Action of the antitumor and antispermatogenic agent lonidamine on electron transport in ehrlich ascites tumor mitochondria

The effect of lonidamine, an antispermatogenic and antitumor drug, on the oxygen consumption, ATPase activity, and redox state of the electron carriers of Ehrlich ascites tumor mitochondria has been studied. Lonidamine inhibits ADP- and uncoupler-stimulated respiration on various NAD- and FAD-linked substrates, but does not affect state 4 respiration. Experiments to determine its site of action showed that lonidamine does not significantly inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c₁ complex, also was unaffected by lonidamine, which failed to inhibit the oxidation of duroquinol. Moreover, inhibition of electron flow through site 2 was also excluded because of the inability of the N,N,N′,N′-tetramethyl-p-phenylenediamine bypass to relieve the lonidamine inhibition of the oxidation of pyruvate + malate. The F₀F₁ATPase activity and vectorial H⁺ ejection are also unaffected by lonidamine. The inhibition of succinate oxidation by lonidamine was found to take place at a point between succinate and iron-sulfur center S3. Spectroscopic experiments demonstrated that lonidamine inhibits the reduction of mitochondrial NAD⁺ by pyruvate + malate and other NAD-linked substrates in the transition from state 1 to state 4. However, lonidamine does not inhibit reduction of added NAD⁺ by submitochondrial vesicles or by soluble purified NAD-linked dehydrogenases. These observations, together with other evidence, suggest that electron transport in tumor mitochondria is inhibited by lonidamine at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state. The action of lonidamine in several respects resembles the selective inhibition of electron transport in tumor cells produced by cytotoxic macrophages.     

Ion transport by ouabain resistant and sensitive ehrlich ascites carcinoma cells

Cell division, net Na⁺-K⁺ and amino-acid transport of cultured Ehrlich ascites is reversibly inhibited by Ouabain at a final concentration of 1 × 10^–3M. A line of Ehrlich ascites cells resistant to the growth inhibiting effects of Ouabain has been developed. These cells behave similarly to Ouabain-sensitive cells in the following respects doubling time, S phase time, chromosome number, cell surface charge density, rate of incorporation of C¹⁴ Uridine and ³H-Thymidine, sensitivity to Digoxin and Digitoxin, steady state Na⁺, K⁺ levels and rate of loss of K⁺ and gain of Na⁺ in cold. Ouabain resistant cells differ from sensitive cells only with respect to the effect of ouabain on active Na⁺, K⁺ transport. Although Ouabain inhibits active Na⁺, K⁺ transport in sensitive cells it has no significant effect in resistant cells.     

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.     

A reduction in energy-dependent amino acid transport by microtubular inhibitors in ehrlich ascites tumor cells

Vincristine, other periwinkle alkaloids, and colchicine partially inhibit the energy dependent transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The properties of this phenomenon were characterized in detail for vincristine. Maximum depression of the steady-state intracellular α-aminoisobutyric acid level was achieved with a vincristine concentration of > 0.5 m̈M. The inhibitory effect of vincristine increases as the extracellular α-aminoisobutyric acid concentration is increased reaching a maximum, however, of only ∼25% at a level of 5 mM, leaving a large gradient for α-aminoisobutyric acid across the cell membrane. Vincristine produced an asymmetrical effect on the bidirectional fluxes decreasing the initial uptake rate, while increasing the efflux of α-aminoisobutyric acid. Inhibition of net α-aminoisobutyric acid transport by vincristine was partially reversible (∼40%). Colchicine (50 m̈M) reduced the steady-state α-aminoisobutyric acid level by 30%, an effect that was not reversible. Inhibition by vinleurosine and vinrosidine was comparable to that of vincristine. Addition of glucose to the medium resulted in a small, but significant, decrease in the inhibitory effects of both vincristine and colchicine. The data indicate that these agents inhibit a small component of the uphill transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The inhibitory effect of vincristine cannot be attributed to an increase in the passive permeability of the cell membrane to this agent. Rather, the data along with other studies from this laboratory suggest that vincristine reduces the energy-dependent transport of α-aminoisobutyric acid by either inhibiting cellular energy metabolism or by inhibiting the coupling of energy-metabolism to the transport of this amino acid and raises the possibility that cellular microtubules play a role in these processes.     

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.     

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.     

Calcium transport by rat brain mitochondria and oxidation of 2-oxoglutarate

Contrary to previous reports brain mitochondria have a substantial capacity for net Ca²⁺ uptake (approx. 1.2 μeq. Ca²⁺ per mg protein) providing succinate is the oxidizable substrate. ATP stimulates calcium uptake (to 1.8 μeq. per mg protein), but is not required. The accumulation of Ca²⁺ with NAD-linked substrates is, however, significantly less. With 2-oxoglutarate, very limited Ca²⁺ uptake occurs before respiration is inhibited. At low concentrations (10 μM), Ca²⁺ stimulates the 2-oxoglutarate dehydrogenase activity of detergent solubilized mitochondria. Millimolar [Ca²⁺] is required for inhibition. Therefore, Ca²⁺ inhibition of 2-oxoglutarate oxidation can explain the low maximum uptake with this substrate, but probably not by directly effecting the dehydrogenase. Hence, the oxidation of 2-oxoglutarate can be either enhanced or suppressed depending upon the net Ca²⁺ accumulated by brain mitochondria.     

Calcium transport and distribution in Ehrlich mouse ascites tumor cells

Data from isotopic uptake experiments were used to measure calcium fluxes and compartment sizes in ascites tumor cells. The data were analyzed with two kinetic models, A and B. In 80% of the experiments model A, which is based on one exchangeable calcium compartment, was rejected in favor of Model B, which predicts two exchangeable compartments. A statistical evaluation of the model's performance, when fit to the experimental data was used to select between the two models. The results show that calcium was distributed between three cellular compartments in the ratio, non-exchangeable (88%): rapidly exchanging (7%): slowly exchanging (5%). The undirectional fluxes suggested that calcium transport could be described as a series system with the temporal sequence: environment ? rapidly exchanging ? slowly exchanging.     

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.     

Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ

The use of digitonin to permeabilize Trypanosoma cruzi plasma membrane enabled us to study Ca2+ transport and oxidative phosphorylation in mitochondria in situ. Addition of Ca2+ to these preparations evoked a cycle of respiratory stimulation. Ca2+ uptake was partially inhibited by ruthenium red, almost totally inhibited by antimycin A, and stimulated by inorganic phosphate. Addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone to digitonin-permeabilized T. cruzi epimastigotes under steady-state conditions was followed by Ca2+ release. Antimycin A- and carbonyl cyanide p-trifluoromethoxyphenylhydrazonein-sensitive Ca2+ uptake was also detected in digitonin-permeabilized epimastigotes. Accordingly, ATP stimulated Ca2+ uptake by preparations de-energized by oligomycin and antimycin A. In conclusion, in contrast to previous reports indicating that a Ca2+ transport system occurs only in mitochondria from vertebrate tissues, T. cruzi epimastigotes also possess a similar system. In addition, these protozoan mitochondria have an extremely high resistance to the deleterious effects of massive Ca2+ loads in comparison with most types of mammalian mitochondria.