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.     

Direct measurement of the membrane potential of ehrlich ascites tumor cells: Lack of effect of valinomycin and ouabain

The Journal of Membrane Biology - 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...     

Movement of thallous ion across the ascites cell membrane

Summary The movement of thallous ion (Tl⁺ across the ascites cell membrane has been characterized. Analogous to previous findings for⁸⁶Rb⁺ (used as a tracer for K⁺),²⁰⁴Tl⁺-influx could be resolved into three components: a ouabain-inhibitable pump flux, a passive flux, and a furosemide- or NO ₃ ^– -sensitive exchange flux. Although Tl⁺ moved approximately nine times faster across the membrane than K⁺, the pump/leak ratio was equal for the two ions. This suggests that the pump- and leak-pathways share a common rate-limiting step. The exchange mechanism was shown to provide close coupling between the Tl⁺- and K⁺-gradients.     

Inorganic phosphate in Ehrlich ascites tumor cells and its distribution across the cell membrane

A regulatory function of the cell membrane in controlling the cytoplasmic level of Pi has been proposed, and in Ehrlich ascites tumor cells an active influx of primary phosphate has been reported in the literature. In the present study, Ehrlich cells were incubated at 1.5--50 mM extracellular Pi at pH 7.4 (Pi mainly secondary phosphate) and at pH 6.0 (mainly primary phosphate), and the measured cell Pi was compared with the value expected from a passive distribution of Pi. At a low extracellular Pi concentration the cell Pi was 3--6 mumol/g or even more. It is suggested that a major part of this cell Pi can be accounted for by enzymic release of Pi during the sampling procedure. If this interpretation is correct, the present results show that both ionic species of Pi are in electrochemical equilibrium across the cell membrane at steady state. Moreover, in vivo the concentration of free Pi in the cytosol will presumably be maintained at a steady-state level of about 0.4 mM, one order of magnitude below the directly measured values. This implies that the ratio [ATP]/[ADP][Pi] which is important in the regulation of energy metabolism, is higher than reported in the literature.     

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 'in situ' mitochondrial membrane potential in Ehrlich ascites tumor cells during aerobic glycolysis

(1) A method is presented for continuous and simultaneous monitoring of the 'in situ' mitochondrial membrane potential (delta psi m) and respiration rate of Ehrlich ascites tumor cells. The method involves permeabilization of the plasma membrane, achieved by treatment with low digitonin concentration, and the use of a TPP+ selective electrode attached to an oxygraph vessel. Binding of the probe inside the cells was analyzed assuming a proportional relationship between the amount of bound TPP+ and the free concentration of the lipophilic cation. (2) Evidence is reported that the addition of glucose to digitonin-permeabilized Ehrlich ascites tumor cells causes a decrease of mitochondrial membrane potential that coincided with a transient enhancement of the respiration rate and remained unchanged during the subsequent Crabtree effect. We have characterized the effect of glucose on delta psi m by determining its dependent on the glycolytic pathway and its sensitivity towards oligomycin. The mutual relationships between glucose and ADP effects on the mitochondrial membrane potential were also studied. A plausible mechanism underlying the depolarization of mitochondrial membrane induced by glucose is presented.     

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% of the total fux. 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% 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 α-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.     

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.     

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.     

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.     

Specific drug sensitive transport pathways for chloride and potassium ions in steady-state ehrlich mouse ascites tumor cells

A major aim of this investigation was to determine whether, in steady-state ascites cells, Cl^? transport can be partitioned into a furosemide-sensitive cotransport with K⁺ and a separate 4,4′-isothiocyanostilbene-2,2′-disulfonic acid (DIDS) sensitive self-exchange. Both Cl^? and K⁺ fluxes were studied. The furosemide- and Cl^? sensitive K⁺ fluxes were equivalent, both in normal ionic media and when the external K⁺ concentration, [K⁺]_o, was varied from 4 to 30 mM. The stoichiometry of the furosemide-sensitive Cl^? and K⁺ fluxes was 2 Cl^?: 1 K⁺ at 0.1 and 0.5 mM drug levels but increased to 3 Cl^? : 1 K⁺ at 1.0 mM furosemide. DIDS at 0.1 mM had no effect on the K⁺ exchange rate but inhibited Cl^? exchange by $\text{39\% ± 2}$ (S.E.). The effects of DIDS and 0.5 mM furosemide on Cl^? transport were additive but 1.0 mM furosemide and DIDS had overlapping inhibitory actions. Thus furosemide acts on components of K⁺ and Cl^? transport which are linked to each other, but the drug also inhibits an additional DIDS-sensitive Cl^? pathway, when present at higher concentrations. The dependence of the furosemide-sensitive K⁺ and Cl^? transport on [K⁺]_o was also studied; both fluxes fell as the [K⁺]_o increased. The latter results recall those in an earlier study by Hempling (Hempling, H.G. (1962) J. Cell. Comp. Physiol. 60, 181–198).     

Temporary suppression of the flash-induced electrical potential across the thylakoid membrane upon energization

Energization of the chloroplast thylakoid membrane causes a temporary decrease in the amplitude of the flash-induced transmembrane electrical potential as monitored by the micro-electrode technique and by the electrochromic absorbance band shift at 518 nm in chloroplasts of Peperomia metallica. This energization-dependent decrease of the flash-induced potential has a relaxation time of recovery in the dark of about 23±4 s. The phenomenon can neither be explained by a decrease of the intrinsic efficiency of photosystem I and II (PSI and PSII) nor by a partial closure of reaction centers of PSI and PSII. This leads us to propose that the energization-dependent decrease of the amplitude of the flash-induced electrical potential is caused by either the formation of a fraction of PSI and/or PSII reaction centers with fast charge recombination or by an increase of the membrane capacitance. The dark recovery after energization of the amplitude of the transmembrane electrical potential and that of non-photochemical fluorescence quenching were found to be comparable, which suggests a common cause for both phenomena.     

Accumulation of thallous ions (Tl+) as a measure of the electrical potential difference across the cytoplasmic membrane of bacteria.

The accumulation of thallous ions (204Tl+) by intact bacteria was investigated. I conclude that Tl+ is a permeant cation, and that it therefore accumulates in response to the electrical potential difference (delta psi) across the cytoplasmic membrane (interior negative). A comparison with other methods shows that the distribution ratio of 204Tl+ serves as a reasonably satisfactory method for measuring the membrane potential of Streptococcus faecalis. Glycolyzing cells of this organism develop membrane potentials of up to 180 mV. Preliminary experiments with Escherichia coli, especially those with a mutant defective in the proton-translocating ATPase, indicate that the Tl+ distribution also serves as a measure of the membrane potential in this organism. The particular advantage of Tl+ over other indicators of the membrane potential is that the cells need not be pretreated in any way. By use of the Tl+ distribution, it was calculated that respiring cells of E. coli develop a membrane potential of 160 mV with D-lactate and 180 mV with glucose as a substrate, respectively.     

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.