digitonin and Carcinoma--Ehrlich-Tumor

Effects of nitric oxide (NO) on oxygen uptake of Ehrlich ascites tumor cells (EATC) were examined in a study of the biological actions of NO on respiration and energy metabolism at the cellular level. Endogenous respiration of EATC was inhibited reversibly by NO in a dose dependent manner. Oxyhemoglobin, an NO trapping agent, restored the respiration promptly. The inhibitory action of NO also depended on oxygen-concentration, and the duration of suppression was prolonged remarkably at low oxygen tension. Similar inhibition was also observed in the presence of glucose. In this case, both lactate production and glucose consumption were promoted by NOC 18, an NO generating agent, and the activation was enhanced by lowering the oxygen-concentration. Furthermore, the membrane potential of EATC was depolarized transiently by adding NO, and the degree of depolarization was decreased in the presence of glucose. These results suggest that at physiologically low oxygen tension in ascites fluid, NO acts not only as a cytotoxic respiratory inhibitor but also as a regulatory factor in the energy metabolism of EATC.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Respiration; Digitonin; Dose-Response Relationship, Drug; Glucose; Glycolysis; Indicators and Reagents; Membrane Potentials; Mice; Mice, Inbred ICR; Nitric Oxide; Oxygen; Oxyhemoglobins; Tumor Cells, Cultured

Preincubation of Ehrlich ascites tumour cells with millimolar concentrations of pantothenic acid, pantothenol or pantethine, but not with homopantothenic acid, at 22 degrees C or 32 degrees C, but not at 0 degrees C, makes the plasma membrane more resistant to the damaging effect of submillimolar concentrations of digitonin. It is proposed that this increased resistance is due to the increased rate of cholesterol biosynthesis. In fact, incorporation of [14C]acetate into cholesterol is by 45% increased in the cells preincubated with pantothenic acid; this probably reflects elevation of the content of CoA in such cells [Slyshenkov, V.S., Rakowska, M., Moiseenok, A.G. & Wojtczak, L. (1995) Free Radical Biol. Med. 19, 767-772].

Topics: Acetic Acid; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Cholesterol; Digitonin; Female; Mice; Pantothenic Acid

The effect of Ca2+ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (delta psi) and alkalinization of the medium. This typical behaviour is drastically changed if Ca2+ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of delta psi, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 microM Ca2+ and a preincubation for 2 min with 0.4-1.0 microM Ca2+. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca2+ concentrations as low as 0.3 microM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca2+] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyz A. Evtodienko YuV. Duszyński J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148-1154; Czyz A. Teplova VV. Sabala P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539-544], the present results suggest that cytoplasmic Ca2+ plays a crucial role in the Crabtree effect.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Deoxyglucose; Digitonin; Glucose; Hydrogen-Ion Concentration; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Pyruvates; Pyruvic Acid; Succinates; Succinic Acid

Ascites tumour cells, cultivated in a serum replacement medium with low mitogenic activity supplemented with free cholesterol or cholesterol complexed to digitonine, were used to study protein kinase C (PKC) activation and its translocation from the cytosol to the membrane compartment. Activity of partially purified PKC was assessed by the Ca2+/phospholipid-dependent phosphorylation of histone as substrate. Staurosporine-sensitive PKC activity was increased by a factor of about three in the membrane compartment as the result of cholesterol supplementation with about 90% of total activity in the membrane, and the remaining 10% in the cytosolic fraction. A translocation of enzyme mass from the cytosol to the membrane compartment was not induced under these conditions, as documented by the enzyme-linked immuno adsorbent assay using a monoclonal anti-PKC antibody. In cells cultivated in the presence of the cholesterol-digitonide complex PKC activation was not observed, suggesting an inadequate utilization of cholesterol as membrane constituent.

Topics: Alkaloids; Animals; Biological Transport; Carcinoma, Ehrlich Tumor; Cell Membrane; Cholesterol; Cytosol; Digitonin; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Humans; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

We have previously observed that extracellular Mg2+ influences the phosphofructokinase (PFK) activity of intact Ehrlich Ascites tumour cells (EATC). In this study we have investigated the mechanism by which Mg2+ modulates this key glycolytic enzyme in EATC made permeable to the cation by either digitonin or dextran sulphate. Results showed that when Mg2+ is freely permeable to the cytosol, the in vivo PFK activity, calculated as FDP/G6P ratio, is not increased as it is in intact cells. We also observed that in permeabilized cells Mg2+ determines the increase of glucose 6 phosphate (G6P), fructose 1,6 bisphosphate (FDP) and lactate production. We hypothesize that extracellular Mg2+ regulates PFK and glycolysis in these neoplastic cells not by entering the cytosol but by a specific interaction with the plasma membrane.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Dextran Sulfate; Digitonin; Glucosephosphates; Glycolysis; Magnesium; Phosphofructokinase-1

Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Line; Cell Membrane Permeability; Digitonin; Electrodes; Energy Metabolism; Glucose; Intracellular Membranes; Membrane Potentials; Mice; Microscopy, Electron; Mitochondria; Onium Compounds; Organophosphorus Compounds

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Digitonin; DNA, Neoplasm; Ethidium; Flow Cytometry; Fluorescent Dyes; Glutamate Dehydrogenase; Mitochondria; Propidium; Rhodamine 123; Rhodamines; Staining and Labeling; Tumor Cells, Cultured

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.

Topics: Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Creatine Kinase; Digitonin; Female; Glycolysis; Indicators and Reagents; Mice; Mitochondria; Oxidation-Reduction; Phosphocreatine; Phosphorylation; Tumor Cells, Cultured

(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.

Topics: Adenosine Diphosphate; Aerobiosis; Animals; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Digitonin; Female; Glucose; Glycolysis; Intracellular Membranes; Membrane Potentials; Mice; Mitochondria; Onium Compounds; Organophosphorus Compounds; Oxygen Consumption

The intracellularly trappable fluorescent Ca2+ indicator quin-2 was used to measure free cytosolic Ca2+, [Ca2+]i, in the two highly dedifferentiated tumor cell lines, Ehrlich and Yoshida ascites carcinomas. It was found that these carcinoma cells can trap quin-2 similarly to normal cells, but [Ca2+]i was apparently significantly lower than in any normal cell tested previously with this method. By using a new lipid-soluble heavy metal chelator TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine), which crosses artificial and natural membranes, it was found that endogenous heavy metals are responsible for partially quenching quin-2 fluorescence trapped inside the cells. Although the quenching of intracellular quin-2 fluorescence is quantitatively more relevant in these ascites carcinomas, TPEN was effective also in normal cells like lymphocytes and granulocytes. Both in the normal and especially in the malignant cell lines [Ca2+]i can be grossly underestimated at low intracellular quin-2 concentrations. Endogenous heavy metal quenching is thus a potential source of artifact when [Ca2+]i is measured with quin-2. When corrected for quin-2 fluorescence quenching by intracellular heavy metals, [Ca2+]i and basic regulatory mechanisms of [Ca2+]i homeostasis in Ehrlich and Yoshida carcinomas are similar to those of nontransformed cells.

Topics: Aminoquinolines; Animals; Calcimycin; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Carcinoma, Ehrlich Tumor; Cell Line; Chelating Agents; Cytosol; Digitonin; Ethers; Ethylenediamines; Homeostasis; Humans; Ionomycin; Liposomes; Mice; Sarcoma, Yoshida

Ribosomal RNA (rRNA) synthesis in the intact Ehrlich ascite carcinoma cells is selectively inhibited by papaverin (ED50 = 0.01 mM), 2,4-dinitrophenol (DPN; ED50 = 5 microM), and actinomycin D (ED50 = 0.1 microgram/ml). The inhibition of rRNA synthesis is not connected with a direct action of these agents on the rRNA synthesis apparatus, and they had no effect on isolated cell nuclei. The rRNA synthesis in cells permeabilized with triton X-100 (0.05%) becomes insensible to the action of papaverine and DPN, but is inhibited by actinomycin D in low doses. In cells permeabilized with digitonin (0.01%) the rRNA synthesis shows no sensibility to the action of low doses of actinomycin D. The results suggest that the action of these agents on the rRNA synthesis may depend on cell integrity and on the permeabilization method employed.

Topics: 2,4-Dinitrophenol; Animals; Antimetabolites; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Dactinomycin; Digitonin; Dinitrophenols; Mice; Neoplasm Transplantation; Papaverine; Polyethylene Glycols; RNA, Neoplasm; RNA, Ribosomal

The digitonin method for the study of cellular compartmentation in mitochondrial and cytosolic fractions was applied to Ehrlich ascites tumor cells. The volume of mitochondrial and cytosolic water spaces are calculated to be 1.62 microliter/30 x 10(6) cells respectively, by the technique of 3H2O permeable and (14C)-sucrose impermeable spaces. The validity of the methods was tested by the distribution of cytosolic (lactate dehydrogenase) and mitochondrial (citrate synthase and glutamate dehydrogenase) marker enzymes. As occurs in normal hepatic cells, an asymmetric distribution of ATP and ADP was observed. The ATP/ADP ratio in the cytosolic fraction was 7 times higher than in the mitochondrial fraction.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Carcinoma, Ehrlich Tumor; Cell Compartmentation; Cell Fractionation; Cell Membrane Permeability; Cytosol; Digitonin; Intracellular Fluid; Mice; Mitochondria

A new, improved procedure for isolating mitochondria from ascites tumor cells is described. The unique feature of this technique is the use of digitonin to make the cells susceptible to disruption by Teflon pestle/glass vessel homogenization. The yield and respiratory control ratios of mitochondria isolated by this method from murine Ehrlich ascites tumor cells and rat AS30-D ascites hepatoma cells are significantly better than those obtained for mitochondria isolated by the commonly employed Nagarse method, which involves the use of proteolytic enzymes. Moreover, mitochondria isolated by this new procedure from three different lines of tumors exhibit respiratory control ratios with both adenosine diphosphate and a respiratory uncoupler comparable to those obtained with mitochondria present in situ within digitonin-permeabilized tumor cells.

Topics: Animals; Ascites; Carcinoma, Ehrlich Tumor; Cell Fractionation; Cell Membrane Permeability; Digitonin; Female; Liver Neoplasms, Experimental; Mice; Mice, Inbred C3H; Mitochondria; Neoplasms, Experimental; Oxygen Consumption; Rats; Rats, Inbred Strains

Topics: Adenine Nucleotides; Animals; Biological Transport, Active; Calcium; Carcinoma, Ehrlich Tumor; Digitonin; Kinetics; Mice; Mitochondria; Oligomycins

The membranolytic activity of 12 chosen detergents upon Ehrlich ascites tumour and Chinese hamster lung cells were studied with the fluorescein diacetate and the dye exclusion tests of cell viability. It was found that all these tests give reproducible and concordant results when non-ionic detergents are used but for ionic detergents only the fluorescein diacetate test can be applied. Membranolytic activity of the detergents appeared to be uncorrelated with their capacity of membrane solubilization. Digitonin and aescin, the horse-chestnut saponin, were found to be much more toxic than sodium dodecylsulphate or Triton X-100. When the detergents with aliphatic hydrophobic residues were compared, a cationic detergent cethylmethylammonium bromide (CTAB) was more toxic than non-ionic or anionic detergents. A hydrophobic part of the aescin molecule, that is its aglycone appeared responsible for the membranolytic activity of this saponin. When the lytic activity of the tested detergents for the two tested types of cells was compared it was found that the horse-chestnut saponin substances show different toxicity for various cells. It is suggested that in the future some of the detergents can be applied for detection and quantitative monitoring of species and tissue specific differences in plasma membrane properties of animal cells.

Topics: Animals; Anions; Ascitic Fluid; Carcinoma, Ehrlich Tumor; Cations; Cell Membrane; Cell Survival; Cricetinae; Detergents; Digitonin; Lung; Saponins

The Mg2+ precipitation procedure of R. D. Palmiter ((1974) Biochemistry 13, 3606) has been used for preparative scale isolation of polysomes from Ehrlich ascites mitochondria. Digitonin-washed metochondria used for isolating the polysomes contain no detectable reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and over 200-fold reduced hexokinase activity. The mitochondrial polysomes exhibit a heterogeneous sedimentation and appear to contain highly aggregated particlses ranging over hexamers. These polysomes are sensitive to RNase, (ethylenedinitrilo)tetraacetic acid and puromycin. Mitochondrial polysomes are active in portein synthesis when supplied with supernatant enzymes from the homologous mitochondrial source or from Escherichia coli. Cytoplasmic enzymes, however, appear to be completely inactive. Protein synthesis by mitochrondrial polysomes is sensitive to chloramphenicol and resistant to cycloheximide and emetine. The procedure yields particles containing intact rRNAs. The extent of cytoplasmic RNA contaminating the total mitochondrial RNA or mitochondrial polysomal RNA has been estimated to be negligible.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Fractionation; Chloramphenicol; Cycloheximide; Cytoplasm; Digitonin; Edetic Acid; Emetine; Escherichia coli; Mice; Mitochondria; Neoplasm Proteins; Polyribosomes; Puromycin; Ribonucleases

Topics: Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Animals; Base Sequence; Carcinoma, Ehrlich Tumor; Centrifugation, Density Gradient; Cytidine; Cytosine Nucleotides; Digitonin; Electrophoresis, Polyacrylamide Gel; Genes; Iodine Radioisotopes; Isotope Labeling; Kinetics; Mice; Mitochondria; Nucleic Acid Hybridization; Pancreas; Polyribosomes; Ribonucleases; RNA, Messenger; RNA, Neoplasm; RNA, Ribosomal; Time Factors; Tritium

Topics: Animals; Borates; Carcinoma, Ehrlich Tumor; Cell Fractionation; Centrifugation; Digitonin; Enzyme Activation; Glutamate Dehydrogenase; Glutaminase; Hydrogen-Ion Concentration; In Vitro Techniques; Isoenzymes; Kinetics; Mice; Mitochondria; Phosphates; Polyethylene Glycols; RNA, Neoplasm; Sonication

Topics: Animals; Carcinoma, Ehrlich Tumor; Centrifugation, Density Gradient; Chloramphenicol; Chromatography, Gel; Cycloheximide; Cytoplasm; Digitonin; Electrophoresis, Polyacrylamide Gel; Emetine; Mice; Mitochondria; Neoplasm Proteins; Phenylalanine; Protein Biosynthesis; Puromycin; Ribosomes; RNA, Ribosomal; RNA, Transfer; Spectrophotometry, Ultraviolet; Streptomycin; Tritium