atractyloside and Carcinoma--Ehrlich-Tumor

In the presence of 3 mM atractyloside, growth of in vitro cultured Ehrlich ascites tumor cells is inhibited by 70% within 24 h. Viability of the cells is not severely affected (dye exclusion test). Incorporation of 2-[14C]-thymidine and U-[14C]-leucine into acid insoluble precipitate were reduced by 80% or 20% respectively as compared to controls. Flow cytometric analysis of cell cycle progression revealed a retardation rather than an arrest of cell growth by atractyloside. Morphological changes of the cells primarily concern mitochondria which are spherical shaped with translucent matrix rid of cristae. After transfer of atractyloside treated cells to normal medium, proliferation and macromolecular synthesis normalized within 3 to 6 h. At 3 mM of atractyloside, glucose consumption of the cells increased by 25%, lactate production by 30%. Lactate/glucose ratio was 1.9 after 24 h. Oxygen uptake was reduced by 35% after 12 h. The [ATP]/[ADP] ratio of the whole cells runs through a maximum between 12 and 18 h. The ratio never falls below 5.0. The ATP/ADP concentration ratio in the mitochondrial and extramitochondrial compartment were increased as compared to controls. delta G of ATP hydrolysis of the intact cells was in a normal range (-50 kJ), energy charge was 0.86 (controls 0.88). Transport of amino acids, uptake of glucose and activity of Na+, K+-ATPase of the plasma membrane were not impaired by 3 mM atractyloside.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Atractyloside; Carcinoma, Ehrlich Tumor; Cell Cycle; Cell Division; Energy Metabolism; Glycosides; Kinetics; Mice; Microscopy, Electron; Mitochondrial ADP, ATP Translocases; Nucleotidyltransferases; Ornithine Decarboxylase; Phosphates

(1) The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.

Topics: Adenosine Diphosphate; Adenosine Triphosphatases; Animals; Atractyloside; Azides; Carcinoma, Ehrlich Tumor; Flavonoids; Glucose; Glucosephosphates; Hexokinase; Hexoses; Hydrogen-Ion Concentration; Kinetics; Mitochondria; Quercetin; Rutamycin

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.

Topics: Adenosine Monophosphate; Animals; Atractyloside; Carcinoma, Ehrlich Tumor; Ethidium; Mitochondria; Poly A; Ribonucleases; RNA, Neoplasm; Uracil Nucleotides

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.

Topics: Adenosine Triphosphate; Animals; Atractyloside; Biological Transport, Active; Calcium; Carcinoma, Ehrlich Tumor; Cell Fractionation; Cell Line; Magnesium; Mitochondria; Oligomycins; Osmolar Concentration; Oxygen Consumption; Phosphates; Rotenone; Succinates; Trypan Blue