title: Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ.
abstract: Digitonin can be used to permeabilize selectively the plasma membrane of Trypanosoma cruzi epimastigotes without significantly affecting the functional integrity of mitochondria. Addition of digitonin at concentrations close to 64 microM caused decrease in the rate of basal respiration of epimastigotes similar to that caused by oligomycin. A further addition of carbonyl cyanide p-trifluorophenylhydrazone (FCCP) brought respiration to the same rate observed prior to the inclusion of digitonin or oligomycin. This suggests that like oligomycin, digitonin is shifting respiration to a nonphosphorylating state probably by depleting the cells from adenine nucleotides due to permeabilization of the plasma membrane. The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of these cells in situ using safranine O. The response of epimastigotes mitochondrial membrane potential to phosphate, FCCP, valinomycin, nigericin, ADP, and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria regarding the properties of their electrochemical proton gradient. In addition, T. cruzi mitochondria are able to build up and retain a membrane potential of a value comparable to that of mammalian mitochondria. The trypanocidal drug crystal violet, as well as other cationic drugs such as dequalinium, induced a rapid dose-related collapse of the inner mitochondrial membrane potential.
authors.affiliation: Department of Veterinary Pathobiology, University of Illinois, Urbana 61801.
authors.forename: A E
authors.initials: AE
authors.lastname: Vercesi
authors.forename: C F
authors.initials: CF
authors.lastname: Bernardes
authors.forename: M E
authors.initials: ME
authors.lastname: Hoffmann
authors.forename: F R
authors.initials: FR
authors.lastname: Gadelha
authors.forename: R
authors.initials: R
authors.lastname: Docampo
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D003868:Dequalinium / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology*; D005840:Gentian Violet / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology; D009550:Nigericin / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D010619:Phenazines / Q000378:metabolism / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000502:physiology*; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D009840:Oligomycins; D010619:Phenazines; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000244:Adenosine Diphosphate; D003868:Dequalinium; D005840:Gentian Violet; D004072:Digitonin; D002122:Calcium Chloride; D009550:Nigericin; C009195:safranine T