Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

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

2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês

10.1074/jbc.m201961200

ISSN

1083-351X

Autores

Sikha Bettina Mukherjee, Manika Das, Ganapasam Sudhandiran, Chandrima Shaha,

Tópico(s)

Cardiac electrophysiology and arrhythmias

Resumo

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 likeN-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. donovanipromastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death. 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 likeN-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. donovanipromastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death. mitochondrial transmembrane potential reactive oxygen species 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide N-acetylcysteine butathione sulfoximine dithiothreitol mitochondrial permeability transition pore permeability transition pore 2′,7′-dichlorofluorescein diacetate cytosolic Ca2+ mitochondrial Ca2+ intracellular Ca2+ sodium-binding benzofuran isophthalate acetoxymethyl ester potassium-binding benzofuran isophthalate acetoxymethyl ester terminal deoxynucleotidyltransferase enzyme (TdT)-mediated dUTP nick-end labeling thapsigargin 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester flufenamic acid fluo-3-acetoxymethyl ester rhod-2/acetoxymethyl ester nonyl acridine orange Mitochondria are pivotal in controlling cell life and death (1Green D.R. Reed J.C. Science. 1998; 258: 1309-1312Crossref Google Scholar). Maintenance of proper mitochondrial transmembrane potential (Δψ m )1 is essential for the survival of the cell as it drives the synthesis of ATP and maintains oxidative phosphorylation (2Gottlieb R.A. Biol. Signals Recept. 2001; 10: 147-161Crossref PubMed Scopus (95) Google Scholar). 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 (2Gottlieb R.A. Biol. Signals Recept. 2001; 10: 147-161Crossref PubMed Scopus (95) Google Scholar). 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 Δψ m constitutes a constant early event of the apoptotic process that precedes nuclear disintegration (3Cossarizza A. Kalashnikova G. Grassilli E. Chiappelli F. Salvioli S. Capri M. Barbieri D. Troiano L. Monti D. Franceschi C. Exp. Cell Res. 1994; 214: 323-330Crossref PubMed Scopus (186) Google Scholar, 4Zamzami N. Marchetti P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-57Crossref PubMed Scopus (387) Google Scholar, 5Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssiere J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1089) Google Scholar). For example, in thymocytes or tumor necrosis factor-stimulated U937 cells (3Cossarizza A. Kalashnikova G. Grassilli E. Chiappelli F. Salvioli S. Capri M. Barbieri D. Troiano L. Monti D. Franceschi C. Exp. Cell Res. 1994; 214: 323-330Crossref PubMed Scopus (186) Google Scholar, 6Li X., Du, L. Darzynkiewicz Z. Exp. Cell Res. 2000; 257: 290-297Crossref PubMed Scopus (52) Google Scholar), thymocytes or imexon-treated myeloma cells (5Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssiere J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1089) Google Scholar, 7Dvorakova K. Waltmire C.N. Payne C.M. Tome M.E. Briehl M.M. Dorr R.T. Blood. 2001; 11: 3544-3551Crossref Scopus (74) Google Scholar), and PC-12 cells (8Satoh T. Enokido Y. Aoshima H. Uchiyama Y. Hatanaka H.J. J. Neurosci. Res. 1997; 50: 413-420Crossref PubMed Scopus (181) Google Scholar), a loss of Δψ m occur as an early change associated with apoptosis. Lymphocytes with low Δψ m show irreversible commitment to apoptosis in comparison to cells with high Δψ m that do not enter the apoptotic pathway (5Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssiere J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1089) Google Scholar). Δψ m loss can be brought about by reactive oxygen species (ROS) added directly in vitro or generated by agents that affect cellular metabolism (6Li X., Du, L. Darzynkiewicz Z. Exp. Cell Res. 2000; 257: 290-297Crossref PubMed Scopus (52) Google Scholar, 7Dvorakova K. Waltmire C.N. Payne C.M. Tome M.E. Briehl M.M. Dorr R.T. Blood. 2001; 11: 3544-3551Crossref Scopus (74) Google Scholar, 8Satoh T. Enokido Y. Aoshima H. Uchiyama Y. Hatanaka H.J. J. Neurosci. Res. 1997; 50: 413-420Crossref PubMed Scopus (181) Google Scholar). A model ROS, H2O2 itself or in combination with Na+ or rotenone can cause a loss of Δψ m in several cell types (9Ehlers R.A. Hernandez A. Bloemendal L.S. Ethridge R.T. Farrow B. Evers B.M. Surgery. 1999; 2: 148-155Abstract Full Text Full Text PDF Scopus (36) Google Scholar, 10Chinopoulos C. Tretter L. Rozsa A. Adam-Vizi V. J. Neurosci. 2000; 20: 2094-2103Crossref PubMed Google Scholar, 11Chinopoulos C. Adam-Vizi V. Ann. N. Y. Acad. Sci. 1999; 893: 269-272Crossref PubMed Scopus (6) Google Scholar). Dissipation of Δψ m primarily occurs because of the permeabilization of the inner mitochondrial membrane resulting in the release of several apoptotic factors (2Gottlieb R.A. Biol. Signals Recept. 2001; 10: 147-161Crossref PubMed Scopus (95) Google Scholar). Because Δψ m is the driving force for mitochondrial ATP synthesis, loss of the Δψ m results in ATP depletion; however, in case of the energy-requiring process of apoptosis as opposed to necrosis, a minimum ATP generation continues (12Lemasters J.J. Qian T. Bradham C.A. Brenner D.A. Cascio W.E. Trost L.C. Nishimura Y. Nieminen A.L. Herman B. J. Bioenerg. Biomembr. 1999; 31: 305-319Crossref PubMed Scopus (336) Google Scholar). Two major alterations in intermediate metabolism have been implicated in the loss of Δψ m and apoptosis. On the one hand, concentration of reduced GSH that largely determines cellular redox state is depleted early during the apoptotic process (13Anderson C.P. Tsai J.M. Meek W.E. Liu R.M. Tang Y. Forman H.J. Reynolds C.P. Exp. Cell Res. 1999; 246: 183-192Crossref PubMed Scopus (98) Google Scholar), and on the other hand, elevation of the cytosolic free Ca2+([Ca2+] c ) level is suggested to participate in the activation of nucleases that are involved in nuclear apoptosis (14Tagliarino C. Pink J.J. Dubyak G.R. Nieminen A.L. Boothman D.A. J. Biol. Chem. 2001; 276: 19150-19159Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The role of elevated [Ca2+] c in bringing about early apoptotic changes including Δψ m loss in a cell is evident from studies showing the ability of intracellular Ca2+ chelators to block apoptosis (15Kruman I.I. Mattson M.P. J. Neurochem. 1999; 72: 529-540Crossref PubMed Scopus (292) Google Scholar) and the proapoptotic changes that can be induced by Ca2+-mobilizing agents like Ca2+ ionophores or thapsigargin, responsible for release of Ca2+ from the endoplasmic reticulum (16Koya R.C. Fujita H. Shimizu S. Ohtsu M. Takimoto M. Tsujimoto Y. Kuzumaki N. J. Biol. Chem. 2000; 275: 15343-15349Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). In some cell systems, it is not only the [Ca2+] c increase but mitochondrial Ca2+ ([Ca2+] m ) overload (17Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar) as well that precipitates a decrease in Δψ m . Among other cations in addition to Ca2+, changes in Na+ and K+homeostasis are known to lead to the loss of Δψ m (18Kim J.A. Kang Y.Y. Lee Y.S. Biochem. Biophys. Res. Commun. 2001; 281: 511-519Crossref PubMed Scopus (19) Google Scholar). Together, the existing literature suggests that, depending on the cell system under investigation, alterations of Ca2+ levels in cell cytoplasm and mitochondria brought about by ROS or other agents can serve as critical signaling components leading to the activation of the apoptotic pathway. The importance of an optimum Δψ m in cell survival has been recognized not only in mammals but in yeast (19Kowaltowski A.J. Vercesi A.E. Rhee S.G. Netto L.E. FEBS Lett. 2000; 473: 177-182Crossref PubMed Scopus (55) Google Scholar) and protozoal parasites as well (20Vercesi A.E. Docampo R. Biochem. J. 1992; 284: 463-467Crossref PubMed Scopus (105) Google Scholar). The species of Leishmania donovani, a protozoal parasite, is the etiologic agent of Kala-azar, a chronic and often fatal form of human visceral Leishmaniasis (21Chang K.P. Int. Rev. Cytol. 1983; 14 (suppl.): 267-305Google Scholar). This parasite has a digenic life cycle residing as flagellated extracellular promastigotes in the gut of the insect vector. Upon transmission to the mammalian host as bloodstream-infective promastigotes, they are engulfed by the macrophages where they reside intracellularly as non-flagellated amastigotes (21Chang K.P. Int. Rev. Cytol. 1983; 14 (suppl.): 267-305Google Scholar). At the time of engulfment of the promastigotes by the macrophages, ROS is generated by the host cell in an attempt to kill the parasites (22Nabi Z.F. Rabinovitch M. Mol. Biochem. Parasitol. 1984; 10: 297-303Crossref PubMed Scopus (13) Google Scholar). ROS is also generated by anti-leishmanial drugs targeting Δψ m of amastigotes to kill them (23Rais S. Perianin A. Lenoir M. Sadak A. Rivollet D. Paul M. Deniau M. Antimicrob. Agents Chemother. 2000; 44: 2406-2410Crossref PubMed Scopus (34) Google Scholar). Existing studies suggest that drug resistance in protozoal parasites may be related to changes in Δψ m (24Basselin M. Robert-Gero M. Parasitol. Res. 1998; 84: 78-83Crossref PubMed Scopus (48) Google Scholar). Even though the importance of ROS and Δψ m is very obvious from these studies, it is not clear how ROS affects the death process in the kinetoplastid parasites. Recent studies (25Das M. Mukherjee S.B. Shaha C. J. Cell Sci. 2001; 114: 2461-2469Crossref PubMed Google Scholar) from this laboratory demonstrate thatL. donovani promastigotes undergo an apoptosis-like death in response to a well established model of a biologically active oxygen-derived intermediate, H2O2. The H2O2-induced death shares many features common to metazoan apoptosis such as nuclear condensation, DNA fragmentation, and cell shrinkage (25Das M. Mukherjee S.B. Shaha C. J. Cell Sci. 2001; 114: 2461-2469Crossref PubMed Google Scholar). The present study was designed to address the functional relationship among free radical levels, Δψ m disruption, GSH concentrations, and alterations in cytosolic Ca2+ and Na+ and mitochondrial Ca2+ in L. donovani induced by H2O2 that lead to apoptosis-like death. It is demonstrated that when death response is activated in L. donovani promastigotes by oxidative stress, a loss of Δψ m occurs. The above findings suggest that elevation of [Ca2+] c due to influx of Ca2+ through non-selective cation channels and release from intracellular stores is tightly linked to the dissipation of Δψ m that is independent of increase in cytosolic Na+, [Ca2+] m , and GSH depletion that accompanies the change in [Ca2+] c . A reduction in ATP generation occurs, but a minimal ATP level is maintained to help the cell enter the apoptosis-like pathway. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1), BAPTA-AM, fluo-3-acetoxymethyl ester (fluo-3/AM), rhod-2/acetoxymethyl ester (rhod-2/AM), 2′,7′-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,o-phthalaldehyde, ruthenium red, medium 199, and any other chemicals unless otherwise mentioned were obtained from Sigma. Promastigotes of L. donovani (UR 6) were obtained from the Cell Biology Laboratory, National Institute of Immunology, New Delhi, India. The promastigotes were cultured in blood agar as described previously (25Das M. Mukherjee S.B. Shaha C. J. Cell Sci. 2001; 114: 2461-2469Crossref PubMed Google Scholar). Briefly, routine cultures were maintained on solid blood agar slants containing 1% glucose, 5.2% brain heart infusion agar extract, and rabbit blood (6% v/v) with gentamycin at a final concentration of 1–1.5 mg/ml of medium at 25 °C. For experimental purposes, cells were recovered from 3-day-old blood agar culture in medium 199 supplemented with 10% fetal calf serum, centrifuged, resuspended in medium, and loaded onto Percoll gradient (20–90%) to remove dead cells. Live cells were collected at the interface between 40 and 90% Percoll. These cells comprising 100% motile promastigotes were washed in medium 199 and resuspended in fresh medium to achieve a culture density of 107 cells/ml. All Δψ m measurements were carried out with a potentiometric probe JC-1. Changes in Δψ m after H2O2 exposure were measured by exposing the cells to 0.1, 1, and 4 mm H2O2 and harvesting the cells at 0, 15, and 60 min and 2, 4, and 8 h for JC-1 staining. A mitochondrial uncoupler, valinomycin (100 nm), was used as a positive control for Δψ m loss. For incubations with NAC, cells were pretreated with 20 mm NAC for 3 h prior to exposure to 4 mmH2O2. Depletion of GSH was achieved by treating the cells with 1 mm BSO for 3 h. For catalase preincubations, cells were exposed to 50 I.U. catalase for 1 h prior to H2O2 exposure. To test if oxidation of cellular thiols was responsible for Δψ m loss, the cells were preincubated with different concentrations of dithiothreitol (DTT) (0.25–8 mm) prior to exposure to H2O2. For intracellular Ca2+measurements under different treatment conditions, cells were pretreated with 1.5 μm thapsigargin or 25 μm BAPTA-AM or 3 mm EGTA or 10 μm Ca2+ ionophore A23187 prior to exposure to H2O2. To determine the route of entry of Ca2+ through plasma membrane channels after H2O2 exposure, bepridil (10–500 μm), verapamil (10 μm), and flufenamic acid (30–240 μm) were used to treat the cells prior to exposure to oxidative stress. Ruthenium red, an inhibitor of mitochondrial Ca2+ uniporter, was used (25, 50 and 100 μm) to preincubate cells prior to H2O2 stress to determine the role of mitochondrial Ca2+ in Δψ m loss. To investigate if induction of mitochondrial permeability transition pore (MPTP) occurred in response to H2O2 exposure, cyclosporin A (5–10 μm), an inhibitor of MPTP, was used to preincubate cells for 1 h prior to exposure to 4 mmH2O2. To determine whether atractyloside could induce MPTP in these cells, cells were treated with atractyloside (5 nm to 5 mm) for different times (1–16 h) following which Δψ m was measured. All measurements of fluorescence were carried out with a LS-50B luminescence spectrometer (PerkinElmer Life Sciences) using FL WinlabTM software package. For confocal microscopy, a Zeiss LSM 510 (Zeiss Inc, Thornwood, NY) confocal system fitted with an upright Axioplan 2 microscope was used. Δψ m was estimated using JC-1 as a probe according to the method of Dey and Moraes (26Dey R. Moraes C.T. J. Biol. Chem. 2000; 275: 7087-7094Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) with slight modifications. JC-1 is a cationic mitochondrial vital dye that is lipophilic and becomes concentrated in the mitochondria in proportion to their Δψ m ; more dye accumulates in mitochondria with greater Δψ m and ATP-generating capacity. Therefore, the fluorescence of JC-1 can be considered as an indicator of relative mitochondrial energy state. The dye exists as a monomer at low concentrations (emission, 530 nm, green fluorescence) but at higher concentrations forms J-aggregates (emission, 590 nm, red fluorescence). JC-1 was chosen because of its reliability for analyzing Δψ m in intact cells, whereas other probes capable of binding mitochondria show a lower sensitivity or a non-coherent behavior due to a high sensitivity to changes in plasma membrane potential (27Bortner C.D. Cidlowski J.A. J. Biol. Chem. 1999; 274: 21953-21962Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 28Eric C. Dietze L. Caldwell E. Grupin S.L. Mariangela M. Seewaldt V.L. J. Biol. Chem. 2001; 276: 5384-5394Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, cells after different treatments were collected and incubated for 7 min with 10 μm JC-1 at 37 °C, washed, resuspended in media, and measured for fluorescence. The ratio of the reading at 590 nm to the reading at 530 nm (590:530 ratio) was considered as the relative Δψ m value. To ensure the viability of cells, SYTOX® Blue (50 nm) nucleic acid stain was used because it did not interfere with the red and green staining obtained with JC-1, and these cells were checked under a Nikon Optiphot fluorescence microscope (Nikon Inc., Japan). For microscopy, JC-1-stained cells were placed on slides and immediately imaged with the confocal microscopy system using plan-neofluar objectives ×40 or 100 with numerical apertures of 0.75 and 1.3, respectively. Pinhole was set at 100 μm. Images were collected after illuminating the J-monomers and -aggregates simultaneously with a 488-nm argon ion laser, and fluorescence was collected with a 500–550 bandpass filter for monomer detection and a 560 long pass filter for aggregate detection. Laser was used at 13% power. Cells were chosen for analysis on a random basis and scanned only once because the laser light could itself induce changes altering cell fluorescence. Collected images were overlapped to visualize the distribution of monomers and aggregates. Mitochondrial mass was measured with NAO, a fluorescent dye that specifically binds to the mitochondrial inner membrane independent of the transmembrane potential and is reported to measure inner mitochondrial cardiolipin content (29Thomas R.L Matsko C.M. Lotze M.T. Amoscato A.A. J. Biol. Chem. 1999; 274: 30580-30588Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Cells were stained with 0.1 μm NAO in medium 199 with 10% fetal calf serum, and fluorescence was measured with excitation at 485 nm and emission at 535 nm. Cellular GSH levels were measured in different treatment groups with the fluorescence probe o-phthalaldehyde (30Senft A.P. Dalton T.P. Shertzer H.G. Anal. Biochem. 2000; 280: 80-86Crossref PubMed Scopus (251) Google Scholar). Briefly, homogenized samples were mixed with trichloroacetic acid-redox quenching buffer (10% trichloroacetic acid in redox quenching buffer, 20 mm HCl, 5 mm diethylenetriaminepentaacetic acid, 10 mm ascorbic acid), and KPi (1:01m in 1:4 ratio) was added followed by addition of 5.5 mmol of o-phthalaldehyde. This mixture was incubated for 5 min at room temperature. o-phthalaldehyde-derived fluorescence was measured at 365-nm excitation and 430 nm emission. ATP was measured by a bioluminescence assay (31Kricka L.J. Anal. Biochem. 1988; 175: 14-21Crossref PubMed Scopus (110) Google Scholar) using an ATP determination kit. The assay is based on the requirement of luciferase for ATP in producing light (emission maximum ∼560 nm at pH 7.8). Briefly, cells (∼5 × 105) after different treatments were resuspended in reaction buffer containing 1 mm DTT, 0.5 mm luciferin, and 12.5 μg/ml luciferase and gently mixed, following which readings were taken in a luminometer (Lumicount, Packard Instrument Co.). ATP standard curves were run in all experiments with different concentrations of ATP, and calculations were made against the curve, and cellular ATP levels were expressed as nmol/106 cells. To monitor the level of ROS, the cell-permeant probe H2DCFDA was used (32Duranteau J. Chandel N.S. Kulisz A. Shao Z. Schumacker P.T. J. Biol. Chem. 1998; 273: 11619-11624Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). H2DCFDA is a nonpolar compound that readily diffuses into cells, where it is hydrolyzed to the nonfluorescent derivative dichlorodihydrofluorescein and is thereby trapped within the cells. In the presence of a proper oxidant, dichlorodihydrofluorescein is oxidized to the highly fluorescent 2,7-dichlorofluorescein. Cells of different treatment groups were resuspended in 500 μl of medium 199 and labeled with H2DCFDA (2 μg/ml) for 15 min in the dark. Fluorimetric analyses was carried out at 507 nm excitation and 530 nm emission. For all measurements, the basal fluorescence was subtracted. Changes in intracellular Ca2+ concentration [Ca2+] i were monitored with the fluorescent probe fluo-3/AM as described by González et al. (33González A. Schulz I. Schmid A. J. Biol. Chem. 2000; 275: 38680-38686Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) with slight modifications. Cells (107/ml) were loaded for 60 min at 25 °C with 5 μm fluo-3/AM containing 1 μm pluronic acid F-127 for proper dispersal, and 0.25 mm sulfinpyrazone, an organic anion transport inhibitor, was used to inhibit the leakage of the fluo-3 dye. Just before use, a sample of loaded cells was washed with medium to remove nonhydrolyzed fluo-3/AM. Fluorescence measurements were performed at 25 °C with excitation at 488 nm and emission at 522 nm. To convert fluorescence values into absolute [Ca2+] i , calibration was performed at the end of each experiment. [Ca2+] i was calculated using the following equation: [Ca2+] i =K d ((F −F min)/(F max −F)), where K d is the dissociation constant of the Ca2+·Fluo 3 complex (400 nm), and F represents the fluorescence intensity of the cells.F max represents the maximum fluorescence (obtained by treating cells with 10 μmA23187), andF min corresponds to the minimum fluorescence (obtained for ionophore-treated cells in the presence of 3 mm EGTA). Fluorescence intensities were expressed as the increase in fluorescence with respect to base-line fluorescence intensity before stimulation. For separate measurement of mitochondrial Ca2+ signals, freshly isolated cells were loaded at 4 °C for 15 min with 8 μm rhod-2/AM, centrifuged for 2 min at 30 ×g, and resuspended in media for incubation at 25 °C for 30 min to allow hydrolysis of rhod-2/AM trapped in mitochondria (33González A. Schulz I. Schmid A. J. Biol. Chem. 2000; 275: 38680-38686Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). After rhod-2 loading, cells were stored at 4 °C, and experiments were performed within 1 h. Rhod-2 fluorescence was measured with excitation at 568 nm and emission at 605 nm. To confirm that rhod-2 fluorescence signals originated from mitochondria only, promastigotes were double-loaded with 8 μm rhod-2/AM, and the fluorescent mitochondrial marker MitoTracker® Green FM (100 nm) for 30 min at room temperature (33González A. Schulz I. Schmid A. J. Biol. Chem. 2000; 275: 38680-38686Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Images of MitoTracker® Green FM fluorescence and red rhod-2 fluorescence were collected using 488 and 568 nm laser lines and detected with a 505–530 bandpass filter and a 590 nm long pass filter, respectively. Changes in intracellular K+ and Na+ ion levels of the promastigotes were monitored by loading the cells with either the potassium or sodium-sensitive fluorescent dyes PBFI-AM and SBFI-AM, respectively (34Bortner C.D. Hughes F.M. Cidlowski J.A. J. Biol. Chem. 1997; 272: 32436-32442Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar), for 60 min to a final concentration of 5 μm. Stock solutions of both dyes (2.5 mm) were prepared fresh by combining equal volumes of a 25% (w/v) pluronic acid F-127 (Molecular Probes) and the dye working solution. Fluorescence for SBFI and PBFI was monitored at excitation of 340/380 nm and emission at 500 nm. In situcalibration of the SBFI fluorescence was performed according to the method described by Zhang and Melvin (35Zhang G.H. Melvin J.E. J. Biol. Chem. 1996; 271: 29067-29072Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) using monensin (5 μm). [Na+] i was calculated according to Zhang and Melvin (35Zhang G.H. Melvin J.E. J. Biol. Chem. 1996; 271: 29067-29072Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) using 18 mm as theK d of SBFI for Na+ ion. Cells were exposed to DNA binding dye Hoechst 33342 (10 μg/ml), and apoptotic cells were monitored under a fluorescence microscope as described previously (25Das M. Mukherjee S.B. Shaha C. J. Cell Sci. 2001; 114: 2461-2469Crossref PubMed Google Scholar). TUNEL staining was performed using the in situ cell death detection kit following the standard protocol provided by the manufacturer (Promega, WI) and as modified earlier (25Das M. Mukherjee S.B. Shaha C. J. Cell Sci. 2001; 114: 2461-2469Crossref PubMed Google Scholar). Briefly, H2O2-treated promastigotes under different treatments were harvested at 6 h, fixed in 4% formaldehyde, and coated onto poly(l-lysine)-covered slides. Permeabilization was done with 0.2% (v/v) Triton X-100 and equilibration buffer (200 mm potassium cacodylate, 25 mm Tris-HCl, 0.2 mm DTT, 0.25 mg/ml bovine serum albumin, 2.5 mmcobalt chloride) for 10 min at room temperature followed by incubation with TdT buffer containing nucleotide mix (50 μmfluorescein-12-dUTP, 100 μm dATP, 10 mmTris-HCl, 1 mm EDTA, pH 7.6) for 1 h at 37 °C. The samples were counter stained with 10 μg/ml propidium iodide and visualized under the confocal microscope using illumination from a 488 nm argon-ion laser, and images were recorded through a bandpass filter 500–550 and a long pass filter at 590 nm. Data are reported as mean ± S.E. unless mentioned. Comparisons were made between different treatments using the unpaired Student's t test. Differences were considered significant at p < 0.05. Recent studies (3Cossarizza A. Kalashnikova G. Grassilli E. Chiappelli F. Salvioli S. Capri M. Barbieri D. Troiano L. Monti D. Franceschi C. Exp. Cell Res. 1994; 214: 323-330Crossref PubMed Scopus (186) Google Scholar, 4Zamzami N. Marchetti P. Castedo M. Hirsch T. Susin S.A. Masse B. Kroemer G. FEBS Lett. 1996; 384: 53-57Crossref PubMed Scopus (387) Google Scholar, 5Zamzami N. Marchetti P. Castedo M. Zanin C. Vayssiere J.L. Petit P.X. Kroemer G. J. Exp. Med. 1995; 181: 1661-1672Crossref PubMed Scopus (1089) Google Scholar) suggest that nuclear features of apoptosis in metazoan cells, like condensation of nuclei and fragmentation of DNA, are preceded by alterations in mitoch

Referência(s)