title: Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ.
abstract: The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of Leishmania donovani promastigotes in situ using safranine O. L. donovani mitochondria were able to build up and retain a membrane potential of a value comparable with that of mammalian mitochondria. The response of promastigotes mitochondrial membrane potential to phosphate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), valinomycin and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria with regard to the properties of their electrochemical proton gradient. When L. donovani promastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM). The presence of 1 microM-FCCP decreased by about 75% the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably by the endoplasmic reticulum, was completely inhibited by 500 microM-vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased by about 25% and the Ca2+ set point was increased to 0.7 microM. The succinate-dependence and FCCP-and Ruthenium Red-sensitivity of the Ca2+ uptake detected in the presence of vanadate indicate that this uptake is probably by the mitochondria. This interpretation was further supported by the Ruthenium Red-sensitive decrease in the mitochondrial membrane potential caused by Ca2+ addition. The anti-leishmanial cationic drugs pentamidine and WR-6026 also induced a rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes.
authors.affiliation: Department of Veterinary Pathobiology, University of Illinois, Urbana 61801.
authors.forename: A E
authors.initials: AE
authors.lastname: Vercesi
authors.forename: R
authors.initials: R
authors.lastname: Docampo
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002413:Cations, Divalent; D004072:Digitonin / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D007893:Leishmania donovani / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D010419:Pentamidine / Q000494:pharmacology*; D012430:Ruthenium Red / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D000634:Aminoquinolines; D002413:Cations, Divalent; D012430:Ruthenium Red; D014634:Valinomycin; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D010419:Pentamidine; D004072:Digitonin; D002118:Calcium; C080436:8-aminoquinoline