Early reactions of cadmium with Ehrlich cells.

Ehrlich ascites tumor cells accumulate cadmium against a concentration gradient in a bisphasic uptake process. There is little efflux of the metal from preloaded cells into a cadmium-free medium. Incorporation of 3H-thymidine into DNA is markedly inhibited by cadmium ion at 5-100 ng atoms of Cd/mg of cell protein, but uptake of the nucleoside label into cells is not depressed in this concentration range. Cell respiration is much less affected by cadmium ion despite the sensitivity of isolated mitochondria to the metal. Model experiments using several cadmium complexes with known conditional formation constants show that bovine heart mitochondria have strong affinity for cadmium ion. The contrast between this result and the resistance of cells to respiratory inhibition with cadmium ion is discussed to illustrate the difficulty in relating in vitro studies to the cell. The behavior of cadmium ion with the Ehrlich cell is compared with data for zinc ion to reveal similarities in inhibition of nucleoside metabolism and respiration but a sharp difference in transport properties.     

Isolation of mitochondria from ascites tumor cells permeabilized with digitonin

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.     

The inhibitory effect of various fatty acids on aerobic glycolysis in Ehrlich ascites tumour cells

Inhibition of glycolysis in Ehrlich ascites tumour cells by saturated fatty acids, added either in form of potassium salts or incorporated into phosphatidylcholine liposomes, increases with the increasing carbon atom chain length and is independent of the concentration within the range of 0.1 to 1.0 mM. In contrast, the inhibition of glycolysis in the cytosolic fraction from Ehrlich ascites cells depends on the concentration of fatty acids. The content of ATP in Ehrlich ascites cells incubated with fatty acids increases with increasing carbon atom chain length, which leads to a crossing-over in the concentrations of pyruvate and 2-phosphoenolpyruvate. Lowering of the sum of both these metabolites by palmitate and stearate points to the inhibition not only of pyruvate kinase but also of other enzymes of early steps of glycolysis. Fatty acids in intact Ehrlich ascites cells inhibit all three key glycolytic enzymes but added to the cytosolic fraction affect mainly the activity of phosphofructokinase. The inhibition of pyruvate kinase by fatty acids is smaller in the cytosolic fraction from tumour cells than from liver and muscles.     

Changes in the SH-group level and respiration during gamma- and Fe2+-induced lipid peroxidation in Ehrlich ascites carcinoma cells

In experiments on Ehrlich ascites tumor cells it was shown that lipid peroxidation induced by gamma-rays and Fe2+ ions was accompanied by a decrease in the endogenous SH-group content at early times after exposure (during 3-hour incubation). It was also established that no significant changes occurred in the oxygen uptake by Ehrlich ascites tumor cells depending on radiation dose of Fe2+ ion concentration. If cells were pre-kept under hypotonic conditions an additional decrease in cell respiration and SH-group content and activation of lipid peroxidation was noted.     

Growth-related changes of oxygen consumption rates of tumor cells grown in vitro and in vivo

Growth-related changes of oxygen consumption rates of tumor cells, grown in vitro or in vivo, were investigated. For in vitro investigations, L929 and DS-carcinosarcoma cells were cultured in artificial media. For in vivo studies, DS-carcinosarcoma cells were implanted into the abdominal cavity of Sprague-Dawley rats (ascites tumor, containing malignant cells, leukocytes, lymphocytes, and macrophages). Oxygen uptake was measured photometrically. Parameters of the extracellular medium judged to possibly influence the respiratory activity of tumor cells were monitored at different growth stages (glucose, lactate, and amino acid levels, oxygen and carbon dioxide partial pressures, and pH values). The results obtained clearly show that the oxygen uptake of tumor cells grown in vitro decreased as quiescence developed. In contrast, the respiratory activity of in vivo DS-carcinosarcoma ascites cells increased as tumor growth reached plateau phase. The differences observed cannot be attributed solely to changes of the environmental conditions monitored. It is likely that an increased respiration rate of activated host cells might profoundly contribute to the elevation of the respiratory capacity of DS-carcinosarcoma ascites tumors grown in vivo. These data provide evidence that solid tumors in vivo can increase their O2 uptake at an enhanced O2 availability not only due to an enlarged tumor volume with adequate O2 supply but also due to an elevation of the respiratory activity of different cell populations within a tumor.     

Membrane transport properties of l-2,4-diaminobutyrate revisited

Summary We explore here the special structural features of certain diamino acid analogs which may account for their intense accumulation into tumor cells, first observed for the Ehrlich ascites tumor cell for in vitro suspensions. This accumulation, which ordinarily occurs mainly by system A for its dipolar substrates, is so intense for these tripolar diamino acids accompanied by the chloride ion as well as by displacement, especially of the cellular potassium ion, that the cells swell to several times their normal volume and osmotic destruction arises. These structural features receive our reconsideration here toward understanding the energization of amino acid transport into cells, also toward identifying among them possible superior ¹¹C-labeled tracers for imaging tumors in situ by positron emission tomography (PET). The possibility of therapeutic, perhaps osmotic, destruction of inoperable terminal gliomas by topical application of such amino acids by microdialysis has also been considered in preliminary tests by one of us (G.R.) and his associatesPresented at Uppsala Symposium on Positron Emission Tomography, 9/13/91     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

Membrane fluidity in Ehrlich ascites tumor cells treated with adriamycin

The incubation of Ehrlich ascites tumor cells with adriamycin resulted in an increase in lipid peroxide content and a decrease in membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the incorporation of [3H]thymidine into tumor cells was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of Ehrlich ascites tumor cells after exposure to a low, but cytotoxic, level of this agent.     

Non-linear relationship between fluorescence and membrane potential

The fluorescence intensity of the cynanine dye DiO-C₆-(3)_in 0.32% suspension of Ehrlich ascites tumor cells was determined under various potassium concentrations. In addition, the fluorescence levels of cell-free buffer solutions and those of supernatants were measured. For every potassium concentration the partition of the dye between cells and medium was calculated and its relation to the potassium gradient was given. A model was developed which assumes the fluorescence of a cell suspension to be the sum of the fluorescence signal of dye in the extracellular medium and that of cell-associated dye. A calibration curve of fluorescence vs. membrane potential was constructed. Neither the fluorescence of the cell suspension nor that of the supernatants was a linear function of the membrane potential. The limitations of membrane potential determination by fluorimetric methods are discussed.     

The role of respiration in tumor cell transition from the noncycling to the cycling state

Resting Yoshida AH130 hepatoma cells, harvested at the plateau of tumor development in vivo, were recruited into the cycling state following transfer to an in vitro system whereby these cells were incubated in the autologous ascites plasma diluted with buffered saline and enriched with glucose. In this system, cell recruitment into the phase of DNA synthesis (S phase) strictly depends on the activity of the respiratory chain and is abolished by anaerobiosis as well as by antimycin A, although the intracellular levels of ATP and the rate of protein synthesis are practically unaffected by these treatments. Furthermore, 2,4-dinitrophenol, at concentrations which uncouple the respiratory phosphorylation and hence enhance both glycolysis and oxygen consumption, does not hinder cell promotion into S phase. Thus, the absolute respiration dependence of cycling resumption by resting ascites cells does not seem to rely on respiratory ATP supply, but rather is linked to the electron flow through the respiratory chain.     

Effect of glucose on pH and potassium ion balance in suspensions of ascites tumor cells subjected to x-irradiation

Glucose (2.14 microM) was introduced into nonirradiated and irradiated (6 and 60 Gy) ascites tumor cell suspension. Irradiation was shown to suppress the consumption of potassium by cells having no influence on the rate of protoxidation of the suspension, induced by glucose, and on the passive release of potassium from cells.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

钾离子通道与神经胶质瘤关系的研究进展

钾离子通道为组织细胞内分布最广、种类最多的离子通道,在细胞增殖、分化及肿瘤细胞的侵袭转移中起着关键作用。神经胶质瘤是颅内最多发的恶性肿瘤,目前其主要治疗方式为手术加术后放化疗,术后五年生存率较低,寻找其相关发病机制及化疗靶点具有重要意义。目前已有多项研究表明,多种钾离子通道在胶质瘤中呈特异性高表达,且与胶质瘤的增殖、分化有密切关系,一些钾离子通道可作为胶质瘤的诊断和预防因子,有望成为未来胶质瘤化疗的新靶点,研究钾离子通道与神经胶质瘤的关系对胶质瘤的诊断、预防和治疗有重要意义。本文主要对近年来钾离子通道与神经胶质瘤关系研究的新进展进行综述。     

Studies on the Movement of Solutes between the Sieve Tubes and Surrounding Tissues3: I. INTERFERENCE BETWEEN SOLUTES AND RATE OF TRANSLOCATION MEASUREMENTS

Certain aspects of the secretion of solutes into, and removalfrom, the sieve tubes of isolated stem segments and rooted cuttingsof Salix viminalis have been studied. Sieve-tube sap was obtainedeither as honeydew from whole individuals or via the severedstylets of the aphid Tuberolachnus salignus (Gmelin). It was shown that interference occurred between the chemicallyunrelated solutes, sucrose and the cations potassium and rubidium.On raising the potassium concentration in the sieve-tube sapby passing a solution of this ion through the xylem, the sucroseconcentration declined. When the sucrose concentration fellover a period of days due to respiratory loss of carbohydratesfrom an isolated stem segment, a concomitant rise in eitherthe potassium or rubidium level in the sap occurred. When a solution of sodium was passed through the xylem, theconcentration of this ion in the sieve-tube sap rose, whilstthat of potassium fell at first, but later rose higher thanits initial value, indicating that both antagonism and synergycan occur between these ions. On introducing both these cationsinto the xylem simultaneously, more sodium than potassium wastaken up by the segment, though the increase in the sodium concentrationin the sieve-tube sap was less than that of the potassium. Perfusingthe xylem with a calcium solution had no effect upon the concentrationof potassium in the sieve tube. It has been shown that the rate of translocation of a solutealong the sieve tube, as measured by the two colony technique,depends upon the rate of removal of this solute from the sievetube. The amount of such lateral loss from the sieve tube isrelated to the potential gradient for a solute between the sievetube and surrounding cells.     

Water-soluble inhibitor(s) of tumor respiration formed from ultraviolet-induced oxidation of linoleic and linolenic acids

Inhibition of Ehrlich ascites carcinoma respiration by aqueous extracts of oxidized linoleic or linolenic acid (aqueous emulsions UV-irradiated, 90 min) was associated entirely with relatively involatile compounds which were both thiobarbituric acid (TBA)-reactive and peroxidase-reactive. Inhibitory compounds were heat stable and migrated in thin-layer chromatography with aldehydes, "hydroperoxides," and TBA-reactive compounds. Peroxidase-catalyzed reduction of the "hydroperoxide" diminished the inhibition. At 4.7 x 10(-5) M "hydroperoxide" concentration, the residues from both linoleic and linolenic acid inhibited tumor oxygen consumption to a similar degree. However, at this concentration of "hydroperoxide" only the dried extract from linolenic acid was able to produce inhibition (100%) of aerobic glucose utilization by tumor cells. No glycolytic inhibition by the dried residue of oxidized linoleic acid was observed. At least 12 compounds (approximate chain length, 7C-13C) containing alpha,Beta-unsaturated carbonyl groups were isolated by gas-liquid chromatography (GLC) of dried extracts of oxidized linolenic acid. No single fraction inhibited tumor respiration, but the recombined mixture of all compounds caused complete respiratory inhibition of ascites tumor cells. Less material was required to inhibit oxygen consumption before than after GLC presumably because the more highly inhibitory components of the extract (along with "hydroperoxides" and TBA-reactive compounds) were lost during GLC. Extracts from oxidized linolenic acid were found to produce in all tumor cells cytoplasmic evaginations which were readily detected by phase microscopy.     

Dynamic changes in the pH and potassium concentration in a suspension of ascitic tumor cells under glucose load

A single addition of glucose to 10% (6 v/v) suspension of the Ehrlich ascite tumor cells results in biphasic changes of pH and potassium ion concentration in the extracellular medium during 120 minutes. Phases of pH and potassium changing do not coincide. Increasing glucose amount from 100 to 400 mumol/10 ml suspension causes no marked modification of ion dynamics. Possible mechanisms of the above phenomena and their relation to cell power are discussed.     

Concanavalin A-induced alterations in sodium and potassium content of Ehrlich ascites tumor cells

Net fluxes of sodium and potassium were studied in Ehrlich mouse ascites tumor cells during contact with the agglutinating protein, concanavalin A. This lectin altered cation transport markedly at concentrations of 20–105 μg/ml (6–47 μg/mg cell protein). Whereas control cells extruded sodium and maintained or accumulated potassium against electrochemical gradients, in the presence of concanavalin A there was rapid net sodium entry and potassium loss. After 10–20 minutes in concanavalin A, sodium extrusion began and potassium loss diminished but these events were prevented by ouabain. The alterations in cation content induced by concanavalin A are unlikely to be the result only of agglutination since soybean agglutinin caused much smaller changes although it agglutinated the cells equally well.     

The effect of exogenous ATP on the electrolyte content of TA3 ascites tumor cells

In the presence of 1 mM ATP in the external medium, TA₃ mouse ascites tumor cells showed a dramatic loss of potassium and gain of sodium down their respective concentration gradients. The volume changes detected by size discrimination with the Coulter counter have been adequately confirmed by densimetric techniques. Further, some experiments were so designed that net losses of both ions occurred and the cell shrank in response to ATP, a response which was predictable if the volume change was produced by a loss of cell water. We believe that ATP produces a major change in the passive permeability of the membrane to these ions and the effect may be due to a response of a contractile protein in the membrane to ATP.     

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.     

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells.

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.