T. Terranova

The role of the α-glycerophosphate shuttle in the reoxidation of cytosolic NADH in ehrlich ascites tumour cells

Abstract 1. Addition of glucose to Ehrlich ascites tumour cells is able to release partially the inhibition by rotenone of the endogenous respiration in the hyperdiploid strain, but has almost no effect on the rotenone-blocked respiration of the hyperdiploid Lettremutant strain. 2. Measurements of redox changes of cytochromes b , c and nicotinamide-adenine nucleotides in the rotenone-inhibited cells indicate that, upon addition of glucose, (a) a translocation of glycolytic reducing equivalents to the mitochondrial cytochromes does occur in the hyperdiploid strain and (b) a reversal of mitochondrial electron transport is stimulated by the increase in the glycolytic phosphate potential in the Lettremutant. 3. Analysis of α-glycerophosphate dehydrogenase activity in the two strains shows that the level of this enzyme is low in the hyperdiploid Lettrecells and much higher (about 20 times) in the hyperdiploid cells. Consequently, upon addition of glucose to rotenone-treated cells, α-glycerophosphate is formed in quantities adequate for the operation of the α-glycerophosphate shuttle only in the latter cells. These findings suggest that the operation of the α-glycerophosphate shuttle is mainly responsible for the aerobic oxidation of glycolytic reducing equivalents found in the wild strain. On the other hand, during glucose breakdown in the presence of rotenone only slight differences are found in the lactate accumulation between the two strains. 4. It is concluded that the high rate of aerobic glycolysis in ascites cells cannot be dependent, as suggested by other authors, on the lack of a system for the transfer of glycolytic NADH to the respiratory chain beyond the first phosphorylation site.

Lack of effect of the Ca2+ ionophore A23187 on tumour cells

The Ca2+ ionophore A23187 increases intracellular calcium content in normal thymic cells, while it is without effect on the corresponding neoplastic cell (Ascites thymoma) and on Ehrlich ascites tumour cells. The A23187-induced total cell calcium increase in normal thymocytes takes place both in control and energy-depleted cells, while it is lacking in neoplastic cells. In addition the ionophore stimulates aerobic glycolysis of normal thymocytes, whereas it is ineffective on neoplastic cells. The study of intracellular calcium exchange properties reveals that in normal cells the ionophore A23187 provokes a 60% increase of the exchangeable pool together with a more significant, 4-fold enlargement of the unexchangeable pool. These effects are lacking in cancer cells. The data give rise to interesting considerations concerning the regulation and compartmentalization of calcium in neoplastic cells. The results will be also discussed in relation to the models that predict altered cell calcium metabolism as a cause of cancer cell high aerobic glycolysis and uncontrolled growth.

Calcium metabolism in Ehrlich ascites tumour cells

Ehrlich ascites tumour cells are able, under the proper experimental conditions, to extrude a substantial amount of Ca2+ from the intracellular space. The Ca2+ extrusion mechanism, probably located at the plasma membrane level, appears to be similar to that found in red blood cells. It is energy-dependent and both respiration and glycolysis are able to drive it. The use of some inhibitors and uncouplers, besides showing that this activity is different from that linked to the mitochondrial Ca2+ pump which acts in the opposite direction, proposes some speculations on the energy compartmentation in the Ehrlich ascites tumour cells.

Effect of pyruvate on the acute cyanide poisoning in mice

Sono stati studiati gli effetti del piruvato di sodio nella intossicazione acuta da cianuro nel topo. I risultati, statisticamente validi, dimostrano che il piruvato iniettato per via endovenosa riduce notevolmente leffetto letale del cianuro di sodio somministrato per via endoperitoneale.

Intracellular calcium and magnesium content and aerobic lactate production in intact Ehrlich ascites tumour cells

The high rate of aerobic glycolysis and the low ‘Pasteur effect’ are the most consistent biochemical features of tumour cell energy metabolism ([ 11, reviewed [2]) but their causes and meaning are still under discussion [3-61. Among various hypotheses proposed to explain the high aerobic glycolysis of cancer cells one, postulating a profound alteration of cell calcium metabolism, seems very attractive as it could explain some other biological features relevant to neoplasia (viz., uncontrolled growth, invasivity ([3], reviewed [7]). This hypothesis implies the existence in cancer cells of a decreased cytosolic Ca2’ concentration resulting from both a low plasma membrane permeability to Ca2+ and a high Ca2+ accumulating capacity of tumour mitochondria. The following alteration of the Ca2’/ Mg2+ ratio would then be responsible for the increased activity of some key glycolytic enzymes [3]. Following our results on Ca2+ extrusion by intact Ehrlich ascites tumour cells (ATC) [8,9] we now report data on the relationship between calcium content and rate of aerobic glycolysis in ATC. For this purpose we utilized, as experimental model, intact ATC suspended in isosmotic, Tris-buffered, mannitol-sucrose solution, since in this medium ATC show an unusual low rate of aerobic glycolysis; in addition we took advantage of the antibiotic ionophore A23 187, which has been shown to strongly increase ATC calcium content [8,9]. The results obtained led us to the conclusion that in intact ATC the intracellular calcium concentration, and the size of its exchangeable pool, do play a

Oxidation of reduced nicotinamide-adenine dinucleotide by the malate- -aspartate shuttle in ehrlich ascites tumour cells.

Abstract The capability of ascites tumour mitochondria to oxidize externally formed NADH has been investigated in intact cells. Lactate has been used as the source of reducing equivalents and the oxidation of this substrate to pyruvate has been estimated. Ascites cells, under conditions of endogenous metabolism, are able to produce pyruvate upon addition of lactate. This effect is prevented by aminooxyacetate, an inhibitor of glutamate—oxalacetate transaminase (EC 2.6.1.1). Half-maximal inhibition by aminooxyacetate is attained at a concentration of approx. 30 μM. Oxidation of lactate is also sensitive to inhibitors of mitochondrial electron and energy transfer and it is enhanced by α-oxoglutarate plus aspartate. These data demonstrate that reducing equivalents can be transported across the mitochondrial membrane of intact Ehrlich ascites tumour cells by the malate—aspartate shuttle.

Shuttles for the transfer of reducing equivalents in ehrlich ascites tumor cells.

Abstract Ehrlich hyperdiploid ascites tumor cells are able to oxidize lactate to pyruvate. Such oxidation is sensitive to inhibitors of mitochondrial electron and energy transfer. Aminooxyacetate, an inhibitor of glutamate-oxaloacetate transaminase (EC 2.6.1.1), does not affect the production of pyruvate under conditions of endogenous metabolism, whereas it partially inhibits in the cells supplemented either with α-oxoglutarate plus aspartate or with dl -isocitrate. Reconstitution experiments of the fatty acid cycle performed in isolated mitochondria show that added NADH is actively oxidized. Whereas the operation of the malateaspartate shuttle under conditions of endogenous metabolism is ruled out, a possible role of the fatty acid shuttle in the transport of reducing equivalents across the mitochondrial membrane is suggested.

Calcium metabolism in intact isolated thymocytes.

SummaryIsolated rat thymocytes incubated under proper metabolic conditions extrude Ca24 previously taken up under metabolically unfavourable conditions.The extrusion can be supported by both respiratory and glycolytic energy but glycolysis seems to be more efficient for this purpose.La3− (50–200 μM) and the ionophore A 23187 inhibit cell Ca2+ extrusion.Ruthenium Red (1–100 μM)) does not influence cell Ca2+ extrusion while it inhibits the in situ mitochondrial cation uptake.All the results are consistent with a cell regulation model of Ca 2+ content in which both plasma membrane and mitochondria co-operate, acting in opposite directions, in order to decrease cytosolic Ca 2+ concentration.The possibility of Na+-Ca2+ hetero-exchange participation to cell Ca2+ homeostasis regulation is also discussed.

Energy metabolism of isolated rat thymus cells

SummaryThe energy metabolism of rat thymus cells has been investigated using preparations of isolated cells obtained by mechanical treatment of whole organs.The addition of glycolytic substrates such as glucose, pyruvate and lactate stimulates the endogenous respiration of these cells by 50%. On the other hand, succinate, glutamate and malate do not produce any effect.Oligomycin (10 µg/ml) inhibits both endogenous and glucose stimulated respiration by about 40%; 2,4-DNP (50 µm) increases by 100% glucose induced respiration.The results obtained by using mitochondrial and glycolytic inhibitors as well as aminoxyacetic acid (AOA) and following pyridine nucleotides redox changes, support the idea that in thymus cells glucose is able to induce a great enhancement of O2 consumption both by raising the level of endogenous pyruvate and feeding the mitochondrial respiratory chain with cytosolic reducing equivalents, through an active malate-aspartate shuttle.Thymus cells exhibit a high Pasteur effect (74%). Both AOA and 2,4 DNP are able to stimulate aerobic lactate accumulation by 200% and 100% respectively, indicating that either the redox or phosphate potential do influence the rate of aerobic glycolysis in isolated thymus cells. Similar experiments are also reported on other cells with well known biochemical characteristics.

The effect of pyruvate on cyanide-inhibited respiration in intact ascites tumor cells

È stato osservato che laggiunta di piruvato a cellule ascite di Ehrlich del topo è capace di rimuovere rapidamente linibizione della respirazione indotta da cianuro, restaurando lintegrità della fosforilazione ossidativa.