Malate-citrate cycle during glycolysis and glutaminolysis in Ehrlich ascites tumor cells

The malate-citrate cycle was studied during aerobic glycolysis and glutaminolysis in a strain of Ehrlich ascites tumor cells which showed a very low malate-aspartate shuttle system activity. The experimental approach includes: estimation of mitochondrial NAD[P]+-dependent malic enzyme activity; respiratory activity of freshly harvested or fasted cells, and of isolated mitochondria; and determination of the metabolites involved in the glycolytic and glutaminolytic pathways. The results suggest that in this strain, the malate-citrate shuttle is not an effective pathway for transferring glycolytic reducing equivalents from cytosol to mitochondria. Less than 15% of the glucose uptake was affected by the 1,2,3-benzenetricarboxylate inhibition of the malate-citrate shuttle. Moreover, in the presence of glucose, the malate-citrate cycle did not appear to play an important role in the glutaminolytic process. The present work supports and extends the finding of previous studies, since the results showed that the glucose metabolism depressed the oxidative processes in Ehrlich ascites tumor mitochondria, not only alone, but also in the presence of glutamine. Interestingly, the high glutamine uptake was maintained in the presence of glucose.     

The effect of magnesium on glycolysis of permeabilized Ehrlich ascites tumor cells.

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.     

Inhibition of lactate transport and glycolysis in Ehrlich ascites tumor cells by bioflavonoids.

Bioflavonoids are potent inhibitors of lactate transport in Ehrlich ascites tumor cells. The most effective bioflavonoids have four to five hydroxyl groups. Sugar substitution at carbon three, or reduction of the double bond between carbons two and three, decreases their inhibitory activity. Quercetin, the most extensively studied of these compounds, inhibits lactate efflux by 50% at 0.1 micrograms/mg of protein. On addition of quercetin to glycolyzing Ehrlich ascites tumor cells, lactate accumulates inside the cell and the intracellular pH drops. Total lactate production is also inhibited. Nigericin prevents the internal acidification that occurs in the presence of quercetin and also reduces the inhibition of glycolysis. Thus, it appears that inhibition of lactate efflux can affect glycolysis through a lowering of the intracellular pH. The inhibitory effect of quercetin on glycolysis can be explained by its effect on lactate efflux and its previously reported effect on the Na+--K+ ATPase [Suolinna, E.--M., et al. (1974) J. Natl. Cancer Inst. 53, 1515].     

The function of redox shuttles during aerobic glycolysis in two strains of Ehrlich ascites tumor cells

The function of glycerophosphate and malate-aspartate shuttles during glucose metabolism in two strains of Ehrlich ascites tumor cells was evaluated by several experimental approaches. The activities of the enzymes involved in these shuttle systems were assayed in the cytosolic and mitochondrial compartments after cell fractionation by the digitonin method. The glycerophosphate shuttle can be ruled out because of the lack of relevant enzymatic activities, and the failure of glucose to increase rotenone-inhibited respiration. Analysis of glycolytic flux in the presence of aminooxyacetate indicates that the activity of malate-aspartate shuttle may be very low. Balance studies of glucose uptake and lactate production suggest the existence of other pathways for the reoxidation of cytosolic NADH, which are acetyl-CoA dependent. Estimation of citrate synthase and ATP citrate lyase, in addition to the observed high activity of malate dehydrogenase, suggests a malate-citrate shuttle.     

Regulation of anaerobic glycolysis in Ehrlich ascites tumour cells.

1) In intact Ehrlich ascites tumour cells the anaerobic glycolytic flux rate and pattern of intermediates have been investigated at different pH values of the extracellular medium. 2) As predicted from the dependence of the lactic acid dehydrogenase equilibrium on pH a strong negative correlation between log ([lactate]/[pyruvate]) and pH has been found. 3) The steady state fluxes of glycolysis at pH 8.0 and 7.4 are rather equal, despite significant differences in the intracellular concentrations of glycolytic intermediates. At pH 8.0 the concentrations of ATP, glucose 6-phosphate, and fructose 6-phosphate are lower, and the concentrations of ADP, AMP, fructose 1,6-bisphosphate, triose phosphates, phosphoglycerates, and phosphoenolpyruvate are higher than at pH 7.4. 4) From the analysis of the pH dependent changes of metabolites it follows that different mechanisms are responsible for maintaining equal actual activities of hexokinase, phosphofructokinase and pyruvate kinase at pH 7.4 and 8.0. 5) From an application of the linear theory of enzymatic chains and a calculation of the control strength of the regulatory important enzymes results that hexokinase is evidently rate-limiting for glycolysis, and phosphofructokinase is also significantly influencing the glycolytic flux. Pyruvate kinase and glyceraldehyde phosphate dehydrogenase, on the other hand, do not significantly affect the rate of the overall glycolytic flux in ascites.     

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.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.     

Evidence for activation of an active electrogenic proton pump in Ehrlich ascites tumor cells during glycolysis

Summary The addition of glucose to a suspension of Ehrlich ascites tumor cells results in rapid acidification of the extracellular medium due to lactic acid production. The nature of the H⁺ efflux mechanism has been studied by measuring the time course of the acidification, the rate of proton efflux, the direction and relative magnitude of the H⁺ concentration gradient, and the voltage across the membrane. Using the pH-sensitive dye acridine orange, we have established that after addition of 10mm glucose an outward-directed H⁺ concentration gradient develops. As the rate of glycolysis slows, the continued extrusion of H⁺ reverses the direction of the H⁺ concentration gradient. Changes in absorbance of the voltagesensitive dye diethyloxadicarbocyanine iodide (DOCC), and changes in the distribution of the lipid permeant cation tetraphenyl phosphonium, showed a dramatic and persistent hyperpolarization of the membrane voltage after glucose addition. The hyperpolarization was prevented by the protonophore tetrachlorosalicylanalide (TCS) and by valinomycin, but not by the neutral-exchange ionophore nigericin. Inhibitors of lactate efflux were found to reduce the rate of acidification after glucose addition but they had no effect on the magnitude of the resulting hyperpolarization. On the basis of these and other data we suggest that an active electrogenic pump mechanism for H⁺ efflux may be activated by glucose and that this mechanism operates independently of the lactate carrier system.     

The transport of chloride in Ehrlich ascites tumor cells.

The steady state transport and distribution of chloride between the intracellular and extracellular phases was investigated when the extracellular chloride concentration was varied by isosmotic replacement with nitrate, bromide and acetate. The results of these experiments show that chloride transport, measured by uptake of 36Cl, is sensitive to the replacement anion. In the presence of nitrate, chloride transport is a linear function of the extracellular chloride concentration. The relationship between chloride transport and extracellular chloride in the presence of bromide is concave upward which suggests that this anion inhibits chloride movement. However, when acetate replaces chloride, the relationship between chloride transport and extracellular chloride is concave downward. The chloride distribution ratio of cells incubated in 145-155mM chloride medium is 0.386 and is not effected by the replacement of chloride with nitrate, bromide or acetate. These findings are consistent with the assertion that chloride transport is composed of two parallel pathways, a diffusional plus a saturating, mediated component. Of the total chloride flux (9.1 mmoles Cl-/kg dry weight per minute) measured in chloride medium (145-155 mM Cl-), the mediated component represents 40% and the diffusional component 60%.     

The role of actin in temperature-dependent gel-sol transformation of extracts of Ehrlich ascites tumor cells

Ehrlich ascites tumor cell extracts form a gel when warmed to 25 degrees C at pH 7.0 in sucrose solution, and the gel rapidly becomes a sol when cooled to 0 degrees C. This gel-sol transformation was studied quantitatively by determining the volume or the total protein of pellets of gel obtained by low-speed centrifugation. The gelation depended on nucleotide triphosphates, Mg2+, KCl, and a reducing agent. Gelation was inhibited reversibly by 0.5 microM free Ca2+, and 25--50 ng/ml of either cytochalasin B or D, but it was not affected by 10 mM colchicine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the gel was composed of six major proteins with mol wt greater than 300,000, 270,000, 89,000, 51,000, 48,000, and 42,000 daltons. The last component was identified as cell actin because it had the same molecular weight as muscle actin and bound with muscle myosin and tropomyosin. The role of actin in gelation was studied by use of actin-inhibitors. Gelation was inhibited by a chemically modified subfragment-1 of myosin, which binds with F-actin even in the presence of ATP, and by bovine pancreatic DNase I, which tightly binds with G-actin. Muscle G-actin neutralized the inhibitory effect of DNase I when added at an equimolar ratio to the latter, and it also restored gelation after its inhibition by DNase I. These findings suggest that gelation depends on actin. However, the extracts showed temperature-dependent, cytochalasin-sensitive, and Ca2+-regulated gelation as did the original extracts when the cell actin in the extracts was replaced by muscle actin, suggesting that components other than cell actin might be responsible for these characteristics of the gelation.