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.
Sánchez Olavarría
J
J
Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá de Henares, Madrid, Spain.
Galindo
C
C
Montero
M
M
Baquero
Y
Y
Victorica
J
J
Satrústegui
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000332:Aerobiosis; D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000503:physiopathology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D005260:Female; D005947:Glucose / Q000494:pharmacology; D006019:Glycolysis; D007425:Intracellular Membranes / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000502:physiology*; D009861:Onium Compounds / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010101:Oxygen Consumption
D009861:Onium Compounds; D009943:Organophosphorus Compounds; D000244:Adenosine Diphosphate; D005947:Glucose; D004072:Digitonin; C013289:tetraphenylphosphonium