Cation flux in the ehrlich ascites tumor cell. Evidence for Na+-for-Na+ and K+-for-K+ exchange diffusion.

In a previous study, evidence was presented for an external Na+-dependent, ouabain-insensitive component of Na+ efflux and an external K+-dependent component of K+ efflux in the Ehrlich ascites tumor cell. Evidence is now presented that these components are inhibited by the diuretic furosemide and that under conditions of normal extracellular Na+ and K+ they represent Na+-for-Na+ and K-+for-K+ exchange mechanisms. Using 86Rb to monitor K+ movements, furosemide is shown to inhibit an ouabain-insensitive component of Rb+ influx and a component of Rb+ efflux, both representing approx. 30 percent of the total flux. Inhibition of Rb+ efflux is greatly reduced by removal of extracellular K+. Furosemide does not alter steady-state levels of intracellular K+ and it does not prevent cells depleted of K+ by incubation in the cold from regaining K+ upon warming. Using 22Na to monitor Na+ movements, furosemide is shown to inhibit an ouabain-insensitive component of unidirectional Na+ efflux which represents approx. 22 percent of total Na+ efflux. Furosemide does not alter steady-state levels of intracellular Na+ and does not prevent removal of intracellular Na+ upon warming from cells loaded with Na+ by preincubation in the cold. The ability of furosemide to affect unidirectional Na+ and K+ fluxes but not net fluxes is consistent with the conclusion that these components of cation movement across the cell membrane represent one-for-one exchange mechanisms. Data are also presented which demonstrate that the uptake of alpha-aminoisobutyrate is not affected by furosemide. This indicates that these components of cation flux are not directly involved in the Na+-dependent amino acid transport system A.     

Phosphorus incorporation in the ehrlich ascites tumor cell

Data from isotopic uptake experiments were used to measure the kinetics of labelling of cellular phosphate, ATP and ADP in the Ehrlich ascites tumor cell. The results show that steady state phosphate exchange flux was 0.333 ± 0.052 (S.E.) μmoles per 10⁷ cells per hour at 37°, and that the specific activity of phosphate was the same as P_γ ATP. Metabolic inhibition reduced the phosphate flux by 30–50%. A model, based on oxidative phosphorylation and the adenylate kinase reaction is used to interpret the labelling sequence of P_β ATP and P_β ADP, and its dependence on P_γ ATP.     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

Measurement of the electrical potential difference across the membrane of the ehrlich mouse ascites tumor cell

The electrical potential difference (PD) across the membrane of the Ehrlich mouse ascites tumor cell has been measured with intracellular microelectrodes. The mean for 111 cells in control Ringer solution was ? 11.2 mV ± 0.29 (SE), interior negative. When sulfate replaced chloride in the external medium the PD fell to ? 2.8 mV if measured as soon as possible after mixing the cells with a sulfate medium, but when nitrate replaced chloride the PD fell only to ? 8.5 mV. Cells equilibrated in nitrate had the same PD as those in control Ringer. These results indicate that the PD is sensitive to changes in the external chloride concentration and that nitrate can substitute for chloride electrically. However, since the PD for chloride, based on the Nernst equation and calculated on the basis of 70% exchangeability of cell chloride, is three times greater than the measured PD, it is hypothesized that sodium contributes significantly to the membrane potential in addition to chloride. On the other hand, potassium does not influence the PD to any great extent.     

KCl loss and cell shrinkage in the ehrlich ascites tumor cell induced by hypotonic media, 2-deoxyglucose and propranolol

Ehrlich ascites tumor cells lose KCl and shrink after swelling in hypotonic media and in response to the addition of 2-deoxyglucose, propranolol, or the Ca²⁺ ionophore, A23187, plus Ca²⁺ in isotonic media. All of these treatments activate cell shrinkage via a pathway with the following characteristics: (1) the KCl loss responsible for cell shrinkage does not alter the membrane potential; (2) NO₃^? does not substitute for Cl^?; (3) the net KCl movements are not inhibited by quinine or DIDS; and (4) early in this study furosemide was effective in inhibiting cell shrinkage but this sensitivity was subsequently lost. This evidence suggests that the KCl loss in these cells occurs via a cotransport mechanism. In addition, hypotonic media and the other agents used here stimulate a Cl^? -Cl^? exchange, a net loss of K⁺ and a net gain of Na⁺ which are not responsible for cell shrinkage. The Ehrlich cell also appears to have a Ca²⁺-activated, quinine-sensitive K⁺ conductive pathway but this pathway is not part of the mechanism by which these cells regulate their volume following swelling or shrink in isotonic media in response to 2-deoxyglucose or propranolol. Shrinkage by the loss of K⁺ through the Ca²⁺ stimulated pathway appears to be limited by Cl^? conductive movements; for when NO₃^?, an anion demonstrated here to have a higher conductive movement than Cl^?, is substituted for Cl^?, the cells will shrink when the Ca²⁺-stimulated K⁺ pathway is activated.     

Immunoprophylaxis with attenuated ehrlich ascites tumor cells in admixture with BCG

Summary During propagation in tissue culture, the Ehrlich ascites carcinoma was found to lose some of its tumor-producing capacity (oncogenicity) when implanted IP or SC into CF-1 mice. On the other hand, attenuated cells retained their immunoprotective capacity; immunization of mice with a single dose (1×10⁴) of these cells induced a high degree of resistance against a challenge 1 month later with virulent Ehrlich cells maintained by IP transplantation. The admixture of BCG (1×10⁶ viable units) with attenuated cells further improved their immunogenicity. The immunogenicity of attenuated cells was almost completely abolished by gamma-irradiation (2,500 rads), but this property was significantly restored by the addition of BCG. Some evidence is presented that suggests that attenuated cells have a higher immunoprotective capacity than the corresponding virulent cells.     

The effect of various seleno-compounds on ehrlich ascites tumor cells

The effect of various concentrations and forms of selenium on in vitro viability of Ehrlich Ascites Tumor Cells (EATC) was investigated. Sodium selenite, selenium dioxide, seleno-dl-cystine, and seleno-dl-methionine, dramatically decreased EATC viability as measured by dye exclusion. Sodium selenate only marginally decreased EATC viability. Cell viabilities decreased with increasing selenium in the incubation media and as a function of time. Viabilities determined by dye exclusion did not correlate with the inhibition of tumor growth observed after treatment with selenium. Intraperitoneal injections of selenite in mice previously inoculated with EATC significantly inhibited tumor development. Delaying intraperitoneal injections of selenite to 5 and 7 days after inoculation of mice with EATC reduced the effectiveness of this nutrient on the inhibition of EATC growth. Incubation of EATC in vitro with supplemental selenium prior to injection of mice completely inhibited EATC development in vivo before any appreciable alteration in cell viability was observed.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Summary The intracellular pH (pH _i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H⁺ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH _i of cells placed in an acidic medium (pH _o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH _o is acutely reduced to 5.5, pH _i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H⁺. The unidirectional influx of H⁺ exhibits saturation kinetics with respect to extracellular [H⁺]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK _m is 0.74 ± 0.09 × 10^–6 m.Steady-state cells with pH _i above 6.8 continuously extrude H⁺ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH _i 6.3) when returned to pH _o 7.3 medium respond by transporting H⁺, resulting in a rapid rise in pH _i . The halftime for this process is 1.09 ± 0.22 min. The H⁺ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH _i to its steady-state value.     

Physical studies on glycoproteins from ascitic fluid of ehrlich ascites tumor

Three glycoproteins, designated as F, M and S glycoproteins were identified in the HClO₄-soluble fraction of ascitic fluid of Ehrlich ascites tumor by 8% polyacrylamide disc gel electrophoresis. They were separated and purified as described previously (Reznick, A.Z. and Winzler, R.J. (1973) Fed. Proc. 32, 368 and Reznick, A.Z., Allen, H.J. and Winzler, R.J. (1973) Anal. Biochem. 52, 395–401) and subjected to physical characterization. Several physical properties such as molecular weights, sedimentation and diffusion coefficients, partial specific volumes, Stoke's radii and frictional ratios were determined. The physical parameters of F and S glycoproteins resemble data that have been reported for orosomucoid and haptoglobin-like glycoproteins, respectively. Properties of M glycoprotein could not be associated with a known glycoprotei.     

The turnover of molecular species of phosphatidylinositol in ehrlich ascites tumor cells

It has been shown previously that ³²P_i is incorporated into phosphatidylinositol 30 times faster than into the other phospholipid classes in Ehrlich ascites tumor cells, whereas [1-¹⁴C]glycerol is incorporated at almost the same rate (Waku, K., Nakazawa, Y. and Mori W. (1976) J. Biochem. 79, 407–411). It was therefore suggested that there is a recirculating system (phosphatidylinositol → diacylglycerol → phosphatidic acid → CDPdiacylglycerol → phosphatidylinositol) of phosphatidylinositol in Ehrlich ascites tumor cells. In this work, ³²P_i or [1-³H]glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells from which the lipids were extracted after selected periods. Phosphatidylinositol was prepared and fractionated in the form of dimethylphosphatidic acid into six molecular species by AgNO₃-impregnated TLC. The specific radioactivities of the fractionated species were determined. ³²P_i was incorporated into diene molecular species and [1-³H]glycerol into monoene species with a higher rate than the other species and both precursors were incorporated into tetraene species rather slowly. ³²P/³H values appeared to be at almost the same for each molecular species, although monoene species showed slightly lower values. These results suggest that there could be a recirculating of the phosphorylinositol moiety in each of the molecular species of phosphatidylinositol.     

Na+/H+ exchange in ehrlich ascites tumor cells: Activation by cytoplasmic acidification and by treatment with cupric sulphate

Summary Exposure of Ehrlich cells to isotonic Na⁺-propionate medium induces a rapid cell swelling. This treatment is likely to impose an acid load on the cells. Cell swelling is absent in K⁺-propionate medium but may be induced by the ionophore nigericin, which mediates K⁺/H⁺ exchange. Cell swelling in Na⁺-propionate medium is blocked by amiloride, but an alternative pathway is introduced by addition of the ionophore monensin, which mediates Na⁺/H⁺ exchange. Consequently, swelling of Ehrlich cells in Na⁺-propionate medium is due to the operation of an amiloride-sensitive, Na⁺-specific mechanism. It is concluded that this mechanism is a Na⁺/H⁺ exchange system, activated by cytoplasmic acidification. We have previously demonstrated that the heavy metal salt CuSO₄ in micromolar concentrations inhibits regulatory volume decrease (RVD) of Ehrlich cells following hypotonic swelling. The present work shows that CuSO₄ inhibits RVD as a result of a net uptake of sodium, of which the major part is sensitive to amiloride. Measurements of intracellular pH show that CuSO₄ causes significant cytoplasmic alkalinization, which is abolished by amiloride. Concomitantly, CuSO₄ causes an amiloride-sensitive net proton efflux from the cells. The combined results confirm that a Na⁺/H⁺ exchange system exists in Ehrlich cells and demonstrate that the heavy metal salt CuSO₄ activates this Na⁺/H⁺ exchange system.     

Saturation behavior of ascites tumor cell chloride exchange in the presence of gluconate

Steady state Cl^? flux across the Ehrlich mouse ascites cell membrane was studied when gluconate replaced Cl^? in the external medium. Saturation behavior was observed; $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was 23.9 mM Cl^? and V was 758 μmol · g^?1 dry weight · h^?1. The cells lost K⁺, Cl^? and H₂O, consistent with relative impermeability to gluconate, and the Cl^? efflux rate coefficient was elevated. The results indicate that a major portion of Cl^? exchange occurs as a membrane transport process and suggest that the process is sensitive to intracellular Cl^? levels.