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.
Highlights
Mitochondria are pivotal in controlling cell life and death (1)
With 4 mM H2O2, there was a significant fall (63%) in ⌬m within the first 15 min as compared with relative ⌬m observed at 0 h. ⌬m values did not significantly change further until about 4 h, and another prominent drop occurred at 8 h postH2O2 treatment
Maximal interference with ⌬m was achieved with 4 mM H2O2, a dose that induces apoptosislike death in the promastigotes (25)
Summary
5,5Ј,6,6Ј-Tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazole carbocyanide iodide (JC-1), BAPTA-AM, fluo-3-acetoxymethyl ester (fluo-3/AM), rhod-2/acetoxymethyl ester (rhod-2/AM), 2Ј,7Ј-dichlorofluorescein diacetate (H2DCFDA), sulfinpyrazone, pluronic acid F-127, MitoTracker Green FM, SYTOX Blue nucleic acid stain, nonyl acridine orange (NAO), potassium-binding benzofuran isophthalate acetoxymethyl ester (PBFI-AM), sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM), and ATP determination kit were obtained from Molecular Probes (Eugene, OR). Terminal deoxynucleotidyltransferase enzyme (TdT)-mediated dUTP nick-end labeling (TUNEL) kit was from Promega (Madison, WI). Cyclosporin A, atractyloside, butathione sulfoximine (BSO), reduced GSH, N-acetylcysteine (NAC), digitonin, ophthalaldehyde, ruthenium red, medium 199, and any other chemicals unless otherwise mentioned were obtained from Sigma
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