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.

Temperature‐induced rapid increase in cytoplasmic free Ca2+ in pathogenic Leishmania donovani promastigotes

“…3A (a)) were affected. In other trypanosomatide, employing permeabilized cells, it was shown earlier that addition of electron transport chain inhibitors prevented uptake of Ca 2+ in mitochondria but did not result in any release of Ca 2+ from the mitochondrial pool [13][14][15]25]. When Mn 2+ (100/aM) was added to the cells that were first exposed to upward shift of temperature and then treated with the inhibitors (Fig.…”

“…More specifically, using digitonin permeabilized cells the presence of a mitochondrial pool and a pH-sensitive pool has been demonstrated in Leishmania spp. [13][14][15][16]. In vitro morphogenetic transformation and flagellar movement are also severely affected in presence of Ca 2÷ channel blockers and calmodulin antagonists [17].…”

Highly Active Anti- Pneumocystis carinii Compounds in a Library of Novel Piperazine-Linked Bisbenzamidines and Related Compounds

“…Loss of these transporters conferred a resistant phenotype. Collapse of mitochondrial membrane potential leading to loss of mitochondrial activity was associated with pentamidine toxicity in Leishmania species (5,36).…”

Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts

“…For example, an initial study showed that when Leishmania amazonensis parasites were treated with pentamidine, the morphology of mitochondria was enormously expanded, followed by condensation and disruption of kinetoplast DNA (kDNA) (23). Later, 2 groups showed that exposure of Leishmania donovani promastigotes to pentamidine rapidly collapsed the parasites' mitochondrial membrane potential, further supporting the hypothesis that the mitochondrion is the target organelle for the pharmacological action of pentamidine (24,25). Through the use of in vitro-generated pentamidine-resistant Leishmania mexicana parasites, Basselin and colleagues demonstrated that the fluorescent analog of pentamidine, 4=,6-diamidino-2-phenylindole (DAPI), is excluded from the kinetoplast of the resistant phenotype, again indicating that the site of action for the diamidine-type inhibitors pentamidine and DAPI is highly associated with mitochondria of Leishmania parasites (26).…”