously characterized ouabain-resistant mutants.     

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.     

Growth and DNA synthesis in embryonic chick heart cells, in vivo and in vitro

On the basis of cell shape, refractility under phase optics, spontaneous beating and nucleolar number, two cell types have been distinguished in embryonic heart cell cultures: M cells (myoblastlike) and F cells (fibroblast-like). However, by different criteria, more than two cell types have been found in the heart in vivo. In the present study, heart cell types are redefined by properties which can be used for identifying cells both in vitro and in vivo. Trypsin-dissociated cells from 7-day chick hearts were cultured for 24 h. PAS staining indicated that all M (thick, refractile) cells contain glycogen (gly⁺), while most F (spread) cells do not (gly⁻). Under the electronmicroscope, only gly⁺ cells contain myofibrils; most of these cells beat spontaneously in culture. Gly⁻ cells do not beat. Gly⁺ cells with myofibrils as well as gly⁻ cells are found in the inoculum and in the heart in vivo. The ultrastructure of gly⁺ cells in vitro and of muscle cells in the myocardium in vivo is similar. Therefore, it is concluded that gly⁺ cells in culture are derived from the myocardium (muscle) of the heart. Gly⁻ cells are apparently derived from the epicardium, the endocardium and the endothelial lining of blood vessels.     

Ion transport by ouabain resistant and sensitive ehrlich ascites carcinoma cells

Cell division, net Na⁺-K⁺ and amino-acid transport of cultured Ehrlich ascites is reversibly inhibited by Ouabain at a final concentration of 1 × 10^–3M. A line of Ehrlich ascites cells resistant to the growth inhibiting effects of Ouabain has been developed. These cells behave similarly to Ouabain-sensitive cells in the following respects doubling time, S phase time, chromosome number, cell surface charge density, rate of incorporation of C¹⁴ Uridine and ³H-Thymidine, sensitivity to Digoxin and Digitoxin, steady state Na⁺, K⁺ levels and rate of loss of K⁺ and gain of Na⁺ in cold. Ouabain resistant cells differ from sensitive cells only with respect to the effect of ouabain on active Na⁺, K⁺ transport. Although Ouabain inhibits active Na⁺, K⁺ transport in sensitive cells it has no significant effect in resistant cells.     

Adaptation of mouse leukemic cells (L5178Y) to anisotonic media : II. Cell growth

Mouse lymphoma cells (L5178Y) exposed to hypertonic media for 1 h behave as osmometers, but in hypotonic media, after initial swelling, they shrink back to normal volume and maintain it for long periods of time. The lower limit of osmolarity at which this “volume adaptation” will occur lies between 140 and 185 mosM. The “volume adaptation” is associated with a loss of cellular K⁺ probably due to a transient increase in K⁺ permeability and to loss of associated anions and osmotically obliged water. Partial dissipation of the large gradient of K⁺ between cells and medium by pre-exposure to ouabain or to K⁺-free medium results in a diminished capacity to adapt. After the shrinking phase is completed, a new steady state is established with a reduced cellular K⁺ content, normal Na⁺, normal K⁺-permeability, and a reduced activity of the Na⁺ − K⁺ transport system. When adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K⁺ content, presumably due to the return to normal activity of the Na⁺ − K⁺ transport system.     

Interactions of cardiac glycosides with cells and membranes Properties and structural aspects of two receptor sites for ouabain in erythrocytes

The existence of two types of binding sites for ouabain in human erythrocyte membranes is described. Receptor sites designated as ‘type I’, which may be identical to the K⁺-insensitive sites of intact cells, were detected at concentrations of ouabain as low as 10⁻⁷ M. The ‘type II’ receptor sites require the inclusion of Mg²⁺ + P_i to form complexes with ouabain; they may be identical to the K⁺-sensitive sites of intact cells. These sites were saturated at approx. 5 · 10⁻⁷ M ouabain but could not be detected at higher concentrations. The range of ouabain concentrations at which ‘type I’ receptors start to predominate (i.e. 5 · 10⁻⁸–5 · 10⁻⁷ M) was termed ‘critical digitalis concentrations’. The process of binding reached equilibrium within 1 and 4 h for ‘type I’ and ‘type II’ sites, respectively. The dissociation constant for ‘type II’ receptor-ouabain complexes was 7.6 · 10⁻⁹ M.Under similar experimental conditions, rat erythrocyte membranes exhibited only non-saturable sites.Alterations in the proportions of the two types of receptors were demonstrated by preincubation of the membranes, in the presence or absence of Mg²⁺ + P_i, prior to the addition of ouabain. In the first case, ‘type II receptor-ouabain’ complexes were stabilized at about 50% of the untreated membranes and ‘type I-ouabain’ complexes slowly approached equilibrium over a period of 24 h. In the latter instance, ‘type I’ receptors were not detected, and only ‘type II-ouabain’ complexes prevailed.     

The effect of Na+-K+ ATPase inhibition by ouabain on histamine release from human cutaneous mast cells

Background: There are controversial reports on the effect of sodium-potassium adenosine triphosphatase (Na⁺-K⁺ ATPase) inhibition on mast cell mediator release. Some of them have indicated that ouabain (strophanthin G), a specific Na⁺-K⁺ ATPase inhibitor, inhibited the release, whereas the others have shown that ouabain had no effect or even had a stimulatory effect on the mediator secretion. Most of these studies have utilized animal-derived mast cells. The aim of this study was to determine the effect of Na⁺-K⁺ ATPase inhibition on human skin mast cells. Methods: Unpurified and purified mast cells were obtained from newborn foreskins and stimulated by calcium ionophore A23187 (1 μM) for 30 min following a 1 hr incubation with various concentrations (10⁻⁴ to 10⁻⁸ M) of ouabain. Histamine release was assayed by enzyme-linked immunosorbent assay (ELISA). Results: The results indicated that ouabain had no significant effect on the non-immunologic histamine release from human skin mast cells, in vitro. Conclusions: Na⁺-K⁺ ATPase inhibition by ouabain had no significant effect on the non-immunologic histamine release from human cutaneous mast cells and suggested differences between human and animal mast cells.     

Protective effect of vitamin E on reduction in colony formation of cultured human cells by bisulfite

Bisulfite had a severe cytotoxic effect on HeLa S-3 cells at a concentration of 10⁻⁴ M, causing complete inhibition of colony formation. A concentration of 10⁻M bisulfite had a moderate cytotoxic effect on the cells, reducing their colony-forming activity to one-third of that of untreated control cultures. Addition of vitamin E at a concentration of 10⁻⁷ M restored the colony-forming activity of 10⁻⁵ M bisulfite-treated cells to 71% of that of control cultures. Addition of 10⁻⁶ M vitamin E resulted in almost complete recovery of the colony-forming activity of bisulfite-treated cells to that of the control cultures. The counteraction of vitamin E to the cytotoxic effect of bisulfite on human cells was discussed.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

Interactions of cardiac glycosides with cultured cardiac cells: II. Biochemical and electron microscopic studies on the effects of ouabain on muscle and non-muscle cells

Electron microscopic and biochemical studies revealed a salient difference in the response to toxic doses of ouabain by cultured cardiac muscle and non-muscle cells from neonatal rats. Progressive cellular injury in myocytes incubated with 1 · 10^?4–1 · 10^?3 M ouabain ultimately leads to swelling and necrosis. The morphological damage in myocytes was accompanied by a drastic decrease in ¹⁴CO₂ formation from ¹⁴C-labeled stearate or acetate but not glucose. Neither morphological nor biochemical impairments were observed in non-muscle cells. The interaction between ouabain and the cultured cells, using therapeutic doses of ouabain (i.e., <1 · 10^?7 M), was characterized. Two binding sites were described in both classes of cells, one site is a saturable K⁺-sensitive site whereas the other is non-saturable and K⁺-insensitive. The complexes formed between the sarcolemma receptor(s) and ouabain, at low concentrations of the drug (e.g., 7.52 · 10^?9 M), had K_d values of 8.9 · 10^?8 and 2.3 · 10^?8 M for muscle and non-muscle cells, respectively. The formation and dissociation of the complexes were affected by temperature and potassium ions.     

Periodate-induced lymphocyte transformation : II. Character of response and comparison with phytohemagglutinin and pokeweed mitogen stimulation

Sodium metaperiodate is mitogenic for human peripheral lymphocytes. Evidence of stimulation can be detected as increased thymidine incorporation at 72 h after only 10 sec of exposure to the IO₄⁻. The degree of response varies with lymphocytes from different donors, but maximum stimulation for the healthy donors studied was obtained at concentrations of IO₄⁻ between 10⁻³ M and 4 × 10⁻³ M. Concentrations of 8 × 10⁻³ M and above are non-stimulatory and toxic. Exposures to optimum concentrations for 1 h or longer result in essentially no stimulation and inincreased cell death. However, a significant response to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) remains. The kinetics of response over a 4 day culture period are similar for IO₄⁻, PHA and PWM. The morphology of the blast cells and the degree of response suggest that the IO₄⁻ responsive lymphocyte population may be more closely related to the PWM stimulated cells than the PHA responsive lymphocytes.     

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

The mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e. a particular mode of death occurring in cells lacking cell-to-extracellular matrix interactions. We tested this hypothesis. Six hour incubation of MDCK cells with ouabain, marinobufagenin or K⁺-free medium almost completely blocked Na⁺,K⁺-ATPase, increased Na_i⁺ content by ∼10-fold and suppressed cell attachment to regular-plastic-plates by up to 5-fold. In contrast, the death of attached cells was observed after 24-h incubation with ouabain but not in the presence of marinobufagenin or K⁺-free medium. Cells treated with ouabain and undergoing anoikis on ultra-low attachment plates exhibited different cell volume behaviour, i.e. swelling and shrinkage, respectively. The pan-caspase inhibitor z-VAD.fmk and the protein kinase C activator PMA rescued MDCK cells from anoikis but did not influence the survival of ouabain-treated cells, whereas medium acidification from pH 7.2 to 6.7 almost completely abolished the cytotoxic action of ouabain, but did not significantly affect anoikis. Our results show that the Na _i⁺,K_i⁺-independent mode of MDCK cell death evoked by ouabain is not mediated by anoikis.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

The Death of Ouabain-Treated Renal Epithelial C11-MDCK Cells is Not Mediated by Swelling-Induced Plasma Membrane Rupture

This study examined the role of cell volume modulation in plasma membrane rupture and death documented in ouabain-treated renal epithelial cells. Long-term exposure to ouabain caused massive death of C11-MDCK (Madin-Darby canine kidney) epithelial cells, documented by their detachment, chromatin cleavage and complete loss of lactate dehydrogenase (LDH), but did not affect the survival of vascular smooth muscle cells (VSMCs) from the rat aorta. Unlike the distinct impact on cell survival, 2-h exposure to ouabain led to sharp elevation of the [Na⁺]_i/[K⁺]_i ratio in both cell types. A similar increment of Na_i⁺ content was evoked by sustained inhibition of Na⁺,K⁺-ATPase in K⁺-free medium. However, in contrast to ouabain, C11-MDCK cells survived perfectly during 24-h exposure to K⁺-free medium. At 3 h, the volume of ouabain-treated C11-MDCK cells and VSMCs, measured by the recently developed dual-image surface reconstruction technique, was increased by 16 and 12%, respectively, whereas 5–10 min before the detachment of ouabain-treated C11-MDCK cells, their volume was augmented by ~30–40%. To examine the role of modest swelling in the plasma membrane rupture of ouabain-treated cells, we compared actions of hypotonic medium on volume and LDH release. We observed that LDH release from hyposmotically swollen C11-MDCK cells was triggered when their volume was increased by approximately fivefold. Thus, our results showed that the rupture of plasma membranes in ouabain-treated C11-MDCK cells was not directly caused by cell volume modulation evoked by Na⁺,K⁺-ATPase inhibition and inversion of the [Na⁺]_i/[K⁺]_i ratio.     

Potentiation by ouabain of bradykinin-induced catecholamine secretion and calcium influx into cultured bovine adrenal chromaffin cells: Evidence for involvement of Na+ influx associated with the bradykinin B2-receptor

The effect of bradykinin (BK), in the presence of ouabain, an inhibitor of Na⁺-K⁺ ATPase, on catecholamine (CA) secretion was studied in cultured bovine adrenal chromaffin cells, to determine whether Na⁺, as well as Ca²⁺, is involved in BK-receptor mediated CA secretion. BK (10⁻⁸–10⁻⁵M)-induced CA secretion was markedly potentiated by addition of ouabain (10⁻⁵M), was blocked by a BK-B₂ receptor antagonist, and was decreased in Ca²⁺-free medium. BK-induced increase in ⁴⁵Ca²⁺ influx was also potentiated by addition of ouabain. The cultured cells were first incubated with BK for 30 min in Ca²⁺-free medium in the presence or absence of ouabain and then kstimulated for 15 min with Ca²⁺-medium without BK or ouabain. Prior stimulation of the cells, BK induced ²²Na⁺ influx and increased Ca²⁺-induced CA secretion and these stimulatory effects of BK were potentiated by added ouabain. When the cells were stimulated with BK and ouabain in Na⁺-free sucrose medium, the Ca²⁺-induced CA secretion was greatly reduced. These results indicated that activation of the BK-B2 receptor and inhibition of the Na⁺ pump both increase the intracellular Na⁺ level, resulting in increase in Ca²⁺ influx and CA secretion.     

The Role of Na+/K+-ATPase during Chick Skeletal Myogenesis

The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na⁺/K⁺-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na⁺/K⁺-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM) and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na⁺/K⁺-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na⁺/K⁺-ATPase.     

Cardiotonic steroid-resistant α1-Na+,K+-ATPase rescues renal epithelial cells from the cytotoxic action of ouabain: evidence for a Na i+ ,K i+ -independent mechanism

Mechanisms underlying the tissue-specific impact of cardiotonic steroids (CTS) on cell survival and death remain poorly understood. This study examines the role of Na⁺,K⁺-ATPase α subunits in death of Madin-Darby canine kidney (MDCK) cells evoked by 24-h exposure to ouabain. MDCK cells expressing a variant of the α1 isoform, CTS-sensitive α1S, were stably transfected with a cDNA encoding CTS-resistant α1R-Na⁺,K⁺-ATPase, whose expression was confirmed by RT–PCR. In mock-transfected and α1R-cells, maximal inhibition of ⁸⁶Rb influx was observed at 10 and 1000 μM ouabain, respectively, thus confirming high abundance of α1R-Na⁺,K⁺-ATPase in these cells. Six-hour treatment of α1R-cells with 1000 μM ouabain led to the same elevation of the [Na⁺]_i/[K⁺]_i ratio that was detected in mock-transfected cells treated with 3 μM ouabain. However, in contrast to the massive death of mock-transfected cells exposed to 3 μM ouabain, α1R-cells survived after 24-h incubation with 1000 μM ouabain. Inversion of the [Na⁺]_i/[K⁺]_i ratio evoked by Na⁺,K⁺-ATPase inhibition in K⁺-free medium did not affect survival of α1R-cells but increased their sensitivity to ouabain. Our results show that the α1R subunit rescues MDCK cells from the cytotoxic action of CTS independently of inhibition of Na⁺,K⁺-ATPase-mediated Na⁺ and K⁺ fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio.     

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.