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 donovaniPromastigotes

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 H 2 O 2 . The present study shows that upon activation of death response by H 2 O 2 , 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 Ca 2؉ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na ؉ and mitochondrial Ca 2؉ . Partial inhibition of cytosolic Ca 2؉ increase achieved by chelating extracellular or intracellular Ca 2؉ 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 Ca 2؉ is an additive result of release of Ca 2؉ from intracellular stores as well as by influx of extracellular Ca 2؉ 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 Nacetylcysteine 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.Mitochondria are pivotal in controlling cell life and death (1). Maintenance of proper mitochondrial transmembrane potential (⌬ m ) 1 is essential for the survival of the cell as it drives the synthesis of ATP and maintains oxidative phosphorylation (2). Recently, the study of mitochondrial potential has become a focus of apoptosis regulation as many investigations demonstrate a major functional impact of mitochondrial alterations on apoptosis (2). Apoptosis is a process of cell death in which the cells undergo nuclear and cytoplasmic shrinkage; the chromatin is condensed and partitioned into multiple fragments, and finally the cells are broken into multiple membrane-bound bodies. In a number of experimental systems, disruption of ⌬ m constitutes a constant early event of the apoptotic process that precedes nuclear disintegration (3-5). For example, in thymocytes or tumor necrosis factor-stimulated U937 cells (3, 6), thymocytes or imexon-treated myeloma cells (5, 7), and PC-12 cells (8), a loss of ⌬ m occur as an early change associated with apoptosis. Lymphocytes with low ⌬ m show irreversible commitment to apoptosis in comparison to cells with high ⌬ m that do...

Antimonial-induced Increase in Intracellular Ca2+ through Non-selective Cation Channels in the Host and the Parasite Is Responsible for Apoptosis of Intracellular Leishmania donovani Amastigotes

“…Exposure of axenic amastigotes of Leishmania infantum to an antimonial compound (SbIII) yields similar results (8). Hydrogen peroxide (H 2 O 2 ) induces nuclear condensation, DNA ladder formation, and activation of caspase-like activity in L. donovani promastigotes that occur through a Ca 2ϩ -dependent mechanism (4,9). The protein kinase inhibitor staurosporine, known to induce apoptosis in all mammalian nucleated cells (10), stimulates apoptosis-like death in the promastigotes of L. major (11).…”

“…1 and related reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), nitric oxide (NO), hydroxyl radicals, and singlet oxygen (18,19). ROS is known to induce apoptosis in metazoans (20) and in unicellular organisms existing outside the host cell (4,9). Therefore, it is possible that ROS generation might have a direct bearing on apoptotic death of intracellular parasites, but this phenomenon has not been examined adequately.…”

Camptothecin induced mitochondrial dysfunction leading to programmed cell death in unicellular hemoflagellate Leishmania donovani

“…18 As shown in Figure 4a, CPT causes 45% decrease in GSH level after 1.5 h and the effect is more pronounced after 2.5 h treatment with CPT. When cells were preincubated with NAC (20 mM) for 1 h, followed by treatment with CPT, GSH level tends to become normal.…”

“…The ratio of the reading at 590 nm to the reading at 530 nm (590 : 530 ratio) was considered as the relative Dc m value. 18 Detection of cytochrome c release…”

“…16 At this stage, numerous different signalling pathways converge through which multiple downstream substrates are cleaved. The importance of an optimum mitochondrial transmembrane potential (Dc m ) in cell survival has been recognized not only in mammals but also in yeast 17 and in protozoan parasites 18,19 as well.…”