monensin and triphenylmethylphosphonium

Cultured rat hepatocytes were treated with potassium cyanide, an inhibitor of cytochrome oxidase; valinomycin, a K+ ionophore; carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophore; and the ATP synthetase inhibitor oligomycin. The effect of these agents on the viability of the cells was related to changes in ATP content and the deenergization of the mitochondria. The ATP content was reduced by over 90% by each inhibitor. All of the agents except oligomycin killed the cells within 4 h. With the exception of oligomycin, the mitochondrial membrane potential as measured by the distribution of [3H]triphenylmethylphosphonium collapsed with each of the agents. Monensin, a H+/Na+ ionophore, potentiated the toxicity of cyanide and CCCP, whereas the toxicity of valinomycin was reduced. The effect of cyanide and monesin on the cytoplasmic pH of cultured hepatocytes was measured with the fluorescent probe, 2',7'-biscarboxyethyl-5,6-carboxyfluorescein. Cyanide promptly acidified the cytosol, and the addition of 10 microM monensin caused a rapid alkalinization of the cytosol. A reduction of pH of the culture medium from 7.4 to 6.6 and 6.0 prevented the cell killing both by cyanide alone and by cyanide in the presence of monensin. However, neither monensin nor extracellular acidosis had any effect on the loss of mitochondrial energization in the presence of cyanide. It is concluded that ATP depletion per se is insufficient to explain the cell killing with cyanide, CCCP, and valinomycin. Rather, cell killing is better correlated with a loss of mitochondrial energization. With cyanide an intracellular acidosis interferes with the mechanism that couples collapse of the mitochondrial membrane potential to lethal cell injury.

Topics: Adenosine Triphosphate; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cells, Cultured; Cytoplasm; Drug Interactions; Energy Metabolism; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Liver; Male; Membrane Potentials; Mitochondria, Liver; Monensin; Oligomycins; Onium Compounds; Potassium Cyanide; Rats; Rats, Inbred Strains; Trityl Compounds; Valinomycin

The membrane potential of L1210 murine leukemia cells was assessed by use of the tritiated lipophilic cation probe triphenylmethylphosphonium bromide. The potassium equilibrium potential of the cells was found to be -71 +/- 7 mV. The resting membrane potential was partly dissipated by the protonophore m-chlorocarbonylcyanidephenylhydrazone (10 microM), but was unaffected by ouabain (1 mM) and apparently by the calcium ionophore A23187 (2.5 microM). Monensin (20 microM) caused a hyperpolarization which, since it was blocked by ouabain, was presumed to be brought about by activation of the Na+K+-ATPase via an elevated cytoplasmic Na+ concentration. Adriamycin at concentrations as high as 5 X 10(-4) M brought about no change in the resting potential of the cells. Also, cytotoxic concentrations of adriamycin, unlike ouabain, had no effect on rubidium-86 transport into L1210 cells, nor upon a monensin-induced increased in rubidium-86 uptake. The results suggest that although adriamycin is capable of interaction with the plasma membrane, and may exert its cytotoxicity at this locus, changes in ion flux mediated by Na+K+-ATPase or those capable of changing the membrane potential do not appear to be implicated in its mechanism of action.

Topics: Animals; Calcimycin; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Line; Cell Membrane; Doxorubicin; Leukemia L1210; Membrane Potentials; Mice; Monensin; Onium Compounds; Ouabain; Sodium-Potassium-Exchanging ATPase; Trityl Compounds