Increase in cytosolic Ca²⁺ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

Abstract

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H₂O₂. The present study shows that upon activation of death response by H₂O₂, a dose-and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca²⁺ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca²⁺. Partial inhibition of cytosolic Ca²⁺ increase achieved by chelating extracellular or intracellular Ca²⁺ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca²⁺ is an additive result of release of Ca²⁺ from intracellular stores as well as by influx of extracellular Ca²⁺ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.