Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

Density-related changes of potassium (86Rb) uptake by amphibian endothelial cells

Potassium influx has been investigated in XTH-2 cells, a line derived from tadpole heart endothelia. In this line, the density at which the cultures become confluent is clearly separated from the density at which growth arrest takes place. Density-related changes in K+ influx were monitored by determining the uptake of 86Rb into well adhering cells kept in culture medium. The main observations were 1) 86Rb uptake is highest in single cells, and on confluency it reaches a low level, which is kept constant at higher cell density regardless of whether the cultures are stationary or still in logarithmic growth phase; 2) the relative amount of 86Rb taken up via the Na+ -K+ -2Cl- cotransport pathway and via the Na+/K+ pump changes from low cell density to confluent cultures; 86Rb uptake of single cells is nearly insensitive to ouabain, a maximum of ouabain sensitivity is reached around confluency, whereas piretanide-sensitive 86Rb uptake is highest in single cells and seems to reach a minimum at the onset of confluency; 3) the variations in Na+/K+ pumping rate reflect neither differences in the amount of enzyme present nor changes in enzyme repartition between apical and basolateral plasma membranes; they seem to result from either "masking" or "unmasking" of the enzyme; 4) no alterations in K+ uptake occur that would be characteristic of the "stationary growth phase." The only changes that seem to be related to arrest of proliferation are concerned with the Na+/K+-ATPase, which achieves an extraordinary susceptibility to stimulation by monensin and exhibits an increase in PNPPase activity.     

Regulation of the number of Na+,K+-pump sites after mitogenic activation of lymphocytes

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.     

Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3.

Long term elevation of the intracellular Na+/K+ ratio inhibits macromolecule synthesis and proliferation in the majority of cell types studied so far, including vascular smooth muscle cells (VSMC). We report here that inhibition of the Na+,K+ pump in VSMC by ouabain or a 1-h preincubation in K+-depleted medium attenuated apoptosis triggered by serum withdrawal, staurosporine, or okadaic acid. In the absence of ouabain, both DNA degradation and Caspase-3 activation in VSMC undergoing apoptosis were insensitive to modification of the extracellular Na+/K+ ratio as well as to hyperosmotic cell shrinkage. In contrast, protection of VSMC from apoptosis by ouabain was abolished under equimolar substitution of Na+o with K+o, showing that the antiapoptotic action of Na+,K+ pump inhibition was caused by inversion of the intracellular Na+/K+ ratio. Unlike VSMC, the same level of increment of the [Na+]i/[K+]i ratio caused by a 2-h preincubation of Jurkat cells with ouabain did not affect chromatin cleavage and Caspase-3 activity triggered by treatment with Fas ligand, staurosporine, or hyperosmotic shrinkage. Thus, our results show for the first time that similar to cell proliferation, maintenance of a physiologically low intracellular Na+/K+ ratio is required for progression of VSMC apoptosis.     

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.     

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR- 106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3- h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)     

Energetic cooperation via ion-permeable junctions in mixed animal cell cultures

Low ouabain concentration (1 x 10(-6) M) is shown to decrease intracellular K+ (K+in) and to increase intracellular Na+ (Na+in) in human fibroblast cell cultures. The same ouabain concentration was without effect upon K+in ad Na+in in rodent cultures such as BHK-21, mouse fibroblasts and rat glyoma C6 cells. K+in and Na+in in the mixed cultures of human and BHK-21 fibroblasts or human and mouse fibroblasts were found to be resistant to 1 x 10(-6) M ouabain whereas that of the mixtures of human and rat glyoma C6 cells proved to be ouabain-sensitive. The gap-junction-mediated dye transfer was revealed between human and BHK-21 cells. Such an effect was very small in the human-C6 cell mixed culture. It is concluded that cells with active ion pumps can support the maintenance of K+ and Na+ gradients in cells with inactive pumps, provided that effective ion transport via gap junctions takes place.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

Anthroylouabain: a specific fluorescent probe for the cardiac glycoside receptor of the Na-K ATPase.

Anthroylouabain (AO) was synthesized by reaction of anthracene-9-carboxylic chloride with ouabain. Nuclear magnetic resonance spectroscopy of AO suggests that the anthracene is esterfied to the rhamnose in the glycoside. AO inhibits Na-K ATPase from human red cells, eel electroplax and rabbit and dog kidney with a KI less than 1muM. AO bound to rabbit or dog kidney Na-K ATPase shows enhanced fluorescence and characteristic spectral shifts. AO binding requires Mg and is optimum in the presence of Mg + Pi or MgATP + Na; ouabain prevents AO binding and fluorescence enhancement if added before AO or reverses it if added after AO is bound. Na inhibits AO binding in the presence of Mg + Pi and K inhibits it in the presence of MgATP + Na. AO binding and dissociation rate constants measured by fluorescence agree qualitatively with reported measurements for ouabain, using other methods, although AO shows faster kinetics than ouabain. Dissociation constants obtained from kinetic measurements are 1.5 X 10(-7) and 1.8 X 10(-7) M for the MgATP + Na complex and Mg + Pi complex, respectively. KD from fluorescence titrations is 2.3 X 10(-7) M for the latter. The enzyme has 2-2.5 nmol of AO binding sites/mg of protein. No differences in the fluorescence parameters of the Mg + Pi or MgATP + Na complexes were observed, suggesting that the same enzyme conformation binds AO under both ligand conditions. Comparison of the AO fluorescence parameters in the enzyme with those of model systems suggests that the binding site is hydrophobic and/or viscous and shielded from H2O. The results indicate that AO is a specific fluorescent probe of the cardiac glycoside receptor of the Na-K ATPase. Possible applications are discussed.     

Dome formation in the human colon carcinoma cell line Caco-2 in culture. Influence of ouabain and permeable supports

We studied formation of domes in cell monolayers of the human colon carcinoma cell line Caco-2 which has been shown to exhibit signs of enterocytic differentiation and transport properties. After a 24 hr incubation with 4 X 10(-8) M ouabain, the number of domes seen on Caco-2 cell monolayers grown on plastic dishes was not significantly altered. After a 90 min preincubation with ouabain, 86rubidium uptake by Caco-2 cells was inhibited by ouabain, indicating that the cells have an ouabain-sensitive Na+, K+-ATPase, while dome formation was unaffected by ouabain. Domes were observed in Caco-2 cell monolayers grown on Nuclepore filters when the pore size was 0.015 micron but not when it was 0.030 micron. Our results suggest that dome formation in the Caco-2 cell line could be independent of Na+, K+-ATPase activity and might be due to accumulation of molecules having an effective hydrodynamic radius comprised between 0.015 and 0.030 micron.     

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Changes in intracellular K+ and Na+ ion concentrations during cell growth and differentiation

We compared intracellular K+ and Na+ ion concentrations during cell growth and differentiation of a mouse myeloid leukemia M1 cell line. Cells undergoing mitosis had higher K+ concentrations than quiescent cells. Treatment with a K+ channel blocker and furosemide enhanced cell growth and produced a slight increase in the intracellular K+ concentration. Treatment with reagents that reduced the intracellular K+ concentration stopped cell growth. Induction of differentiation in this cell line produced a decrease in the K+ concentration, which always was accompanied by an increase in the Na+ concentration. Treatment with ouabain, which decreased the intracellular K+ concentration, did not, however, induce differentiation in the M1 cell line. The data suggest that cell growth and differentiation in the M1 cells are accompanied by changes in the intracellular K+ and Na+ concentrations but that the changes in the contents of these monovalent cations do not necessarily induce differentiation in this cell line.     

Sodium and rubidium uptake in cells transformed by Rous sarcoma virus

Rates of uptake and intracellular concentrations of monovalent cations were measured in virus-transformed and nontransformed chick embryo (CE) cells. Uptake of 22Na+ into cells transformed by the BH strain of Rous sarcoma virus (RSV-BH) (CE-BH) was about double the rate of uptake into CE cells, or cells transformed by the Schmidt-Ruppin strain (RSV-SR): CE-SR. Likewise, the rate of efflux of 22Na+ was greater in CE-BH cells than in CE or CE-SR cells. The greater permeability of CE-BH cells to Na+ was apparent in higher intracellular Na+ concentrations. Experiments with cells exhibiting temperature-dependent transformation showed that new RNA and protein synthesis was a requirement for the acquisition of increased Na+ permeability, suggesting that the change is an indirect effect of the virus-coded transformation-inducing protein. Rates of 86Rb+ uptake, used as a measure of K+ influx, were indistinguishable in CE, CE-BH, and CE-SR cells. Also, equilibrium intracellular levels of 86Rb+ were similar in transformed and nontransformed cells, as were observed concentrations of K+. Also, no differences in ATPase activity, as indicated by ouabain binding or temperature sensitivity, were observed. We conclude that monovalent cations play no direct role in RSV-induced transformation, although the higher levels of Na+ in CE-BH cells may be responsible for other distinguishing biochemical features of these cells.     

Direct measurement of the membrane potential of Ehrlich ascites tumor cells: lack of effect of valinomycin and ouabain.

The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+, K+ and Cl- showed that valinomycin induced net losses of K+ and Cl- and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.     

Regulation of cellular growth by sodium pump activity.

Cellular growth has been found to be directly related to the amount of sodium pumping activity in mouse lymphoblasts (L5178-Y) cultured in varying concentrations of the cardiac glycoside, ouabain. No short-term adaptation (within one generation) occured; i.e., neither growth rate nor (Na+ + K+)-ATPase activity increased in cells cultured for 1-2 days in ouabain. Growth inhibition commenced after two hours, occurring concomitantly with decreased 3H-leucine incorporation into protein. The time course of this inhibition of protein synthesis, measured by leucine incorporation was similar to, but slightly slower than the time course or the dissipation of the sodium gradient. On the other hand, 3H-thymidine incorporation is unaffected by ouabain treatment over the same period. The uptake of 3H-alanine, a neutral amino acid thought to be transported via a Na+-dependent carri-r, was depressed concurrently with the sodium gradient dissipation. It is suggested, therefore, that ouabain inhibition of cellular growth results primarily from the dissipation of the sodium gradient leading to decreased Na+-dependent transport of amino acids (e.g., alanine) and, therefore, decreased protein synthesis, as observed by leucine incorporation. A sensitive and rapid method for determining ouabain inhibition of cell volume regulation is also described, which may prove potentially useful for assaying Na pump activity.     

Effect of acute serum depletion on Na+-K+ homeostasis in cultured human skin fibroblasts

In order to elucidate changes in cell transport behavior of cultured human skin fibroblasts in response to acute serum depletion, we performed uptake and washout of 22Na+ and 86Rb+ as well as measurements of the intracellular Na+ and K+ levels in the presence and absence of ouabain. Pronounced and lasting increase in cellular Na+ and decrease in K+ were observed after removal of fetal bovine serum (FBS) from the medium. The sum of the Na+ and K+ contents (nEq/10(5) cells) was lower in FBS-free medium (mean +/- SD; 17.3 +/- 2.2) than in FBS-containing medium (26.2 +/- 3.8; P less than .02). Simultaneously, a decrease in cellular water volume was detected in the FBS-free medium. The cation uptake and washout data suggest that FBS removal primarily renders the cells more permeable to Na+ and K+ with a secondary stimulation of the ouabain-sensitive Na+ extrusion mechanism. FBS at a concentration of 0.2% prevented approximately 50% of the maximal increase in the 86Rb+ washout rate constant associated with FBS depletion. Ouabain (2 microM) produced an increase in the 86Rb+ washout rate constant. This effect was substantially larger in cells subjected to medium without FBS (from 0.0303 to 0.2500 min-1) than in fibroblasts incubated in medium with FBS (from 0.0107 to 0.0487 min-1). The cellular K+ content was drastically reduced by ouabain to a level not different in medium with or without FBS (33.9 +/- 4.5 to 1.75 +/- 0.38 and 16.7 +/- 1.4 to 1.4 +/- 0.13 nEq/10(5) cells, respectively). The 22Na+ washout data exhibited a three-exponential pattern. Analytical solutions of the washout data by means of two models (serial and parallel) with three compartments showed that FBS depletion resulted in increase of the size of all three compartments. It is concluded that in cultured human skin fibroblasts, FBS is essential to the maintenance of a normal Na+ and K+ homeostasis. The removal of FBS results in dramatic permutation of this homeostasis that develops within minutes and lasts for hours.