Increase in Cytosolic Ca2� Levels through the Activation ofNon-selective Cation Channels Induced by Oxidative StressCauses Mitochondrial Depolarization Leading to Apoptosis-likeDeath in Leishmania donovani Promastigotes*

Received for publication, February 27, 2002, and in revised form, April 30, 2002Published, JBC Papers in Press, April 30, 2002, DOI 10.1074/jbc.M201961200

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

From the National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India

Reactive oxygen species are important regulators ofprotozoal infection. Promastigotes of Leishmania dono-vani, the causative agent of Kala-azar, undergo an apo-ptosis-like death upon exposure to H2O2. The presentstudy shows that upon activation of death response byH2O2, a dose- and time-dependent loss of mitochondrialmembrane potential occurs. This loss is accompanied bya depletion of cellular glutathione, but cardiolipin con-tent or thiol oxidation status remains unchanged. ATPlevels are reduced within the first 60 min of exposure asa result of mitochondrial membrane potential loss. Atight link exists between changes in cytosolic Ca2� ho-meostasis and collapse of the mitochondrial membranepotential, but the dissipation of the potential is inde-pendent of elevation of cytosolic Na� and mitochondrialCa2�. Partial inhibition of cytosolic Ca2� increaseachieved by chelating extracellular or intracellularCa2� by the use of appropriate agents resulted in signif-icant rescue of the fall of the mitochondrial membranepotential and apoptosis-like death. It is further demon-strated that the increase in cytosolic Ca2� is an additiveresult of release of Ca2� from intracellular stores as wellas by influx of extracellular Ca2� through flufenamicacid-sensitive non-selective cation channels; contribu-tion of the latter was larger. Mitochondrial changes donot involve opening of the mitochondrial transitionpore as cyclosporin A is unable to prevent mitochon-drial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochon-drial membrane potential and prevent apoptosis-likedeath. Together, these findings show the importance ofnon-selective cation channels in regulating the responseof L. donovani promastigotes to oxidative stress thattriggers downstream signaling cascades leading to apo-ptosis-like death.

Mitochondria are pivotal in controlling cell life and death (1).Maintenance of proper mitochondrial transmembrane poten-tial (��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 afocus of apoptosis regulation as many investigations demon-strate a major functional impact of mitochondrial alterationson apoptosis (2). Apoptosis is a process of cell death in whichthe cells undergo nuclear and cytoplasmic shrinkage; the chro-matin is condensed and partitioned into multiple fragments,and finally the cells are broken into multiple membrane-boundbodies. In a number of experimental systems, disruption of ��m

constitutes a constant early event of the apoptotic process thatprecedes nuclear disintegration (3–5). For example, in thymo-cytes or tumor necrosis factor-stimulated U937 cells (3, 6),thymocytes or imexon-treated myeloma cells (5, 7), and PC-12cells (8), a loss of ��m occur as an early change associated withapoptosis. Lymphocytes with low ��m show irreversible com-mitment to apoptosis in comparison to cells with high ��m thatdo not enter the apoptotic pathway (5). ��m loss can be broughtabout by reactive oxygen species (ROS) added directly in vitroor generated by agents that affect cellular metabolism (6–8). Amodel ROS, H2O2 itself or in combination with Na� or rotenonecan cause a loss of ��m in several cell types (9–11). Dissipationof ��m primarily occurs because of the permeabilization of theinner mitochondrial membrane resulting in the release of sev-eral apoptotic factors (2). Because ��m is the driving force formitochondrial ATP synthesis, loss of the ��m results in ATPdepletion; however, in case of the energy-requiring process ofapoptosis as opposed to necrosis, a minimum ATP generationcontinues (12). Two major alterations in intermediate metabo-lism have been implicated in the loss of ��m and apoptosis. Onthe one hand, concentration of reduced GSH that largely de-termines cellular redox state is depleted early during the apo-ptotic process (13), and on the other hand, elevation of thecytosolic free Ca2� ([Ca2�]c) level is suggested to participate inthe activation of nucleases that are involved in nuclear apo-ptosis (14). The role of elevated [Ca2�]c in bringing about earlyapoptotic changes including ��m loss in a cell is evident fromstudies showing the ability of intracellular Ca2� chelators toblock apoptosis (15) and the proapoptotic changes that can beinduced by Ca2�-mobilizing agents like Ca2� ionophores orthapsigargin, responsible for release of Ca2� from the endo-

* This work was supported by the Indian Council of Medical Re-search, Department of Biotechnology, Government of India, and a fel-lowship from the Council of Scientific and Industrial Research (toS. B. M.). The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ To whom correspondence should be addressed: National Institute ofImmunology, Aruna Asaf Ali Marg, New Delhi 110067, India. Fax:11-616-2125, E-mail: cshaha@nii.res.in.

1 The abbreviations used are: ��m, mitochondrial transmembranepotential; ROS, reactive oxygen species; JC-1, 5,5�,6,6�-tetrachloro-1,1�,3,3�-tetraethylbenzimidazole carbocyanide iodide; NAC, N-acetyl-

cysteine; BSO, butathione sulfoximine; DTT, dithiothreitol; MPTP, mi-tochondrial permeability transition pore; PTP, permeability transitionpore; H2DCFDA, 2�,7�-dichlorofluorescein diacetate; [Ca2�]c, cytosolicCa2�; [Ca2�]m, mitochondrial Ca2�; [Ca2�]i, intracellular Ca2�; SBFI-AM, sodium-binding benzofuran isophthalate acetoxymethyl ester;PBFI-AM, potassium-binding benzofuran isophthalate acetoxymethylester; TUNEL, terminal deoxynucleotidyltransferase enzyme (TdT)-mediated dUTP nick-end labeling; TG, thapsigargin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N�,N�-tetraacetic acid-acetoxymethylester; FFA, flufenamic acid; fluo-3/AM, fluo-3-acetoxymethyl ester;rhod-2/AM, rhod-2/acetoxymethyl ester; NAO, nonyl acridine orange.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 27, Issue of July 5, pp. 24717–24727, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 24717

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

plasmic reticulum (16). In some cell systems, it is not only the[Ca2�]c increase but mitochondrial Ca2� ([Ca2�]m) overload(17) as well that precipitates a decrease in ��m. Among othercations in addition to Ca2�, changes in Na� and K� homeosta-sis are known to lead to the loss of ��m (18). Together, theexisting literature suggests that, depending on the cell systemunder investigation, alterations of Ca2� levels in cell cytoplasmand mitochondria brought about by ROS or other agents canserve as critical signaling components leading to the activationof the apoptotic pathway. The importance of an optimum ��m

in cell survival has been recognized not only in mammals but inyeast (19) and protozoal parasites as well (20).

The species of Leishmania donovani, a protozoal parasite, isthe etiologic agent of Kala-azar, a chronic and often fatal formof human visceral Leishmaniasis (21). This parasite has adigenic life cycle residing as flagellated extracellular promas-tigotes in the gut of the insect vector. Upon transmission to themammalian host as bloodstream-infective promastigotes, theyare engulfed by the macrophages where they reside intracellu-larly as non-flagellated amastigotes (21). At the time of engulf-ment of the promastigotes by the macrophages, ROS is gener-ated by the host cell in an attempt to kill the parasites (22).ROS is also generated by anti-leishmanial drugs targeting ��m

of amastigotes to kill them (23). Existing studies suggest thatdrug resistance in protozoal parasites may be related tochanges in ��m (24). Even though the importance of ROS and��m is very obvious from these studies, it is not clear how ROSaffects the death process in the kinetoplastid parasites.

Recent studies (25) from this laboratory demonstrate thatL. donovani promastigotes undergo an apoptosis-like death inresponse to a well established model of a biologically activeoxygen-derived intermediate, H2O2. The H2O2-induced deathshares many features common to metazoan apoptosis such asnuclear condensation, DNA fragmentation, and cell shrinkage(25). The present study was designed to address the functionalrelationship among free radical levels, ��m disruption, GSHconcentrations, and alterations in cytosolic Ca2� and Na� andmitochondrial Ca2� in L. donovani induced by H2O2 that leadto apoptosis-like death. It is demonstrated that when deathresponse is activated in L. donovani promastigotes by oxidativestress, a loss of ��m occurs. The above findings suggest thatelevation of [Ca2�]c due to influx of Ca2� through non-selectivecation channels and release from intracellular stores is tightlylinked to the dissipation of ��m that is independent of increasein cytosolic Na�, [Ca2�]m, and GSH depletion that accompaniesthe change in [Ca2�]c. A reduction in ATP generation occurs,but a minimal ATP level is maintained to help the cell enter theapoptosis-like pathway.

EXPERIMENTAL PROCEDURES

Reagents

5,5�,6,6�-Tetrachloro-1,1�,3,3�-tetraethylbenzimidazole carbocyanideiodide (JC-1), BAPTA-AM, fluo-3-acetoxymethyl ester (fluo-3/AM),rhod-2/acetoxymethyl ester (rhod-2/AM), 2�,7�-dichlorofluorescein diac-etate (H2DCFDA), sulfinpyrazone, pluronic acid F-127, MitoTracker�Green FM, SYTOX� Blue nucleic acid stain, nonyl acridine orange(NAO), potassium-binding benzofuran isophthalate acetoxymethyl es-ter (PBFI-AM), sodium-binding benzofuran isophthalate acetoxymethylester (SBFI-AM), and ATP determination kit were obtained from Mo-lecular Probes (Eugene, OR). Terminal deoxynucleotidyltransferase en-zyme (TdT)-mediated dUTP nick-end labeling (TUNEL) kit was fromPromega (Madison, WI). Cyclosporin A, atractyloside, butathione sul-foximine (BSO), reduced GSH, N-acetylcysteine (NAC), digitonin, o-phthalaldehyde, ruthenium red, medium 199, and any other chemicalsunless otherwise mentioned were obtained from Sigma.

Promastigote Culture

Promastigotes of L. donovani (UR 6) were obtained from the CellBiology Laboratory, National Institute of Immunology, New Delhi, In-

dia. The promastigotes were cultured in blood agar as described previ-ously (25). Briefly, routine cultures were maintained on solid blood agarslants containing 1% glucose, 5.2% brain heart infusion agar extract,and rabbit blood (6% v/v) with gentamycin at a final concentration of1–1.5 mg/ml of medium at 25 °C. For experimental purposes, cells wererecovered from 3-day-old blood agar culture in medium 199 supple-mented with 10% fetal calf serum, centrifuged, resuspended in medium,and loaded onto Percoll gradient (20–90%) to remove dead cells. Livecells were collected at the interface between 40 and 90% Percoll. Thesecells comprising 100% motile promastigotes were washed in medium199 and resuspended in fresh medium to achieve a culture density of107 cells/ml.

Cell Treatments

All ��m measurements were carried out with a potentiometric probeJC-1. Changes in ��m after H2O2 exposure were measured by exposingthe cells to 0.1, 1, and 4 mM H2O2 and harvesting the cells at 0, 15, and 60min and 2, 4, and 8 h for JC-1 staining. A mitochondrial uncoupler,valinomycin (100 nM), was used as a positive control for ��m loss. Forincubations with NAC, cells were pretreated with 20 mM NAC for 3 h priorto exposure to 4 mM H2O2. Depletion of GSH was achieved by treating thecells with 1 mM BSO for 3 h. For catalase preincubations, cells wereexposed to 50 I.U. catalase for 1 h prior to H2O2 exposure. To test ifoxidation of cellular thiols was responsible for ��m loss, the cells werepreincubated with different concentrations of dithiothreitol (DTT) (0.25–8mM) prior to exposure to H2O2. For intracellular Ca2� measurementsunder different treatment conditions, cells were pretreated with 1.5 �M

thapsigargin or 25 �M BAPTA-AM or 3 mM EGTA or 10 �M Ca2� iono-phore A23187 prior to exposure to H2O2. To determine the route of entryof Ca2� through plasma membrane channels after H2O2 exposure, bepri-dil (10–500 �M), verapamil (10 �M), and flufenamic acid (30–240 �M) wereused to treat the cells prior to exposure to oxidative stress. Rutheniumred, an inhibitor of mitochondrial Ca2� uniporter, was used (25, 50 and100 �M) to preincubate cells prior to H2O2 stress to determine the role ofmitochondrial Ca2� in ��m loss. To investigate if induction of mitochon-drial permeability transition pore (MPTP) occurred in response to H2O2

exposure, cyclosporin A (5–10 �M), an inhibitor of MPTP, was used topreincubate cells for 1 h prior to exposure to 4 mM H2O2. To determinewhether atractyloside could induce MPTP in these cells, cells were treatedwith atractyloside (5 nM to 5 mM) for different times (1–16 h) followingwhich ��m was measured.

Detection of Fluorescence

All measurements of fluorescence were carried out with a LS-50Bluminescence spectrometer (PerkinElmer Life Sciences) using FL Win-labTM software package. For confocal microscopy, a Zeiss LSM 510(Zeiss Inc, Thornwood, NY) confocal system fitted with an uprightAxioplan 2 microscope was used.

Measurement of Mitochondrial Changes

Mitochondrial Membrane Potential Determinations—��m was esti-mated using JC-1 as a probe according to the method of Dey and Moraes(26) with slight modifications. JC-1 is a cationic mitochondrial vital dyethat is lipophilic and becomes concentrated in the mitochondria inproportion to their ��m; more dye accumulates in mitochondria withgreater ��m and ATP-generating capacity. Therefore, the fluorescenceof JC-1 can be considered as an indicator of relative mitochondrialenergy state. The dye exists as a monomer at low concentrations (emis-sion, 530 nm, green fluorescence) but at higher concentrations formsJ-aggregates (emission, 590 nm, red fluorescence). JC-1 was chosenbecause of its reliability for analyzing ��m in intact cells, whereas otherprobes capable of binding mitochondria show a lower sensitivity or anon-coherent behavior due to a high sensitivity to changes in plasmamembrane potential (27, 28). Briefly, cells after different treatmentswere collected and incubated for 7 min with 10 �M JC-1 at 37 °C,washed, resuspended in media, and measured for fluorescence. Theratio of the reading at 590 nm to the reading at 530 nm (590:530 ratio)was considered as the relative ��m value. To ensure the viability ofcells, SYTOX� Blue (50 nM) nucleic acid stain was used because it didnot interfere with the red and green staining obtained with JC-1, andthese cells were checked under a Nikon Optiphot fluorescence micro-scope (Nikon Inc., Japan).

For microscopy, JC-1-stained cells were placed on slides and immedi-ately imaged with the confocal microscopy system using plan-neofluarobjectives �40 or 100 with numerical apertures of 0.75 and 1.3, respec-tively. Pinhole was set at 100 �m. Images were collected after illuminat-ing the J-monomers and -aggregates simultaneously with a 488-nm argon

Depolarization Leads to Apoptosis-like Death in L. donovani24718

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

ion laser, and fluorescence was collected with a 500–550 bandpass filterfor monomer detection and a 560 long pass filter for aggregate detection.Laser was used at 13% power. Cells were chosen for analysis on a randombasis and scanned only once because the laser light could itself inducechanges altering cell fluorescence. Collected images were overlapped tovisualize the distribution of monomers and aggregates.

Measurement of Mitochondrial Mass—Mitochondrial mass wasmeasured with NAO, a fluorescent dye that specifically binds to themitochondrial inner membrane independent of the transmembrane po-tential and is reported to measure inner mitochondrial cardiolipin con-tent (29). Cells were stained with 0.1 �M NAO in medium 199 with 10%fetal calf serum, and fluorescence was measured with excitation at 485nm and emission at 535 nm.

GSH Levels

Cellular GSH levels were measured in different treatment groupswith the fluorescence probe o-phthalaldehyde (30). Briefly, homoge-nized samples were mixed with trichloroacetic acid-redox quenchingbuffer (10% trichloroacetic acid in redox quenching buffer, 20 mM HCl,5 mM diethylenetriaminepentaacetic acid, 10 mM ascorbic acid), and KPi

(1:01 M in 1:4 ratio) was added followed by addition of 5.5 mmol ofo-phthalaldehyde. This mixture was incubated for 5 min at room tem-perature. o-phthalaldehyde-derived fluorescence was measured at365-nm excitation and 430 nm emission.

ATP Measurements

ATP was measured by a bioluminescence assay (31) using an ATPdetermination kit. The assay is based on the requirement of luciferasefor ATP in producing light (emission maximum �560 nm at pH 7.8).Briefly, cells (�5 � 105) after different treatments were resuspended inreaction buffer containing 1 mM DTT, 0.5 mM luciferin, and 12.5 �g/mlluciferase and gently mixed, following which readings were taken in aluminometer (Lumicount, Packard Instrument Co.). ATP standardcurves were run in all experiments with different concentrations ofATP, and calculations were made against the curve, and cellular ATPlevels were expressed as nmol/106 cells.

ROS Levels

To monitor the level of ROS, the cell-permeant probe H2DCFDA wasused (32). H2DCFDA is a nonpolar compound that readily diffuses intocells, where it is hydrolyzed to the nonfluorescent derivative dichlorodi-hydrofluorescein and is thereby trapped within the cells. In the pres-ence of a proper oxidant, dichlorodihydrofluorescein is oxidized to thehighly fluorescent 2,7-dichlorofluorescein. Cells of different treatmentgroups were resuspended in 500 �l of medium 199 and labeled withH2DCFDA (2 �g/ml) for 15 min in the dark. Fluorimetric analyses wascarried out at 507 nm excitation and 530 nm emission. For all meas-urements, the basal fluorescence was subtracted.

Measurement of Cytosolic and MitochondrialFree Ca2� Concentrations

Changes in intracellular Ca2� concentration [Ca2�]i were monitoredwith the fluorescent probe fluo-3/AM as described by Gonzalez et al.(33) with slight modifications. Cells (107/ml) were loaded for 60 min at25 °C with 5 �M fluo-3/AM containing 1 �M pluronic acid F-127 forproper dispersal, and 0.25 mM sulfinpyrazone, an organic anion trans-port inhibitor, was used to inhibit the leakage of the fluo-3 dye. Justbefore use, a sample of loaded cells was washed with medium to removenonhydrolyzed fluo-3/AM. Fluorescence measurements were performedat 25 °C with excitation at 488 nm and emission at 522 nm. To convertfluorescence values into absolute [Ca2�]i, calibration was performed atthe end of each experiment. [Ca2�]i was calculated using the followingequation: [Ca2�]i � Kd((F � Fmin)/(Fmax � F)), where Kd is the dissoci-ation constant of the Ca2��Fluo 3 complex (400 nM), and F representsthe fluorescence intensity of the cells. Fmax represents the maximumfluorescence (obtained by treating cells with 10 �M A23187), and Fmin

corresponds to the minimum fluorescence (obtained for ionophore-treated cells in the presence of 3 mM EGTA). Fluorescence intensitieswere expressed as the increase in fluorescence with respect to base-linefluorescence intensity before stimulation.

For separate measurement of mitochondrial Ca2� signals, freshlyisolated cells were loaded at 4 °C for 15 min with 8 �M rhod-2/AM,centrifuged for 2 min at 30 � g, and resuspended in media for incuba-tion at 25 °C for 30 min to allow hydrolysis of rhod-2/AM trapped inmitochondria (33). After rhod-2 loading, cells were stored at 4 °C, andexperiments were performed within 1 h. Rhod-2 fluorescence was meas-ured with excitation at 568 nm and emission at 605 nm. To confirm that

rhod-2 fluorescence signals originated from mitochondria only, promas-tigotes were double-loaded with 8 �M rhod-2/AM, and the fluorescentmitochondrial marker MitoTracker� Green FM (100 nM) for 30 min atroom temperature (33). Images of MitoTracker� Green FM fluorescenceand red rhod-2 fluorescence were collected using 488 and 568 nm laserlines and detected with a 505–530 bandpass filter and a 590 nm longpass filter, respectively.

Measurement of Intracellular Na� and K� Level

Changes in intracellular K� and Na� ion levels of the promastigoteswere monitored by loading the cells with either the potassium or sodium-sensitive fluorescent dyes PBFI-AM and SBFI-AM, respectively (34), for60 min to a final concentration of 5 �M. Stock solutions of both dyes (2.5mM) 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 340/380nm and emission at 500 nm. In situ calibration of the SBFI fluorescencewas performed according to the method described by Zhang and Melvin(35) using monensin (5 �M). [Na�]i was calculated according to Zhang andMelvin (35) using 18 mM as the Kd of SBFI for Na� ion.

Assessment of Apoptosis by Hoechst and TUNEL Staining

Cells were exposed to DNA binding dye Hoechst 33342 (10 �g/ml),and apoptotic cells were monitored under a fluorescence microscope asdescribed previously (25). TUNEL staining was performed using the insitu cell death detection kit following the standard protocol provided bythe manufacturer (Promega, WI) and as modified earlier (25). Briefly,H2O2-treated promastigotes under different treatments were harvestedat 6 h, fixed in 4% formaldehyde, and coated onto poly(L-lysine)-coveredslides. Permeabilization was done with 0.2% (v/v) Triton X-100 andequilibration buffer (200 mM potassium cacodylate, 25 mM Tris-HCl, 0.2mM DTT, 0.25 mg/ml bovine serum albumin, 2.5 mM cobalt chloride) for10 min at room temperature followed by incubation with TdT buffercontaining nucleotide mix (50 �M fluorescein-12-dUTP, 100 �M dATP,10 mM Tris-HCl, 1 mM EDTA, pH 7.6) for 1 h at 37 °C. The samples werecounter stained with 10 �g/ml propidium iodide and visualized underthe confocal microscope using illumination from a 488 nm argon-ionlaser, and images were recorded through a bandpass filter 500–550 anda long pass filter at 590 nm.

Statistical Analyses

Data are reported as mean � S.E. unless mentioned. Comparisonswere made between different treatments using the unpaired Student’st test. Differences were considered significant at p 0.05.

RESULTS

Effect of H2O2 on Mitochondrial Membrane Potential,GSH and ATP Levels, and Cardiolipin Content

H2O2 Induces Dose- and Time-dependent Loss of ��m—Re-cent studies (3–5) suggest that nuclear features of apoptosis inmetazoan cells, like condensation of nuclei and fragmentationof DNA, are preceded by alterations in mitochondrial structureand transmembrane potential. Previous observations (25) fromthis laboratory showed that oxidative stress induced DNA frag-mentation 6 h after H2O2 treatment in L. donovani promasti-gotes. In this study, we sought to dissect the cells early re-sponse to H2O2-induced stress in terms of mitochondrialchanges. Simultaneous measurement of J-aggregate (indica-tive of intact mitochondria) and J-monomer (indicative of de-energized mitochondria) formation expressed as the ratio of590:530 fluorescence showed a progressive loss of ��mvalueswith increasing dose and time of exposure to H2O2 (Fig. 1).With 4 mM H2O2, there was a significant fall (63%) in ��m

within the first 15 min as compared with relative ��m observedat 0 h. ��m values did not significantly change further untilabout 4 h, and another prominent drop occurred at 8 h post-H2O2 treatment. The dose of 0.1 mM did induce a slight fall of��m, but this alteration was not significant, although 1 mM

H2O2 by 4 h was able to induce a notable fall in ��m. Theconcomitant increase in JC-1 monomeric fluorescence observedwith the loss of J-aggregate fluorescence suggests that the lossof JC-1 aggregates was not due to an overall loss of this dye

Depolarization Leads to Apoptosis-like Death in L. donovani 24719

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

from the cell. As a positive control we used valinomycin, amitochondrial uncoupler (36) where aggregate/monomer ratiowas 1.5 � 0.3 (n � 4) in comparison to control values of 6.5 �0.8 (n � 4). From the above data it can be inferred that the ��m

in promastigotes is sensitive to increasing the degree of oxida-tive stress similar to what was observed in metazoan cells(6–11). The early loss of potential is indicative of considerablecellular changes taking place soon after the application of oxi-dative stress. In these studies, maximal interference with ��m

was achieved with 4 mM H2O2, a dose that induces apoptosis-like death in the promastigotes (25). For all further experi-ments, 4 mM H2O2 was used to determine early changes leadingto ��m loss and the subsequent apoptosis-like death.

Because spectrofluorimetric results represent an averaged orintegrated estimate, we analyzed the JC-1-stained promasti-gote mitochondria with a confocal microscope over a period oftime. Fig. 2A, a and b, shows the distribution of JC-1 dye inintact mitochondria of a L. donovani promastigote. Fig. 2A, c–f,shows changes that occur in the promastigote mitochondriaafter exposure to oxidative stress. Mitochondria fluoresce redbefore exposure to H2O2, indicating an intact ��m (Fig. 2A, c).Sixty min and 4 h after exposure to 4 mM H2O2, an increasednumber of mitochondria show a heterogeneous staining pat-tern (Fig. 2A, d and e), and at 8 h most of the mitochondriafluoresce green (Fig. 2A, f). Under higher magnification it wasevident that although mitochondria from control promastigotesshowed red fluorescence throughout the mitochondria (Fig. 2B,a–c), treated promastigote mitochondria had a distinct stainingpattern with areas of both red and green that at some placesoverlapped as yellow, indicating a very close placement of re-gions with high and low ��m (Fig. 2B, d–f). An actual count ofcells exposed to H2O2 for different periods of time show that thenumber of cells with mitochondria showing heterogeneousstaining pattern increased from 30 min to 4 h, and finally at 8 hthe number of cells with complete green fluorescent mitochon-dria became prevalent (Table I). It can be speculated that thisheterogeneous staining pattern in the H2O2-treated mitochon-dria as opposed to mitochondria before treatment or untreatedmitochondria indicated variations in membrane potential (and

corresponding level of activity) imposed by H2O2 treatment.Yellow or red fluorescence separated by areas of green fluores-cence along the length of mitochondria has been reported innormal human fibroblasts (37) and in the processes of oligo-dendrocytes (38). Cells with red mitochondria or with hetero-geneous staining pattern did not show significant number ofblue nuclei with vital dye SYTOX� Blue showing that cellswere viable under these conditions of mitochondrial staining.In controls, Sytox blue staining was confined to 3% cells only.Sytox blue-positive cells showed green mitochondria under anormal fluorescence microscope (data not shown). From theabove data it was evident that the single mitochondrion ofL. donovani undergoes an early dissipation of ��m upon expo-sure to H2O2 and turns to a lower activity state. This change is

FIG. 1. Dissipation of ��m occurs in L. donovani promastigotesafter exposure to H2O2. L. donovani promastigotes (107/ml) wereexposed to different doses of H2O2 (0.1, 1, and 4 mM) in vitro for thetimes indicated and were subsequently stained with the potentiometricprobe JC-1 (10 �M). Dose and time-dependent changes of relative ��mvalues are expressed as the ratio of the reading at 590 nm (aggregate)to the reading at 530 nm (monomer). Data are � S.D. of four experi-ments. **, p 0.001 in comparison to 0-h values.

FIG. 2. During the early phase of H2O2 exposure the ��m loss isnot uniform throughout the mitochondria. Promastigotes wereharvested after 60 min of exposure to 4 mM H2O2 and stained with 10�M JC-1 for 7 min at 37 °C, washed, and placed on slides. The dye wasexcited with a 488 nm argon-ion laser, and monomer and aggregatedetection was carried out with a 500–550 nm bandpass filter and a 560long pass filter, respectively. A, visualization of mitochondria at differ-ent time points after treatment with 4 mM H2O2 showing localization ofJ-aggregates and J-monomers. a, J-aggregate distribution in a promas-tigote mitochondria; b, phase contrast image of “a” overlapped withJ-aggregate staining; c, mitochondria from cells without exposure toH2O2; d, mitochondria from cells after 60 min exposure to H2O2; e,mitochondria from cells after exposure to H2O2 for 4 h; f, mitochondriafrom cells after 8 h of exposure to H2O2 (m, mitochondria; n, nucleus; t,tail). B, enhanced photomicrograph of one representative mitochon-drion. a–c, mitochondrion from a cell before treatment with H2O2; a,visualization of J-monomers; b, visualization of J-aggregates; c, co-localization of J-aggregates and J-monomers. d–f, mitochondrion from acell exposed to 4 mM H2O2; d, visualization of J-monomers; e, visualiza-tion of J-aggregates; f, co-localization of J-aggregates and J-monomers.

TABLE IStatus of mitochondria during exposure to H2O2

Table represents the percent of mitochondria with different stainingpatterns from promastigotes before and after treatment with H2O2showing the number of mitochondria with a particular staining pattern(n � 6).

Time of exposureto H2O2

Staining pattern

Red Red/green Green

min

0 97 � 12 2 � 0.18 1 � 0.130 40 � 5a 50 � 4.5a 10 � 160 19 � 2a 75 � 6.7a 6 � 0.4

240 15 � 2a 80 � 7.5a 5 � 0.5480 5 � 1 20 � 1.8 75 � 8.0a

a p 0.01.

Depolarization Leads to Apoptosis-like Death in L. donovani24720

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

dependent on the dose and duration of H2O2 exposure. Visualevidence suggests that there is a regional heterogeneity in ��m

loss in the mitochondria upon exposure to oxidative stress thatis indicative of partially active mitochondria.

H2O2 Induces GSH Depletion—Because H2O2 was inducing aloss of ��m, we examined whether there was a depletion ofantioxidant molecules like GSH that was in turn contributingtowards the dissipation of the potential. Even a partial deple-tion of GSH, an important molecule for protecting organismsincluding kinetoplastids and metazoans from ROS or toxiccompounds (39), would be detrimental to the cell and mayinduce a loss of ��m (40). We also wanted to see if antioxidantslike NAC could inhibit the loss of mitochondrial function. NACis a unique compound, which with its multiple activities caneither work via its ROS scavenging ability by increasing intra-cellular GSH levels or by serving as a reducing agent. GSHlevels decreased concomitantly with the fall of ��m (Fig. 3A,inset). Preincubation of cells with 20 mM NAC prior to exposureto 4 mM H2O2 increased cellular GSH levels (Fig. 3A) andsignificantly inhibited the loss of ��m (Fig. 3B). It was specu-

lated from the above data that the protective action of NAC waspossibly through a replenishment of intracellular GSH levelsas it is seen in mammalian cells exposed to different ROSgenerating agents like imexon (7) and perfluoro-octanoic acid(41). Arguably, if this was the case, a cell depleted of GSHshould also show a dissipation of ��m. To substantiate this, weused BSO, a �-glutamylcysteine synthetase inhibitor (42), tolower promastigote GSH levels, and by 60 min achieved a 77%depletion (Fig. 3A). Interestingly, this depletion did not bringabout a ��m loss (Fig. 3B) indicating that a disturbance in GSHlevel was not sufficient to cause a fall in ��m. Our results areconsistent with earlier observations in L. donovani where a lossof GSH was shown to be merely cytostatic (43). We also ob-served that H2O2 treatment did not make the cells more sus-ceptible to BSO-induced GSH depletion, and NAC preincuba-tion in the presence of BSO did not increase GSH levels. NACin certain instances activates synthesis of relevant proteins(44) that help in the protection of a cell. This was clearly not themode of action of NAC as NAC was still able to inhibit the fallof ��m induced by H2O2 by about 60% in the presence ofcycloheximide (1 �g/ml), a protein synthesis inhibitor. Inhibi-tion of the loss of ��m by NAC was 70%, and in the presence ofcycloheximide this inhibition was 60%. The remaining possi-bility was that NAC acted via its ROS scavenging ability sothat cellular changes associated with increased ROS were pre-vented. ROS levels measured with H2DCFDA, a dye that isconverted to a fluorescent product upon oxidation to H2O2 (32),showed that 20 mM NAC could completely quench the increaseof intracellular ROS when cells were preincubated with it (Fig.4A). Clearly, there was no secondary increase in ROS levels dueto lowered GSH levels, as depletion of GSH affected by BSO didnot increase the intracellular level of ROS further (Fig. 4B).The ability of catalase to prevent the fall in ��m (Table II)reiterates that a decreased level of H2O2 within the cells couldprevent a fall in ��m (Fig. 4C). H2O2 can induce oxidation ofsulfhydryl groups (45), but preincubation of the cells with dif-ferent concentrations of DTT, a sulfhydryl-reducing agent priorto treatment with H2O2, could not effect an inhibition of theloss of ��m (Table II). Inhibition of OH� radical formation bysodium formate (1 mM) did not give any protection to the fall of��m (Table II); therefore, it was H2O2 itself rather than OH�

radicals generated by it that affected mitochondrial function.The above experiments suggested that a lowered GSH level inresponse to H2O2 was not related to the lowering of ��m. NACwas able to prevent the fall of ��m, but its ability to act as anintracellular antioxidant (46) was more important than itscapability to function as a modifier to intracellular thiol levels(47) in preventing H2O2 induced ��m loss.

Fall in Cellular ATP Content Occurs following ��m Loss—Because disruption in the function of mitochondria translatesinto reduced ATP generation, levels of ATP were measuredafter exposure to H2O2. ATP level is important as progressionto necrosis or apoptosis depends on the availability of ATP (12).There was a gradual fall in the ATP levels resulting to about80% loss by 60 min (Fig. 5A). Because more than 95% of cellsare viable at a 60-min post-H2O2 exposure, it can be speculatedthat generation of ATP during the 1st h is sufficient to sustaincellular activity and direct the cell toward an apoptotic path-way. The first significant drop in ATP levels at 20 min showsthat lowering of cellular ATP occurs after ��m loss is initiated.Preincubation of cells with 20 mM NAC prior to H2O2 treatmentprevented the fall in ATP levels as measured at 60 min, pre-sumably an outcome of reduced ROS within the cells thatprevented ��m loss (Fig. 5B). BSO treatment did not induce afall in ATP levels showing that depletion of GSH did not inter-fere with ATP generation.

FIG. 3. NAC is able to prevent the loss of ��m, but depletion ofGSH by BSO is unable to induce any alterations in ��m. A,intracellular GSH levels as measured using o-phthalaldehyde with cellsexposed to different treatments measured at 60-min time point. a,control; b, 4 mM H2O2; c, cells preincubated with 20 mM NAC prior totreatment with 4 mM H2O2; d, cells treated with 20 mM NAC only; e,cells treated with 1 mM BSO only; f, cells treated with 1 mM BSO priorto treatment with 4 mM H2O2; g, cells treated with 1 mM BSO and 20 mM

NAC only; h, cells preincubated with 20 mM NAC and 1 mM BSO priorto treatment with 4 mM H2O2. Inset shows changes in GSH levels withinthe first 60 min after H2O2 exposure. Results are mean � S.D. of threeexperiments. **, p 0.001 as compared with a. B, ��m was measuredusing JC-1 potentiometric probe at 60 min after various treatments.Groups are same as A. Results are mean � S.D. of three experiments.**, p 0.001 as compared with a.

Depolarization Leads to Apoptosis-like Death in L. donovani 24721

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

Preincubation of Cells with NAC Reduces the Number of Apo-ptotic Cells and DNA Fragmentation—Because it was evidentfrom the data described so far that one of the early events asso-ciated with apoptosis-like death in L. donvani following H2O2

exposure was the loss of ��m followed by a reduction in ATPgeneration, the next question was whether this change was re-lated to cellular apoptosis and if so whether NAC can salvagecellular apoptosis as it prevented the loss of ��m. NAC was ableto reduce significantly the number of cells with a nuclei showing

apoptosis-like changes as observed by Hoechst staining of thenuclei (Fig. 5C). Even though the effect of NAC on kinetoplastiddeath is not known, it protects several types of mammalian cellsfrom undergoing apoptosis (46, 47). Depletion of GSH by BSO didnot increase apoptosis-like death (Fig. 5C), and these data are inagreement with observations showing that depletion of GSH doesnot precipitate cell death in L. donovani (43). In agreement withour observations that NAC pretreatment prior to H2O2 exposurereduced ��m loss, restored ATP levels, and reduced the numberof apoptotic cells, no DNA fragmentation was observed in NAC-treated groups (Fig. 5D, lane c) in comparison to the DNA frag-mentation observed in H2O2-treated promastigotes (Fig. 5D, laneb). Taken together, the above data suggested that H2O2 didinduce a decrease in ATP generation, but sufficient levels weremaintained until apoptosis-like changes were initiated. NAC wasable to inhibit ATP loss and DNA fragmentation and conse-quently to reduce the number of cells undergoing apoptosis-likedeath.

Status of Cardiolipin Does Not Change after H2O2 Treat-ment—Caridolipin, a major mitochondrial lipid, is often alteredfollowing exposure to reactive oxygen species (48) that resultsin mitochondrial dysfunction. There was no alteration in car-diolipin content as measured by NAO within the 1st h afterexposure. Both the control and the treated groups showedreadings that did not differ significantly (readings at excitation485 nm and emission at 535 nm; control, 641 � 56 (n � 3);treated, 651 � 60 (n � 3)). Therefore, mitochondrial mass orcardiolipin content was not affected by H2O2.

H2O2 Induces Elevation of Cytosolic Calcium

Fall in ��m Is Related to an Increase in Cytosolic Ca2�—Within first 10 min of exposure to H2O2, cytosolic Ca2� levelsbegan to increase. In the presence of extracellular EGTA or theabsence Ca2� in the medium a partial elevation in [Ca2�]ioccurred, but this was not as high as the levels achieved whenextracellular Ca2� was present (Fig. 6A). [Ca2�]c increase wasalso seen with thapsigargin (Fig. 6A) and Ca2� ionophoreA23187 (data not shown) in the absence of H2O2, but theincrease achieved with thapsigargin was not additive in thepresence of H2O2, showing that H2O2 also released Ca2� fromthapsigargin-sensitive intracellular stores. There was an in-crease in cytosolic Na� level post-treatment that occurredalong with the increase in [Ca2�]c, and the increase was com-parable with monensin-induced levels (Fig. 6B). There was noincrease in K� levels as measured by PBFI fluorescence (datanot shown). When ��m loss induced by H2O2 was checked

FIG. 4. ROS levels are scavenged by NAC and catalase, andBSO does not induce generation of ROS. ROS levels were meas-ured with the fluorescent dye H2DCFDA (2 �g/107 cells) preloaded ontocells. A shows complete scavenging of ROS levels in cells preincubatedwith 20 mM NAC prior to 4 mM H2O2 treatment at the time pointsindicated. B, ROS levels in cells treated with 1 mM BSO only and with4 mM H2O2 showing no increased generation of ROS when cells weredepleted of GSH. C, figure showing complete scavenging of ROS levelsin cells pretreated with 50 I.U. of catalase prior to exposure to 4 mM

H2O2. Results are mean � S.D. of three experiments.

TABLE IIEffect of different treatments on the mitochondrial membrane

potential of L. donovani as measured by JC-1 fluorescenceChanges in mitochondrial membrane potential after different treat-

ments to the promastigotes of L. donovani.

Groups �m

Control 6.3 � 0.9 (n � 6)4 mM H2O2 1.6 � 0.4 (n � 3)a

4 mM H2O2 � catalase (50 I.U.) 7.1 � 0.9 (n � 3)4 mM H2O2 � DTT (2 mM) 1.8 � 0.7 (n � 3)a

4 mM H2O2 � sodium formate (1 mM) 2.0 � 0.4 (n � 4)a

4 mM H2O2 � BAPTA-AM (25 �M) 5.9 � 0.9 (n � 3)4 mM H2O2 (in sodium-free media) 2.2 � 0.4 (n � 3)a

4 mM H2O2 � ruthenium red (100 �M) 1.6 � 0.6 (n � 3)a

4 mM H2O2 � verapamil (10 �M) 2.0 � 0.3 (n � 3)a

4 mM H2O2 � bepridil (500 �M) 1.9 � 0.6 (n � 3)a

a All data represent � S.E. of four experiments, p 0.001. DTT wasused in doses of 500 �M, 1 mM, and 2 mM; sodium formate was used atdoses of 100 and 500 �M and 1 mM; BAPTA-AM was used at 10 and 25�M; bepridil was used in the doses of 10, 50, 100, 250, and 500 �M;ruthenium red was used in the doses of 10, 25, 50, and 100 �M. Dataobtained at the highest doses are represented.

Depolarization Leads to Apoptosis-like Death in L. donovani24722

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

under different conditions of altered Ca2� uptake, in the pres-ence of 3 mM EGTA or the absence of Ca2� in the medium, H2O2

could induce only a 25% fall of ��m (Fig. 7, A and B) incomparison to above 65% that it could induce when Ca2� waspresent in the extracellular media. Addition of 5 mM CaCl2 toCa2� free media in the presence of H2O2 precipitated a 70% fallin ��m (Fig. 7A). Further confirmation of a role of cytosolicCa2� came from experiments where thapsigargin that mobi-lizes Ca2� from intracellular stores (16) could induce a loss of��m (Fig. 7A). Incubations of the cells with BAPTA-AM, anintracellular Ca2� chelator, was successful in preventing thefall of ��m induced by H2O2 (Table II). Because both Ca2� andNa� increased in response to H2O2, it was of interest to see ifboth ions were important for the dissipation of ��m. H2O2 couldinduce a 70% fall in ��m in media devoid of Na� (Table II)showing that Na� was not mandatory for the loss of ��m.Mitochondria showed heterogeneous staining pattern (Fig. 7B,b and c) as compared with controls (Fig. 7B, a) when H2O2

stress was given in the presence of EGTA or in Ca2�-freemedia. Treatment with thapsigargin alone or with H2O2 orwith A23187 showed predominantly green mitochondria indi-cating significant loss of potential. From the above observa-tions it is clear that ��m loss induced by H2O2 is independentof [Na�]c levels but sensitive to an increase in [Ca2�]c level thatforms part of a critical signaling pathway responsible for themitochondrial changes.

To see if the above changes in Ca2� levels translated intoapoptosis-like death, we checked the number of apoptotic cells

under manipulated Ca2� conditions. Treatment with H2O2 in thepresence of EGTA or in the absence of extracellular Ca2� couldalso prevent apoptosis-like death as was evident from a reducednumber of TUNEL-positive cells (Fig. 7C, b and c) as comparedwith cells treated with H2O2 only (Fig. 7C, d). Thapsigargin andA23187 could also induce apoptosis-like death as evident fromTUNEL-positive nuclei of the cells (Fig. 7C, e and f).

The Fall in ��m Is Not Related to Increase in MitochondrialCa2�—Mitochondrial Ca2� overload in response to [Ca2�]c in-crease is known to lead to the dissipation of ��m (49). Wedetected a significant [Ca2�]m increase as measured by rhod-2fluorescence within the first 10 min of H2O2 stress after whichthe levels reached a plateau (Fig. 8A). This increase in [Ca2�]mcould be prevented by ruthenium red (Fig. 8A), an inhibitor ofmitochondrial uniporter (50), but ruthenium red was unable toreduce the loss of ��m (Table II). The presence of EGTA in theextracellular medium prevented the increase in [Ca2�]m clearlyshowing that there was a direct transfer between the cytosoland the mitochondria. Even though H2O2 could induce releaseof Ca2� from intracellular stores, we did not find any increasein [Ca2�]m when EGTA was present in the medium. A possibleexplanation for the lack of an increase of [Ca2�]m under theseconditions would be that [Ca2�]c did not reach the thresholdlevels where it would cause an increase in [Ca2�]m. The appar-ent increase in the level of mitochondrial Ca2� in all the groupsreflects the normal uptake during in vitro incubations thatplateaus after 60 min. In the treated group, the Ca2� uptakereached a plateau after 10 min possibly because the drop in

FIG. 5. H2O2 exposure leads to a decrease in ATP levels that is salvaged by NAC, and NAC can prevent apoptosis-like deathinduced by H2O2. A, relative ATP levels in cells at different time points after exposure to 4 mM H2O2 over a period of 60 min. Results are expressedas percentage of control values (0.60 � 0.08 nmol of ATP/106 cells (mean � S.D. for three experiments). B, ATP levels in different treatment groupsat a 60-min time point. a, control; b, 4 mM H2O2; c, cells preincubated with 20 mM NAC prior to treatment with 4 mM H2O2; d, cells treated with20 mM NAC only; e, cells treated with 1 mM BSO only; f, cells treated with 1 mM BSO prior to treatment with 4 mM H2O2; g, cells treated with 1mM BSO and 20 mM NAC only; h, cells preincubated with 20 mM NAC and 1 mM BSO prior to treatment with 4 mM H2O2. Results are mean � S.D.of four experiments. **, p 0.001 as compared with a. C, number of apoptotic cells as counted by Hoechst staining of nuclei at 4 h in response tothe various treatments. a, control; b, 4 mM H2O2; c, cells preincubated with 20 mM NAC prior to treatment with 4 mM H2O2; d, cells treated with20 mM NAC only; e, cells treated with 1 mM BSO only; f, cells treated with 1 mM BSO prior to treatment with 4 mM H2O2; g, cells treated with 1mM BSO and 20 mM NAC only; h, cells preincubated with 20 mM NAC and 1 mM BSO prior to treatment with 4 mM H2O2. Results are mean � S.D.of four experiments. **, p 0.001 as compared with a. D, DNA fragmentation at 6 h in various treatments. a, control; b, 4 mM H2O2; c, cellspreincubated with 20 mM NAC prior to treatment with 4 mM H2O2; d, cells treated with 1 mM BSO only; f, cells treated with 1 mM BSO prior totreatment with 4 mM H2O2.

Depolarization Leads to Apoptosis-like Death in L. donovani 24723

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

��m forced the mitochondria to stop taking up Ca2� becausethis uptake was powered by the ��m. Fig. 8B shows the co-localization of MitoTracker� Green FM and rhod-2 indicatingthat rhod-2 fluorescence was originating solely from the mito-chondria. Therefore, the above data suggest that the fall in ��m

under oxidative stress was possibly not totally dependent on[Ca2�]m increase.

H2O2 Induces the Opening of Non-selectiveCation Channels

Inhibition of Non-selective Cation Channels Could PartiallyBlock the Loss of ��m—Because the above data clearly indi-cated that the primary source of increased [Ca2]c was fromextracellular sources, experiments were conducted to establishthe route of entry. Verapamil preincubation of cells prior toH2O2 exposure could not reduce the Ca2� influx (Table II)showing that the entry did not involve any verapamil-sensitivechannel. Bepridil, a blocker of Ca2�-Na� exchanger (51), couldnot prevent the fall in ��m loss induced by H2O2 indicating thatthe reverse mode of Ca2�-Na� exchanger (51) was not triggeredby H2O2 exposure that was contributing to Ca2� accumulation.FFA, a non-selective cation channel blocker (52), was able toinhibit significantly the H2O2-induced ��m loss (Fig. 9A). Thisability to inhibit was dependent on the dose of FFA, with 240�M being the most effective. Because FFA was able to induce an

inhibition of ��m loss, it was likely that the cation influxthrough FFA-sensitive channels upon H2O2 exposure was re-duced when FFA was present in the medium. It was observedthat the increase of Ca2� and Na� levels in the cells post-H2O2

treatment was significantly abrogated by the presence of FFAduring the treatment (Fig. 9, B and C). Because we observedthat the ��m loss was independent of Na� elevation earlier, themost likely possibility was that the inhibition of Ca2� influxwas responsible for the ability of FFA to prevent ��m loss.

Loss of ��m Could Not Be Prevented by Cyclosporin A or In-duced by Atractyloside—In mammals, permeabilization of theouter mitochondrial membrane and/or inner membrane is, atleast in part, mediated by the permeability transition pore com-plex in mammals (1). Specific inhibitors of PTP such as cyclo-sporin A (a ligand of cyclophilin D) can prevent apoptosis bypreventing PTP opening in different models, whereas openingPTP by different pharmacological agents like atractyloside, anagonistic ligand of adenine nucleotide translocator (ANT) (1), canprecipitate a fall in ��m. We preincubated cells with cyclosporinA prior to H2O2 exposure, but it was unable to prevent the loss of��m (Table III). Atractyloside treatment for 30 min to 3 h couldnot induce MPT (Table III). The possibilities are that the type ofchannel that are present in the L. donovani are either insensitiveto cyclosporin A or atractyloside or that the permeability transi-tion pore complex composition is very different from mammaliancells.

DISCUSSION

Our earlier studies showed that a bolus addition of H2O2 toL. donovani promastigote cultures induces an apoptosis-likedeath that shares several characteristics of metazoan apoptosis(25). In the present report we demonstrate that one of the earlyevents associated with the apoptosis-like promastigote death isthe induction of functional dysfunction of the single long mito-chondrion brought about by major cellular biochemicalchanges. Key elements in the dysfunction of the mitochondriawhen oxidative stress is imposed on the promastigotes are asfollows: 1) loss of ��m; 2) depletion of GSH; 3) a fall in the ATPlevel; and 4) a deregulation of Ca2� and Na� homeostasis. Inthe interpretation of these observations, the following ques-tions have to be addressed: 1) what are the underlying mech-anisms and the sequence of these changes? 2) what is therelevance of 2–4 to ��m loss where oxidative stress is assumedto have a pivotal role?

Our earlier studies (25) demonstrated that activation ofcaspase-like proteins took place between 30 and 60 min afterH2O2 exposure, but cell death occurred much later. It wastherefore speculated that early changes were most likely totake place within the first 60 min that initiate signaling path-ways leading to apoptosis-like death. Mitochondrial activitywas investigated, and the biphasic pattern of loss of ��m and aminimal ATP generation during the first 60 min evidentlyindicated that the mitochondria maintained a reduced functionsufficient to initiate changes required for the cell to enter theapoptosis-like pathway. Biphasic loss of potential is known inmammalian mitochondria where a partial dissipation of ��m

after ROS generation occurs followed by a complete collapse ata later time point (53). Evidently, our studies show that similarevents can also occur in the L. donovani. It is interesting thatafter the first initial fall of ��m, the mitochondria went into alower activity state and remained in such a situation for anextended period. The presence of areas of high and low ��m

values within the single mitochondrion of the promastigotes isof obvious importance for the survival of these parasites in theface of oxidative stress. Metazoan cells with multiple mitochon-dria have the advantage of containing these organelles in dif-ferent energy states, giving the cell the opportunity to survive

FIG. 6. H2O2 induces an increase in cytosolic Ca2� and Na� ionlevels. A, increase in intracellular Ca2� levels in response to 4 mM H2O2over a period of 60 min showing that in the presence of Ca2� chelatorEGTA or in Ca2�-free media there was less increase in intracellularCa2� levels in comparison to the levels achieved in the presence ofextracellular Ca2�. Thapsigargin was able to induce an increase incytosolic Ca2�. Inset, a, fluo-3/AM fluorescence in cells treated withH2O2 in the presence of EGTA; b, cells loaded with fluo-3/AM andexposed to H2O2 in normal medium containing Ca2�. B, increase incytosolic Na� in response to 4 mM H2O2 exposure and monensin over aperiod of 50 min. HP, H2O2; TH, thapsigargin.

Depolarization Leads to Apoptosis-like Death in L. donovani24724

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

based on the ATP generated by the active ones (54, 55). How-ever, in a single celled organism with a single mitochondrion,total loss of potential would result in immediate death. There-fore, the single mitochondria of L. donovani may adopt a vari-able energy-generating state to sustain viability. Studies onfibroblast mitochondria that are long show heterogeneity instaining with JC-1 along the length (37). Therefore, it is alsopossible that longer mitochondria may behave in this mannerwhen exposed to oxidative stress irrespective of whether theyare unicellular or multicellular organisms. In a cell where ��m

is lowered, cell energy supply would progressively decline be-cause it depends upon the proton gradient. Here the ATP levelsbegan to drop to about 50% at a time at which the first prom-inent drop of ��m has already occurred. Therefore, depletion ofATP is a result of the dissipation of mitochondrial potential.The minimal ATP generation observed is consistent with thedetection of areas alternating between the active and inactivestates of oxidative phosphorylation in the mitochondria, andthis ATP level ensures the entry of the cell into the apoptoticpathway.

The next question was whether the energy deficit created inresponse to H2O2 stress was due to a direct action of H2O2 onthe mitochondria or it occurred through the modulation of somecellular messenger like GSH. GSH plays a critical role in me-diating apoptosis in some cell systems by influencing the redoxstatus (39) as well as the ��m (47, 56). The protection given byNAC generated several possibilities as follows: first, a replen-ishment of the GSH supply; second, up-regulation of synthesisof some protective proteins; and third, the direct scavenging offree radicals. Arguing against the first possibility was the in-tact ��m of BSO-treated cells where GSH levels were loweredto the levels precipitated by H2O2. It is possible that in this cell

type other antioxidant molecules (58) compensate for the cel-lular stress generated by the GSH loss. New protein synthesisinduced by NAC was ruled out as cycloheximide, an inhibitor ofprotein synthesis, was unable to prevent the protective effect ofNAC. It was therefore likely that NAC directly scavenged ROS,and this was reiterated when catalase, an H2O2-scavengingenzyme, was also able to prevent ��m loss. Mitochondrial massas measured by NAO remained unaltered, ruling out any majordisruption of mitochondrial lipids. Therefore, it was clear fromthe above data that H2O2 did not interfere with mitochondrialmass, and the fall in ��m was independent of cellular GSHlevel or thiol oxidation status.

Mitochondrial dysfunction may also result through a changein cation homeostasis brought about by high ROS levels (59).Experiments in this study substantiated that the increasedlevel of [Ca2�]c and not [Na�]c affected ��m after H2O2 expo-sure. There are several lines of evidence to support this con-cept. First, the ability of EGTA, a Ca2� chelator, or absence ofCa2� in the media to significantly prevent ��m loss than whatcould be induced by H2O2 in a medium containing normalCa2�; second, the capacity of subsequent readdition of Ca2� toCa2�-free media to induce a prominent fall in ��m; third, thecapability of H2O2 to induce ��m loss in a medium containingCa2� but not Na�; and fourth, the competency of agents thatincreased intracellular Ca2� levels like thapsigargin (TG), asarcoplasmic reticulum Ca2�-ATPase pump inhibitor (16), orCa2� ionophore A23187 to bring about a fall in ��m as well asapoptosis-like death, all argued strongly for a role of Ca2�. Ifincreased [Ca2�]c can cause ��m loss, chelating of [Ca2�]ishould prevent it. BAPTA-AM, an intracellular Ca2� chelator,could reduce the fall in ��m reinforcing this notion. The pres-ence of TG with H2O2 did not cause an additional release

FIG. 7. Status of ��m in L. donovani promastigotes after exposure to various agents affecting cellular Ca2� and apoptotic statusof the cells. A, treatment of cells with different Ca2�-modulating agents showing the status of ��m. a, control; b, 4 mM H2O2; c, 3 mM EGTA �4 mM H2O2; d, Ca2�-free media � 4 mM H2O2; e, Ca2�-free media � 4 mM H2O2 � 5 mM CaCl2; f, 1.5 �M thapsigargin; g, 1.5 �M thapsigargin �4 mM H2O2; h, A23187 (10 �M). B, microphotographs showing the status of mitochondria under different treatments for 60 min. a, confocalmicrograph showing intact ��m in mitochondria from control cells; b, cells treated with 4 mM H2O2 in the presence of 3 mM extracellular EGTAshowing mitochondria with heterogeneous staining; c, cells treated with 4 mM H2O2 in Ca2�-free media showing heterogeneous staining; d, cellstreated with 1.5 �M thapsigargin showing mitochondria with reduced ��m; e, cells treated with 1.5 �M thapsigargin and 4 mM H2O2 showingmitochondria with reduced ��m; f, cells treated with 10 �M Ca2� ionophore A23187 showing reduced ��m. C, TUNEL staining of cells at 6 h underdifferent treatments. a, control; b, cells treated with 4 mM H2O2 in Ca2�-free media; c, cells treated with 4 mM H2O2 in the presence of 3 mM

extracellular EGTA; d, cells treated with 4 mM H2O2; e, cells treated with 1.5 �M thapsigargin; f, cells treated with 10 �M Ca2� ionophore A23187.

Depolarization Leads to Apoptosis-like Death in L. donovani 24725

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

indicating that H2O2 was inducing release from thapsigargin-sensitive stores as well. If H2O2 did not release Ca2� fromintracellular stores then addition of TG would incur a furtherincrease in [Ca2�]c. The above observations clearly confirmedthat influx of extracellular Ca2� through plasma membranechannels, coupled with release of Ca2� from intracellular pools,provide a signal for the loss of ��m. Although this establishedthe role of [Ca2�]c increase, the involvement of [Ca2�]m was notruled out. In many metazoan systems, a transient depolariza-tion of the ��m values under pathological conditions of [Ca2�]coverload, particularly in association with oxidative stress,cause an increase in [Ca2�]m leading to cell death (10). How-ever, in our model this was clearly not happening as it appearsthat the mitochondria are trying to counter the large Ca2� loadimposed by Ca2� influx in the cytosol by sequestering extraCa2�. We suggest that the mitochondrial depolarizations ob-served in this study may be understood in terms of a directeffect of [Ca2]c as translocation of proteins from the cytosol tothe mitochondria cannot be ruled out in the face of mountingevidence that in metazoan cells increased [Ca2�]

c can activatethe translocation of proapoptotic proteins to mitochondria thatbring about early apoptotic changes (1, 2). We attempted tolook at cytochrome c release in response to H2O2 stress byconfocal microscopy, but we could not detect a significant loss ofcytochrome c staining from the mitochondria in the treatedgroups (data not shown). Because cardiolipin status remainedunaltered, it is likely that cytochrome c may not have beenreleased as cardiolipin is bound to cytochrome c in the innermitochondrial membrane, and its oxidation results in the re-lease of cytochrome c (29). An alternative explanation of the

��m loss, associated with an increase in [Ca2�]c, is that H2O2 isplacing stress on cellular energy store by driving down the ATPlevels that would drive ATP synthesis and run down the elec-trochemical gradient. Because ATP levels do not fall signifi-cantly before the ��m loss, this explanation is unlikely, andsuch changes are not very quick. Our study, therefore, indi-cates that the change in ��m induced by H2O2 is dominated by[Ca2�]c increase followed by a decrease in ATP levels.

There is no clear consensus in the literature indicating thelikely mechanism by which H2O2 causes an increase in intra-cellular cation levels. Suggestions include influx of cationsthrough non-selective channels, alteration in Na�-Ca2� ex-change, influx through voltage-gated Ca2� channels, orchanges in Ca2� release from intracellular stores. The inabilityof verapamil, sometimes used to reverse drug resistance inL. donovani and bepridil, a blocker of Na�-Ca2� exchanger, to

FIG. 8. Mitochondrial Ca2� increases in response to H2O2stress. A, increase in mitochondrial Ca2� levels after H2O2 exposure asmeasured by rhod-2 loading of the cells. HP, H2O2; RR, ruthenium red.B, co-localization of rhod-2 and MitoTracker� Green FM showing thatrhod-2 staining primarily reflected mitochondrial uptake of the dye. a,rhod-2 staining; b, MitoTracker� Green FM staining; c, overlay of greenand red channel showing co-localization of the two colors confirmingthat the rhod-2 staining was originating from the mitochondria only.

FIG. 9. FFA preincubation can prevent H2O2-induced ��m lossand increase in Ca2� and Na� ions. Preincubation of cells withdifferent concentrations of FFA, a nonspecific cation channel blocker,inhibited the loss of ��m as measured at 60 min after H2O2 exposure. A,a, control; b, 4 mM H2O2; c, 30 �M FFA � 4 mM H2O2; d, 60 �M FFA �4 mM H2O2; e, 120 �M FFA � 4 mM H2O2; f, 240 �M FFA � 4 mM H2O2.Data are means � S.E. of four experiments. **, p 0.001 as comparedwith b. B, Ca2� levels after H2O2 treatment and treatment with FFAand H2O2 as measured at 60 min after H2O2 exposure. Groups are thesame as A. Data are means � S.E. of three experiments. **, p 0.001as compared with b. C, Na� ion levels after H2O2 treatment and treat-ment with FFA and H2O2 as measured at 60 min after H2O2 exposure.Groups are same as A. Data are mean � S.E. of three experiments. **,p 0.001 as compared with b.

Depolarization Leads to Apoptosis-like Death in L. donovani24726

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

rescue the loss of ��m induced by H2O2 rules out influx of Ca2�

through voltage-gated channels or through the activation ofreverse mode of Na�-Ca2�-ATPase. As FFA, a non-selectivecation channel blocker, was able to prevent significantly a fallin ��m values, it was clear that cations were entering throughnonspecific channels that were activated by high H2O2 stress.Evidently, in the presence of FFA total rescue of ��m does notoccur due to accumulation of Ca2� from intracellular storesreleased by H2O2 that was sufficient to cause a partial fall. It isnot clear whether [Na�]i increase in the presence of the anti-oxidant could be the result from impaired extrusion of Na�

from the cytosol by Na�-K-ATPase. It is possible that themitochondria with impaired respiratory capacity during oxida-tive stress that is unable to generate sufficient amounts of ATPto fuel the Na�-K-ATPase activated by a small increase in[Na�]i induce accumulation of Na� (18).

Even though promastigote mitochondria are known to be-have similarly to vertebrate mitochondria with regard to prop-erties of their electrochemical proton gradient (26), we found noevidence for the involvement of a cyclosporin A-sensitive per-meability transition in the collapse of the potential. It is pos-sible that in the Leishmania promastigotes the composition ofMPTP may be very different as compared with higher eu-karyotes or the pore may be cyclosporin A-insensitive like ratliver mitochondria that open under certain conditions (57).

We propose that the influx of extracellular Ca2� throughnon-selective cation channels activated under oxidative stressrepresents a novel pathway for entry of Ca2� in the promas-tigotes. This Ca2� influx coupled with release of Ca2� fromintracellular pools provides a signal for the loss of ��m thateventually leads to apoptosis-like death in L. donovani. There-fore, this study demonstrates for the first time that non-selec-tive cation channels in the promastigotes of L. donovani con-stitute very important components in modulating the responseof the parasites to oxidative stress. Because mitochondrialchanges brought about by ROS generated by macrophagescould be a crucial event in the pathogenesis of the disease, itwill be interesting to see if some formulations of leishmanicidaldrugs can open these channels to precipitate death. Finally, theidentification of the possible modulators released from the mi-tochondria to cause nuclear disruption may lead to identifica-tion of novel pathways relevant for interception.

REFERENCES

1. Green, D. R., and Reed, J. C. (1998) Science 258, 1309–13122. Gottlieb, R. A. (2001) Biol. Signals Recept. 10, 147–1613. Cossarizza, A., Kalashnikova, G., Grassilli, E., Chiappelli, F., Salvioli, S.,

Capri, M., Barbieri, D., Troiano, L., Monti, D., and Franceschi, C. (1994)Exp. Cell Res. 214, 323–330

4. Zamzami, N., Marchetti, P., Castedo, M., Hirsch, T., Susin, S. A., Masse, B.,and Kroemer, G. (1996) FEBS Lett. 384, 53–57

5. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssiere, J. L., Petit,P. X., and Kroemer, G. (1995) J. Exp. Med. 181, 1661–1672

6. Li, X., Du, L., and Darzynkiewicz, Z. (2000) Exp. Cell Res. 257, 290–297

7. Dvorakova, K., Waltmire, C. N., Payne, C. M., Tome, M. E., Briehl, M. M., andDorr, R. T. (2001) Blood 11, 3544–3551

8. Satoh, T., Enokido, Y., Aoshima, H., Uchiyama, Y., and Hatanaka, H. J. (1997)J. Neurosci. Res. 50, 413–420

9. Ehlers, R. A., Hernandez, A., Bloemendal, L. S., Ethridge, R. T., Farrow, B.,and Evers, B. M. (1999) Surgery 2, 148–155

10. Chinopoulos, C., Tretter, L., Rozsa, A., and Adam-Vizi, V. (2000) J. Neurosci.20, 2094–2103

11. Chinopoulos, C., and Adam-Vizi, V. (1999) Ann. N. Y. Acad. Sci. 893, 269–27212. Lemasters, J. J., Qian, T., Bradham, C. A., Brenner, D. A., Cascio, W. E., Trost,

L. C., Nishimura, Y., Nieminen, A. L., and Herman, B. (1999) J. Bioenerg.Biomembr. 31, 305–319

13. Anderson, C. P., Tsai, J. M., Meek, W. E., Liu, R. M., Tang, Y., Forman, H. J.,and Reynolds, C. P. (1999) Exp. Cell Res. 246, 183–192

14. Tagliarino, C., Pink, J. J., Dubyak, G. R., Nieminen, A. L., and Boothman,D. A. (2001) J. Biol. Chem. 276, 19150–19159

15. Kruman, I. I., and Mattson, M. P. (1999) J. Neurochem. 72, 529–54016. Koya, R. C., Fujita, H., Shimizu, S., Ohtsu, M., Takimoto, M., Tsujimoto, Y.,

and Kuzumaki, N. (2000) J. Biol. Chem. 275, 15343–1534917. Ward, M. W., Rego, A. C., Frenguelli, B. G., and Nicholls, D. G. (2000)

J. Neurosci. 20, 7208–721918. Kim, J. A., Kang, Y. Y., and Lee, Y. S. (2001) Biochem. Biophys. Res. Commun.

281, 511–51919. Kowaltowski, A. J., Vercesi, A. E., Rhee, S. G., and Netto, L. E. (2000) FEBS

Lett. 473, 177–18220. Vercesi, A. E., and Docampo, R. (1992) Biochem. J. 284, 463–46721. Chang, K. P. (1983) Int. Rev. Cytol. 14, (suppl.) 267–30522. Nabi, Z. F., and Rabinovitch, M. (1984) Mol. Biochem. Parasitol. 10, 297–30323. Rais, S., Perianin, A., Lenoir, M., Sadak, A., Rivollet, D., Paul, M., and Deniau,

M. (2000) Antimicrob. Agents Chemother. 44, 2406–241024. Basselin, M., and Robert-Gero, M. (1998) Parasitol. Res. 84, 78–8325. Das, M., Mukherjee, S. B., and Shaha, C. (2001) J. Cell Sci. 114, 2461–246926. Dey, R., and Moraes, C. T. (2000) J. Biol. Chem. 275, 7087–709427. Bortner, C. D., and Cidlowski, J. A. (1999) J. Biol. Chem. 274, 21953–2196228. Eric, C., Dietze, L., Caldwell, E., Grupin, S. L., Mariangela, M., and Seewaldt,

V. L. (2001) J. Biol. Chem. 276, 5384–539429. Thomas, R. L, Matsko, C. M., Lotze, M. T., and Amoscato, A. A. (1999) J. Biol.

Chem. 274, 30580–3058830. Senft, A. P., Dalton, T. P., and Shertzer, H. G. (2000) Anal. Biochem. 280,

80–8631. Kricka, L. J. (1988) Anal. Biochem. 175, 14–2132. Duranteau, J., Chandel, N. S., Kulisz, A., Shao, Z., and Schumacker, P. T.

(1998) J. Biol. Chem. 273, 11619–1162433. Gonzalez, A., Schulz, I., and Schmid, A. (2000) J. Biol. Chem. 275,

38680–3868634. Bortner, C. D., Hughes, F. M., and Cidlowski, J. A. (1997) J. Biol. Chem. 272,

32436–3244235. Zhang, G. H., and Melvin, J. E. (1996) J. Biol. Chem. 271, 29067–2907236. Savel’ev, A. S., Novikova, L. A., Kovaleva, I. E., Luzikov, V. N., Neupert, W.,

and Langer, T. (1998) J. Biol. Chem. 273, 20596–2060237. Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith,

T. W., Steele, G. D., Jr., and Chen, L. B. (1991) Proc. Natl. Acad. Sci.U. S. A. 88, 3671–3675

38. Simpson, P. B., and Russell, J. T. (1998) J. Physiol. (Lond.) 508, 413–42639. Fairlamb, A. H., and Cerami, A. (1992) Annu. Rev. Microbiol. 46, 695–72940. Bojes, H. K., Feng, X., Kehrer, J. P., and Cohen, G. M. (1999) Cell Death Differ.

1, 61–7041. Panaretakis, T., Shabalina, I. G., Grander, D., Shoshan, M. C., and DePierre,

J. W. (2001) Toxicol. Appl. Pharmacol. 173, 56–6442. Nicole, A., Santiard-Baron, D., and Ceballos-Picot, I. (1998) Biomed. Pharma-

cother. 52, 349–35543. Weldrick, D. P., Chodacka, B., Vogt, R., and Steenkamp, D. J. (1999) FEMS

Microbiol. Lett. 173, 139–14644. Oka, S., Kamata, H., Kamata, K., Yagisawa, H., and Hirata, H. (2000) FEBS

Lett. 472, 196–20245. Pariente, J. A., Camello, C., Camello, P. J., and Salido, G. M., (2001) J. Membr.

Biol. 179, 27–3546. Geller, H. M., Cheng, K. Y., Goldsmith, N. K., Romero, A. A., Zhang, A. L.,

Morris, E. J., and Grandison, L. (2001) J. Neurochem. 78, 265–27547. Yang, C. F., Shen, H. M., and Ong, C. N. (2000) Arch. Biochem. Biophys. 374,

142–15248. Ostrander, D. B., Zhang, M., Mileykovskaya, E., Rho, M., and Dowhan, W.

(2001) J. Biol. Chem. 276, 25262–2527249. Lemasters, J. J., Nieminen, A. L., Qian, T., Trost, L. C., Elmore, S. P.,

Nishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A.,and Herman, B. (1998) Biochim. Biophys. Acta 1366, 177–196

50. Sparagna, G. C., Gunter, K. K., Sheu, S. S., and Gunter, T. E. (1995) J. Biol.Chem. 270, 27510–27515

51. Kim, J. A., Kang, Y. S., Lee, S. H., and Lee, Y. S. (2000) Free Radic. Res. 33,267–277

52. Meyer, T. N., Gloy, J., Hug, M. J., Greger, R., Schollmeyer, P., and Pavenstadt,H. (1996) Kidney Int. 49, 388–395

53. Huser, J., and Blatter, L. A. (1999) Biochem. J. 343, 311–31754. Diaz, G., Setzu, M. D., Zucca, A., Isola, R., Diana, A., Murru, R., Sogos, V., and

Gremo, F. (1999) J. Cell Sci. 112, 1077–108455. Overly, C. C., Rieff, H. I., and Hollenbeck, P. J. (1996) J. Cell Sci. 109, 971–98056. Anuradha, C. D., Kanno, S., and Hirano, S. (2001) Free Radic. Biol. Med. 31,

367–37357. Sultan, A., and Sokolove, P. M. (2001) Arch. Biochem. Biophys. 386, 37–5158. Ariyanayagam, M. R., and Fairlamb, A. H. (2001) Mol. Biochem. Parasitol.

115, 189–19859. Duchen, M. R. (2000) J. Physiol. (Lond.) 529, 57–68

TABLE IIIEffect of cyclosporin A and atractyloside on the mitochondrial

membrane potential of L. donovani (n � 6)Table represents changes in mitochondrial membrane potential after

treatment with atractyloside and cyclosporin A. Atractyloside wastested at doses between 5 nM and 5 mM. Results for dosage of only 5 mM

are represented because no change was found in any of the doses.Cyclosporin A was tested at doses between 5 and 20 �M. Data for onlythe 5 �M dose are shown, as results were similar with other doses. Alldata represent � S.D. of four experiments.

Groups �m

Control 6.35 � 0.99H2O2 (4 mM) 0.99 � 0.33Cyclosporin A (5 �M) 6.39 � 0.72Cyclosporin A (5 �M) � H2O2 (4 mM) 0.91 � 0.35Atractyloside (5 mM) 6.15 � 0.83

Depolarization Leads to Apoptosis-like Death in L. donovani 24727

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from

Sikha Bettina Mukherjee, Manika Das, Ganapasam Sudhandiran and Chandrima ShahaPromastigotes Leishmania donovanito Apoptosis-like Death in

LeadingChannels Induced by Oxidative Stress Causes Mitochondrial Depolarization Levels through the Activation of Non-selective Cation2+Increase in Cytosolic Ca

doi: 10.1074/jbc.M201961200 originally published online April 30, 20022002, 277:24717-24727.J. Biol. Chem. 

  10.1074/jbc.M201961200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/277/27/24717.full.html#ref-list-1

This article cites 59 references, 23 of which can be accessed free at

by guest on January 20, 2020http://w

ww

.jbc.org/D

ownloaded from