Positively Charged Ceramide Is a Potent Inducer ofMitochondrial Permeabilization*

Received for publication, October 14, 2004, and in revised form, February 11, 2005Published, JBC Papers in Press, February 18, 2005, DOI 10.1074/jbc.M411707200

Sergei A. Novgorodov‡, Zdzislaw M. Szulc§, Chiara Luberto§, Jeffrey A. Jones§,Jacek Bielawski§, Alicja Bielawska§, Yusuf A. Hannun§, and Lina M. Obeid‡¶�

From the ¶Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29401 andthe Departments of ‡Medicine and §Biochemistry and Molecular Biology, Medical University of South Carolina,Charleston, South Carolina 29425

Ceramide-induced cell death is thought to be mediatedby change in mitochondrial function, although the pre-cise mechanism is unclear. Proposed models suggest thatceramide induces cell death through interaction with la-tent binding sites on the outer or inner mitochondrialmembranes, followed by an increase in membrane perme-ability, as an intermediate step in ceramide signal prop-agation. To investigate these models, we developed a newgeneration of positively charged ceramides that readilyaccumulate in isolated and in situ mitochondria. Accumu-lated, positively charged ceramides increased inner mem-brane permeability and triggered release of mitochon-drial cytochrome c. Furthermore, the positively chargedceramide-induced permeability increase was suppressedby cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide(90%). These observations suggest that the inner mem-brane permeability increase is due to activation of spe-cific ion transporters, not the generalized loss of lipidbilayer barrier functions. The difference in sensitivity ofceramide-induced ion fluxes to inhibitors of mitochon-drial transporters suggests activation of at least twotransport systems: the permeability transition pore andthe electrogenic H� channel. Our results indicate thepresence of specific ceramide targets in the mitochon-drial matrix, the occupation of which triggers permeabil-ity alterations of the inner and outer mitochondrial mem-branes. These findings also suggest a novel therapeuticrole for positively charged ceramides.

Ceramide is a pleiotropic lipid messenger that regulates adiverse range of cellular processes, including apoptosis, cellgrowth, and differentiation (1–5). Multiple studies show intimateconnections between ceramide-induced cell death and mitochon-drial function. Thus, several studies have implicated changes inmitochondrial function as an intermediate step in transduction ofceramide signals, especially with respect to ceramide-dependentapoptotic and necrotic cell death (4, 6). Ceramide has been shownto alter mitochondrial function by two major pathways, indirectand direct. Indirectly, ceramide modifies the activity of pro-apoptotic and anti-apoptotic members of the Bcl-2 family of pro-

teins, which, in turn, alter the outer mitochondrial membranepermeability for cytochrome c and other pro-apoptotic molecules.In this pathway, targets for ceramide are non-mitochondrial mol-ecules: cathepsin D, which triggers translocation of Bax to themitochondria (5, 7, 8), and serine/threonine protein phosphatase2A, which dephosphorylates Bcl-2, thereby decreasing its anti-apoptotic activity (9). An additional substrate for protein phos-phatase 2A is the serine/threonine kinase Akt/protein kinase B(5). Protein phosphatase 2A inactivation of Akt results in dephos-phorylation and activation of pro-apoptotic Bad, an Akt immedi-ate substrate (10). The overall effect of ceramide on this pathwayis an increase in pro-apoptotic proteins bound to mitochondria.

Evidence is also accruing to implicate direct actions of cer-amide on mitochondria. Specifically, the selective hydrolysisof a mitochondrial pool of sphingomyelin by bacterial sphingo-myelinase targeted to the mitochondrial matrix results in apo-ptosis, whereas production of ceramide in the plasma mem-brane, endoplasmic reticulum, nucleus, and Golgi apparatus bybacterial sphingomyelinase targeted to these compartmentshas no effect on cell viability (11). This study therefore pointedto a local action of ceramide on mitochondria in intact cells.

In this context, modulation by ceramide of mitochondrialfunctions at the level of isolated organelles has provided fur-ther evidence in support of this second mechanism. It has beenreported that ceramides directly suppress respiratory chainactivity (12–14). Moreover, current research is focused on theability of ceramides to release cytochrome c from the intermem-brane space and to induce permeability of the inner mitochon-drial membrane, permitting passage of low molecular solutes.In the models of Siskind et al. (15, 16) and Ghafourifar et al.(17), the outer mitochondrial membrane is considered a pri-mary target for ceramides when inducing cytochrome c release,whereas the inner membrane is viewed as being ceramide-insensitive. In contrast, Pastorino et al. (18) and Szalai et al.(19) suggested that the opening of a PTP1 of the inner mito-chondrial membrane could be a primary event in initiation ofcytochrome c release and in increased solute permeability ofthe inner membrane in the presence of ceramides. Yet anotherreport by Di Paola et al. (14) provides evidence for the role ofceramide as a nonspecific modulator of ionic permeability of thelipid component of the inner membrane. Thus, the proposed

* This work was supported in part by National Institutes of HealthGrants IPO1CA097132 (to A. B.), AG16583 (to L. M. O.), and DK59340(to Y. A. H.). The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

� To whom correspondence should be addressed: Dept. of Medicine,Medical University of South Carolina, 114 Doughty St., P. O. Box250779, Charleston, SC 29425. Tel.: 843-876-5169; Fax: 843-876-5172;E-mail: obeidl@musc.edu.

1 The abbreviations used are: PTP, permeability transition pore;TMRM, tetramethylrhodamine methyl ester; D-erythro-C6 pyridinium-DMAS-ceramide, D-erythro-2-N-[6�-[1��-[4���-(N,N-dimethylamino-styryl)-pyridinium]-hexanoyl]]-sphingosine; C6-NBD-ceramide, N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine; TPP�, tetraphenylphosphonium; FCCP, carbonyl cyanidep-trifluoromethoxyphenylhydrazone; CSA, cyclosporin A; DCCD,1,3-dicyclohexylcarbodiimide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 16, Issue of April 22, pp. 16096–16105, 2005Printed in U.S.A.

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mechanism by which ceramides alter mitochondrial membranepermeabilization are varied, and the localization of function-ally significant ceramide targets (outer mitochondrial mem-brane, inner membrane, or matrix space) is also unclear.

Given these intimate and direct connections between ceram-ide and mitochondria, we sought to develop a strategy by whichceramide can be selectively delivered to the mitochondrial ma-trix to probe its mechanisms of action. In this study, we reporton the development of derivatives of ceramide with a fixedpositive charge (Fig. 1). These molecules are expected to accu-mulate in the mitochondrial matrix based on their electrochem-ical potential and thus would serve as direct probes of ceramidefunctions on mitochondria. Our results show that these novelagents do localize selectively in mitochondria in living cells. Wenext investigated the effects of these ceramides on permeabilityof the inner and outer mitochondrial membranes. Our studyshows that these positively charged ceramides increase perme-ability of the inner membrane (decrease in �� and largeamplitude swelling), which, in turn, results in release of cyto-chrome c. In addition, we provide evidence that ceramide-induced permeabilization of the inner mitochondrial mem-brane is mediated by specific ion transport systems, viz. thePTP and electrogenic H� transporter.

EXPERIMENTAL PROCEDURES

Materials—RPMI 1640 medium, Dulbecco’s modified Eagle’s me-dium, and fetal bovine serum were from Invitrogen. TMRM was fromMolecular Probes, Inc. C6-NBD-ceramide was from Matreya. Ceramidesand their derivatives were from the Lipidomics Core of the MedicalUniversity of South Carolina. All other reagents were from Sigma.

Preparation of Mitochondria from Rat Liver—Mitochondria wereprepared from livers of male Sprague-Dawley rats (220–250 g) fastedovernight. Livers from two rats were homogenized in 100 ml of isolationmedium containing 230 mM mannitol, 70 mM sucrose, 2 mM EDTA, and10 mM HEPES (pH 7.4 adjusted with KOH). The homogenate wascentrifuged at 579 � gmax for 10 min to pellet the nucleus and unbrokencells. The supernatant from the previous step was centrifuged at 8000 �gmax for 10 min to pellet the mitochondria. The mitochondrial pellet waswashed with 25 ml and then with 12.5 ml of isolation medium withoutEDTA. The final mitochondrial pellet was resuspended in the abovemedium to provide a protein concentration of 60 mg/ml. Mitochondrialprotein concentration was determined by the BCA assay using bovineserum albumin as the standard (20).

Mitochondrial Incubation Medium—Unless otherwise specified, in-cubations of isolated mitochondria were conducted at 25 °C with 1mg/ml protein in medium containing 250 mM sucrose, 10 mM HEPES(pH 7.4 adjusted with KOH), 10 mM succinate, 5 mM KH2PO4, and 2 �M

rotenone. Deviations from this medium and other reagents employedare described in the figure legends.

Mitochondrial Respiration—Oxygen consumption by mitochondriawas measured in a chamber equipped with a Clark-type oxygen elec-trode (Instech Laboratories) under the conditions described under “Mi-tochondrial Incubation Medium.”

Synthesis of Mitochondrially Targeted Ceramide Molecules—The mi-tochondrially targeted compounds consisted of the lipophilic cationpyridinium covalently linked to ceramide. These pyridinium-ceramideswere prepared by N-acylation of D-erythro-sphingosine with �-bromo

acid chlorides following quaternization of pyridine with the formed�-bromoceramides. The detailed synthesis of pyridinium-ceramides hasbeen described.2

Measurement of Mitochondrial Permeabilization—Inner membranepermeabilization was assayed by measurements of �� and mitochon-drial swelling and by changes in mitochondrial ultrastructure. �� wasestimated from the accumulation of TPP� in the mitochondrial matrixas described by Kamo et al. (22). TPP� (2 �M) was added to the incu-bation medium as indicated in the figure legends. Mitochondrial swell-ing was measured by changes in absorbance at 520 nm using a Brink-mann PC 900 probe colorimeter and fiberoptic probe.

Changes in mitochondrial ultrastructure were examined by electronmicroscopy. Mitochondria were fixed with 3% glutaraldehyde for 15min, followed by sedimentation and additional fixation overnight. Thefixed mitochondria were washed three times with 0.1 M sodium caco-dylate (pH 7.4), treated with 2% osmium tetroxide for 1 h, dehydratedthrough a graded ethanol series, and embedded in Embed 812 resin.Thin sections (70 nm) were stained with uranyl acetate and lead citrateand subsequently examined using a Jeol/JEMI 1010 electronmicroscope.

Cytochrome c Release from Mitochondria—Aliquots of mitochondrialsuspension were taken as indicated in the figure legends and centri-fuged at 15,000 � g for 3 min. The supernatant and mitochondrial pelletwere frozen and stored at �20 °C. Cytochrome c in the supernatantsand pellets was quantified using the Quantikine cytochrome c enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN).

Cell Culture—HepG2 cells (obtained from American Type CultureCollection) were cultured in Eagle’s minimal essential medium supple-mented with 10% fetal bovine serum, 2 mM L-glutamine, nonessentialamino acids, 1 mM sodium pyruvate, and 1.5 g/liter sodium bicarbonatein humidified air (5% CO2) at 37 °C. For confocal microscopy, cells wereplated onto poly-D-lysine-coated 35-mm glass bottom microwell dishesat a density of 20,000–25,000/cm2 and were grown for 2 days. MCF7cells (obtained from American Type Culture Collection) were culturedin RPMI 1640 medium supplemented with 10% fetal bovine serum and2 mM glutamine in humidified air (5% CO2) at 37 °C. All media weresupplemented with 100 units/ml penicillin and 100 �g/ml streptomycin.

Isolation of Mitochondria from HepG2 Cells—For studies with mito-chondria isolated from HepG2 cells, cells were cultured in the mediumdescribed under “Cell Culture” for 3 days in 75-cm2 flasks until 70%confluent. Cells were detached by treatment with 3 ml of 0.05% trypsinand 0.53 mM EDTA, diluted to 13 ml with incubation medium, andsedimented at 900 � g for 10 min. The pellet was washed with 1 ml ofice-cold phosphate-buffered saline, and cells were resuspended in 300 �lof isolation medium and then disrupted by 20 passages through a28-gauge needle (1⁄2 inch). The homogenate was centrifuged at 900 � gfor 10 min to pellet the nucleus and unbroken cells. The supernatantfrom the previous step was centrifuged at 10,000 � g for 10 min to pelletthe mitochondria, which were then resuspended in incubation mediumto provide a protein concentration of �10 mg/ml.

Measurement of Cell Viability—HepG2 or MCF7 cells were plated ata density of 104 cells/well in 96-well plates in the medium describedunder “Cell Culture.” After 24 h of incubation, the cells were treatedwith ceramides for 46 h in 2% fetal bovine serum. Cell viability wasdetermined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (Sigma) following the manufacturer’s instructions.

2 Szulc, Z. M., Gustilo, M., Mayroo, N. I. El-Zahwary, A., Bielawski,J., S.-Gracz, H., Hannun, Y. A., Obeid, L. M., and Bielawska, A. (2005)Bioorg.Med.Chem., manuscript in preparation.

FIG. 1. Chemical structures of theceramides employed in this study.

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Confocal Microscopy—Plated cells were washed once with serum-freemedium and treated with 2 ml of 100 nM TMRM, 2 �M C6-pyridinium-DMAS-ceramide, or 2 �M C6-NBD-ceramide dissolved in the culturemedium supplemented with 2% fetal bovine serum. After 30 min, un-bound dyes were washed out, and images were collected using a ZeissLSM 510 META system equipped with a krypton/argon laser and a �63oil objective (numerical aperture of 1.4). In parallel experiments, afterinitial loading of cells with TMRM or ceramides, cells were treated withuncoupling mixture (10 �M FCCP, 5 �g/ml antimycin A, and 10 �g/mloligomycin A) for an additional 30 min to the discharge mitochondrialinner membrane potential. The TMRM images were collected by exci-tation at 543 nm and emission at 560 nm with a long path emissionfilter. The C6-pyridinium-DMAS-ceramide and C6-NBD-ceramide im-ages were collected by excitation at 488 nm and emission at 505 nmwith a long path emission filter.

Analysis of Ceramides by Mass Spectroscopy—Accumulated ceram-ides in mitochondria were analyzed by mass spectroscopy using re-verse-phase high performance liquid chromatography couple with anelectrospray triple quadruple mass spectrometer, operating at positiveionization in the multiple reaction monitoring mode. Mass separationswere performed using a ThermoFinnigan TSQ 7000 mass spectrometeraccording to the methodology described by Bielawski et al. (45).

Statistical Analysis—Standard curves and the data for cytochrome crelease were computed by generation of a four-parameter logistic curvefit. The values for ceramide accumulation and cytochrome c release areexpressed as the means � S.E. Differences between data were analyzedfor significance by Student’s t test. The results were considered signif-icant at p 0.05.

RESULTS

C6-pyridinium-ceramide Accumulates in Intact Cell Mito-chondria in an Energy-dependent Manner—To determinewhether whole cells will accumulate exogenous pyridinium-ceramides in the mitochondrial matrix, we used a fluorescentanalog of C6-pyridinium-ceramide, C6-pyridinium-DMAS-cer-amide. Fig. 2 shows the distribution pattern of C6-pyridinium-DMAS-ceramide (panels C and D) and the specific mitochon-drial reporter TMRM (panels E and F), which is known toaccumulate inside the mitochondrial matrix. Both C6-pyridin-ium-DMAS-ceramide- and TMRM-treated cells demonstrated asimilar punctate pattern of staining, characteristic of mito-chondria. Thus, C6-pyridinium-DMAS-ceramide accumulatesselectively in mitochondria in living cells.

Subsequent addition of the uncoupler FCCP in combinationwith inhibitors of the respiratory chain and ATPase (antimycin

A and oligomycin, respectively) resulted in diffuse staining ofthe cytoplasm for both fluorophores, indicating that mitochon-drial accumulation of C6-pyridinium-DMAS-ceramide in intactcells is indeed energy-dependent. In the presence of uncou-plers, the diffuse staining of C6-pyridinium-DMAS-ceramideprobably reflects equilibration of this molecule in cell mem-branes without specific concentration in any one compartment.

In contrast to C6-pyridinium-DMAS-ceramide, cells treatedwith a fluorescent analog of neutral C6-ceramide, viz. C6-NBD-ceramide, developed prominent fluorescence in a perinuclearregion (Fig. 2A), whereas mitochondrial staining was minimal.These results are consistent with several previous studies thathave identified this compartment as the Golgi apparatus, andindeed, C6-NBD-ceramide has been accepted as a specificmarker of this compartment (23, 24). Also, in agreement withprevious observations that accumulation of C6-NBD-ceramidein the Golgi apparatus is energy-independent (24), Fig. 2Bdemonstrates that uncouplers of oxidative phosphorylation didnot affect staining of the perinuclear compartment by C6-NBD-ceramide. Taken together, these experiments provide evidencethat exogenously added pyridinium-ceramide localizes prefer-entially to mitochondria and that its mitochondrial accumula-tion in situ is energy-dependent.

To demonstrate definitively that C6-pyridinium-ceramidepreferentially accumulates in mitochondria, HepG2 cells weretreated with equal concentrations of C6-ceramide and C6-pyri-dinium-ceramide (3 �M) for 30 min; mitochondria were thenisolated; and ceramide values were determined by mass spec-troscopy. The amount of C6-pyridinium-ceramide in mitochon-dria was �7-fold higher compared with the amount of C6-ceramide (985 and 142 pmol/mg of protein, respectively).

C6-pyridinium-ceramide Is a Potent Effector of Cell Viabili-ty—Next, we tested whether C6-pyridinium-ceramide is a morepotent cell-killing agent compared with its uncharged analog.

FIG. 3. Dose-response curves of the effect of ceramides on theviability of HepG2 and MCF7 cells. HepG2 (A) and MCF7 (B) cellswere incubated under the conditions described under “ExperimentalProcedures.” C6-ceramide (C-6; traces 1) and C6-pyridinium-ceramide(C-6 pyr; traces 2) at the concentrations indicated were present from thebeginning of the experiment. Cell viability was assessed 46 h after theaddition of ceramides. Data are expressed as the means � S.E. (n 3).

FIG. 2. C6-pyridinium-ceramide accumulates in the mitochon-dria of HepG2 cells in an energy-dependent manner. HepG2 cellswere treated as described under “Experimental Procedures.” A, C, andE show fluorescent images of the cells stained for 30 min with C6-NBD-ceramide (C-6-NBD cer), C6-pyridinium-DMAS-ceramide (C-6-pyr-DMAS cer), and TMRM, respectively. In B, D, and F, cells were pre-treated with fluorophores for 30 min and washed, and �� wasdissipated for additional 30 min by a combination of inhibitors ofoxidative phosphorylation (see “Experimental Procedures”).

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Indeed, C6-pyridinium-ceramide readily induced killing ofhepatocarcinoma HepG2 cells (IC50 � 8 �M) (Fig. 3A, trace 2),whereas electroneutral C6-ceramide was much less effective atthe same range of concentrations (IC50 � 31 �M) (trace 1). Theeffect of C6-pyridinium-ceramide is not unique for HepG2 cells.MCF7 breast cancer cells also responded to this compound (Fig.3B). MCF7 cells appeared to be more sensitive to C6-ceramidecompared with HepG2 cells (IC50 � 16 �M) and demonstrated aconsiderable increase in sensitivity to C6-pyridinium-ceramide(IC50 � 2 �M).

Accumulation of C6-pyridinium-ceramide in Isolated RatLiver Mitochondria Is Energy-dependent—Next, we set out todetermine whether the accumulation of C6-pyridinium-ceram-ide by isolated mitochondria is energy-dependent. The additionof C6-pyridinium-ceramide (10 �M) to mitochondria resulted in95% association with mitochondria (Fig. 4). Dissipation of mi-tochondrial �� by simultaneous addition of the complex IIIinhibitor antimycin A and the protonophore FCCP suppressedaccumulation of C6-pyridinium-ceramide by 66.8% (Fig. 4). Thedifference in the amount of ceramide bound in the absence andpresence of uncouplers of oxidative phosphorylation providesthe amount of ceramide accumulated by mitochondria in anenergy-dependent manner, whereas the component resistant touncouplers indicates the ceramide that may be partitioned intothe lipid phase of mitochondrial membranes or associated withnonspecific binding sites. Calculating the approximate mito-chondrial matrix volume as 1.6 �l/mg of protein (25) and the��-dependent uptake of pyridinium-ceramide as 6.28 nmol/mgof protein, the concentration of pyridinium-ceramide in thematrix space can reach 3.9 mM. On the other hand, the additionof uncharged C6-ceramide (10 �M) also resulted in its consid-erable association (79.3%) with mitochondria. The striking dif-ference between the association of positively charged C6-pyri-dinium and electroneutral C6-ceramides is that the associationof the latter is insensitive to dissipation of ��. Thus, theassociation of C6-ceramide with mitochondria is exclusivelyrelated to its partitioning into the lipid phase of mitochondriaand/or its association with nonspecific mitochondrial bindingsites. Therefore, C6-ceramide is evenly redistributed betweenthe lipid phase of the inner and outer membranes with equalconcentration of free ceramide in the intermembrane space andthe matrix. In contrast, C6-pyridinium-ceramide is highly en-

riched in the inner membrane of energized mitochondria, andits free concentration in the matrix space is considerably ele-vated compared with that in the intermembrane space.

C6-pyridinium-ceramide Is a Potent and Specific Inducer ofInner Mitochondrial Membrane Permeabilization—The resultsshown above suggest that, because of its greater accumulationin the mitochondrial matrix, C6-pyridinium-ceramide shouldaffect mitochondrial function more potently compared withneutral ceramides. To this end, we compared the effects ofC6-pyridinium-ceramide and its neutral derivative on perme-ability of the inner mitochondrial membrane for low molecularmass solutes. Respiring liver mitochondria, which contain�10–15 nmol of endogenous Ca2�/mg of protein, maintainedaccumulated TPP�, an index of ��, for �30 min (Fig. 5A, trace1). Only slight decreases in the absorbance of the mitochondrialsuspension (indicative of swelling) were observed under theseconditions (Fig. 5B, trace 1), consistent with previous results onisolated mitochondria.

The addition of 40 �M C6-pyridinium-ceramide induced abiphasic release of accumulated TPP� (Fig. 5A, trace 2). Theinitial partial release of TPP� was accomplished within 4 minand was followed by a slower phase of total TPP� release

FIG. 4. Accumulation of C6-ceramide and C6-pyridinium-cer-amide in isolated rat liver mitochondria. Mitochondria were incu-bated under the conditions described under “Experimental Procedures,”except that 1 �M CSA and 1 mM EGTA were present from the beginningof the experiment, and 10 �M C6-ceramide or C6-pyridinium-ceramidewas added 2 min later after the addition of mitochondria. The plotshows the amounts of C6-ceramide (C-6) and C6-pyridinium-ceramide(C-6 pyr) that accumulated in energized and de-energized mitochon-dria. First and third bars (controls), binding of ceramides to mitochon-dria that developed high �� as a result of succinate oxidation understandard conditions; second and fourth bars (�FCCP), dissipation of ��by the addition of FCCP (1 �M) and antimycin A (0.5 �g/mg of protein)at the beginning of the experiment. Results are expressed as themeans � S.E. (n 3). *, p 0.01 versus the control.

FIG. 5. Effects of ceramides on �� (A) and mitochondrial largeamplitude swelling (B and C). Mitochondria were incubated underthe conditions described under “Experimental Procedures,” except that2 �M TPP� was present from the beginning of the experiment. Alam-ethicin (ALA; 7 �g/mg of protein), a pore-forming peptide, was added asindicated to induce permeabilization and to determine the full extent ofthe potential changes in the parameters of interest. Where indicated,ceramides (40 �M) were present from the beginning of the experiment.A and B, time courses of ceramide effects on �� and mitochondrialswelling. Traces 1, no addition; traces 2, C6-pyridinium-ceramide (C-6pyrid); traces 3, C6-ceramide (C-6); traces 4, C2-pyridinium-ceramide(C-2 pyrid). RLM, rat liver mitochondria. C, dose-response curves ofceramide effects on mitochondrial swelling. The degree of mitochondrialswelling was determined 30 min after ceramide treatment. Trace 1,C6-pyridinium-ceramide; trace 2, C6-ceramide.

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reflecting complete dissipation of ��. This later phase wasaccompanied by a rapid decrease in absorbance, which indi-cates stimulation of large amplitude swelling caused by in-creased permeability of the inner membrane to the componentsof the incubation medium (Fig. 5B, trace 2). Indeed, the effectsof C6-pyridinium-ceramide were very similar to those of thepore-forming peptide alamethicin, the addition of which to themitochondrial suspension produced essentially the same light-scattering response as C6-pyridinium-ceramide (Fig. 5B, trace3), suggesting that this ceramide enhances pore formation.Importantly, examination of mitochondrial ultrastructure byelectron microscopy before (Fig. 6A) and 30 min (Fig. 6B) afterthe addition of C6-pyridinium-ceramide revealed the typicalpicture of large amplitude mitochondrial swelling, whereas inthe absence of C6-pyridinium-ceramide, mitochondria re-mained in the aggregated configuration characterized by ashrunken matrix space and a large intracristal space (Fig. 6A).Incubation of mitochondria with C6-pyridinium-ceramide re-sulted in an extensive increase in matrix volume and unfoldedcristae, characteristic of colloid/osmotic swelling (Fig. 6B). Theinner membrane remained apparently intact, whereas theouter membrane was mostly ruptured and detached from theinner membrane. Thus, the results show that C6-pyridinium-ceramide exerts significant effects on isolated mitochondria,which are characterized by a relatively specific increase inpermeability of the inner membrane. In contrast to C6-pyridin-ium-ceramide, which induced dissipation of �� (Fig. 5A, trace2) as well as mitochondrial swelling (Fig. 5C, trace 1) with IC50

� 27.5 �M, neutral C6-ceramide failed to induce dissipation of�� (Fig. 5A, trace 3) or mitochondrial permeabilization (Fig. 5,B, trace 3; and C, trace 2) at concentrations up to 60 �M.

Structural Specificity of C6-pyridinium-ceramide Action—Toverify that the effect of C6-pyridinium-ceramide is due to itsacting as a ceramide analog, we compared its effect with theaction of a number of structurally related and unrelated posi-tively charged compounds. First, we determined the effect ofthe pyridinium moiety on mitochondrial permeabilization. To

this end, the effects of short chain C2-pyridinium-ceramide andcetylpyridinium were evaluated. Fig. 5 (A, trace 4; and B, trace4) shows that, employed at the same concentration as C6-pyridinium-ceramide (40 �M), C2-pyridinium-ceramide causedonly minor changes in the magnitude of mitochondrial swellingand the value of �� compared with the control. Even employedat 60 �M (binding of 29.8 � 1.4 nmol/mg of protein at 4 min)(Fig. 7A, trace 2), C2-pyridinium-ceramide failed to induce thesame degree of swelling that was observed with 30 �M C6-pyridinium-ceramide (binding of 5 � 0.8 nmol/mg of protein at4 min) (Fig. 8A, trace 2).

When used at 60 �M, cetylpyridinium provided only moder-ate mitochondrial swelling (Fig. 7B, trace 4). In line with thisnotion, two other hydrophobic cations (viz. TPP� and TMRM)that readily accumulated in the mitochondrial matrix driven by�� (negative inside) failed to induce large amplitude swellingeven at concentrations twice as high as C6-pyridinium-ceram-ide (TMRM (Fig. 7, A, trace 3) and TPP� (Fig. 7B, trace 5),concentration of 60 �M, binding of 50 � 2.6 nmol/mg of proteinat 4 min; and C6-pyridinium-ceramide (Fig. 8A, trace 2), con-centration of 30 �M, binding of 5 � 0.8 nmol/mg of protein at 4min). To the contrary, inhibition of the basal swelling rate wasobserved.

To further confirm that the effect of C6-pyridinium-ceramideis specific with respect to the structure of this molecule, weinvestigated the permeabilizing properties of its structural an-alog, viz. C6-pyridinium-dihydroceramide, which differs only bythe lack of a 4,5-trans-double bond in the sphingoid backbone.Fig. 8A shows an �3-fold increase in the lag period of theinduction of mitochondrial swelling in the presence of C6-pyr-idinium-dihydroceramide (trace 3; concentration of 30 �M,binding of 9.8 � 0.7 nmol/mg of protein at 4 min) comparedwith C6-pyridinium-ceramide (trace 2; concentration of 30 �M,binding of 5 � 0.8 nmol/mg of protein at 4 min). Moreover, thedose-response curves (Fig. 8B) demonstrate that increasesin the different ceramide concentrations shortened the lag pe-riods of C6-pyridinium- and C6-pyridinium-dihydroceramide-

FIG. 6. C6-pyridinium-ceramide induces ultrastructural changes in isolated rat liver mitochondria characteristic of colloid/osmotic swelling. Mitochondria were incubated under the conditions described under “Experimental Procedures”, fixed, and examined using aelectron microscope. Photographs show mitochondria images before (A) and 30 min after (B) the addition of 40 �M C6-pyridinium-ceramide.

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induced swelling. These data indicate that the unsaturatedpyridinium-ceramide analog is somewhat more effective thanthe pyridinium-dihydroceramide analog. Overall, these resultsindicate that C6-pyridinium-ceramide can be considered as ananalog of the uncharged ceramide and that its action does notreflect nonspecific mitochondrial perturbation that could beexpected with any cationic hydrophobic compound.

Inhibitors of Mitochondrial Ion Transporters (CSA and DCCD)Suppress the C6-pyridinium-ceramide-induced MitochondrialPermeability Increase—The permeability increase observed inthe presence C6-pyridinium-ceramide could arise from the forma-tion of lipid channels as a result of perturbation of the hydropho-bic portion of the inner membrane, or alternatively, C6-pyridin-ium-ceramide could regulate specific transport pathways,resulting in equilibration of small molecules and ions across theinner membrane, large amplitude swelling, and dissipation of��. To discriminate between these two possibilities and to ad-dress the mechanism by which C6-pyridinium-ceramide inducesmitochondrial permeability, we investigated the effects of thetranspotent PTP inhibitor CSA and the mitochondrial ion trans-porter nonselective inhibitor DCCD on C6-pyridinium-ceramide-induced permeabilization of the inner membrane.

As shown in Fig. 9, CSA substantially suppressed (60%) anddelayed the pyridinium-ceramide-induced decreases in �� andlarge amplitude swelling (A, trace 3; and B, trace 3, respec-tively). Chelation of the PTP activator Ca2� by EDTA as well as

the use of another PTP inhibitor, bongkrekic acid, resulted in asimilar degree of suppression of ceramide-induced mitochon-drial alterations (data not shown). Although not as specific asCSA, the carboxylic group modifier DCCD is also known to bean inhibitor of PTP opening (26–28). DCCD also suppressedthe permeability increase induced by C6-pyridinium-ceramideby 90% (Fig. 10A, trace 2). This inhibition reached a maximumat a DCCD concentration of �40 nmol/mg of protein (Fig. 10,inset).

In contrast to the slow phase of �� discharge, the initial fastphase was insensitive to CSA (Fig. 9A, traces 1 and 3) and wasaccompanied by the shrinkage of mitochondria rather than bylarge amplitude swelling (Fig. 9B, traces 1 and 3). This rapiddischarge of �� could be explained by ceramide-induced sup-pression of respiratory chain activity that could occur directlyas described (12–14) or indirectly as a result of cytochrome crelease from the intermembrane space (17, 29, 30). However,the addition of 10 �M cytochrome c (an amount exceeding thatfor maximum activation of respiratory chain activity (30)) tothe incubation medium did not modify the mitochondrial re-sponse to C6-pyridinium-ceramide (data not shown). Moreover,measurement of oxygen consumption of the mitochondrial sus-pension showed nearly maximum acceleration of respirationwithin the first minutes after C6-pyridinium-ceramide addition(Fig. 10B). These data strongly suggest activation of an elec-trogenic H� leak across the inner membrane as a cause of

FIG. 7. Effect of hydrophobic cations on the time course ofmitochondrial large amplitude swelling. A and B, mitochondriawere incubated under the conditions described under “ExperimentalProcedures.” Alamethicin (ALA; 7 �g/mg of protein), a pore-formingpeptide, was added as indicated to induce permeabilization and todetermine the full extent of mitochondrial swelling. Where indicated,cations (60 �M) were added to the incubation medium. Traces 1, noaddition (A and B); trace 2 (A), C2-pyridinium-ceramide (C-2 pyr); trace3 (A), TMRM; trace 4 (B), cetylpyridinium (cetyl-pyr); trace 5, TPP� (B).Trace 3 was corrected for the absorbance of TMRM. For determinationof cation and C2-pyridinium-ceramide binding to mitochondria, themitochondria were incubated under essentially the same conditions,but 100 �M DCCD was present from the beginning of the experiment.Four minutes after the addition of ceramides, mitochondria were sedi-mented, and the amount of ceramides in the pellet was determined bymass spectroscopy. TPP� binding was determined using a TPP�-selec-tive electrode as described under “Experimental Procedures.” BecauseTMRM (similar to TPP�) rapidly equilibrates across the inner mem-brane according to its electrochemical potential, its accumulatedamount was assumed to be equal to that of TPP�. RLM, rat livermitochondria.

FIG. 8. Time course (A) and dose-response curves (B) of theeffect of C6-pyridium-ceramide versus C6-pyridinium-dihydro-ceramide on mitochondrial large amplitude swelling. Mitochon-dria were incubated under the conditions described under “Experimen-tal Procedures.” Alamethicin (ALA; 7 �g/mg of protein), a pore-formingpeptide, was added as indicated to induce permeabilization and todetermine the full extent of mitochondrial swelling. Where indicated,ceramides (30 �M) were added to the incubation medium. A, timecourses of ceramide effects on mitochondrial swelling. Trace 1, no ad-dition; trace 2, C6-pyridinium-ceramide (C-6 pyr); trace 3, C6-pyridini-um-dihydroceramide (C-6 Dh pyr). B, dose-response curves of ceramideeffects on mitochondrial swelling. The degree of mitochondrial swellingwas determined 15 min after ceramide treatment. Trace 1, C6-pyridin-ium-dihydroceramide; trace 2, C6-pyridinium-ceramide. Determinationof C6-pyridinium-ceramide and C6-pyridinium-dihydroceramide bind-ing to mitochondria was performed as described in the legend to Fig. 7for C2-pyridinium-ceramide. RLM, rat liver mitochondria.

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decreased ��. Similar to the PTP opening described above,electrogenic ion fluxes arising in the first minutes after C6-pyridinium-ceramide addition were sensitive to DCCD, as canbe seen from suppression of acceleration of oxygen consumption(Fig. 10B). Therefore, electrogenic H� flux activated by ceram-ide is mediated by some specific transporter, not by disturbanceof the lipid phase of the inner membrane.

Next, we set out to investigate the possible mitochondrialsites of ceramide action. The results in Figs. 2 and 3 indicatethat uncoupling of mitochondria correlates with loss of C6-pyridinium-ceramide from the mitochondrial matrix. Underthese same conditions, the addition of FCCP suppressed theswelling phase of the mitochondrial response to ceramide (Fig.9B, trace 4). These observations therefore indicate that thesites of action of C6-pyridinium-ceramide for activation of PTPare localized in the inner membrane or matrix space of themitochondria.

C6-pyridinium-ceramide Induces Cytochrome c Release in anEnergy-dependent Manner—Studies aimed at elucidating themechanisms of ceramide-induced cell death showed that cer-amide acts at least in part by inducing the release of cyto-chrome c from mitochondria. Formation by ceramides of spe-cific pores for cytochrome c and molecules of up to 60 kDa in theouter mitochondrial membrane was suggested as a preferentialmechanism for cytochrome c release (16). Yet induction byceramides of the classical permeability transition of mitochon-

dria, which is accompanied by the their osmotic swelling, rup-ture of the outer membrane, and, as a result, release of cyto-chrome c from the intermembrane space, was proposed as analternative model (18, 19). To determine whether C6-pyridini-um-ceramide is able to release cytochrome c from mitochondriaand to address the mechanism by which this occurs, experi-ments were conducted to evaluate its effect on cytochrome crelease under conditions that result in mitochondrial swellingversus conditions when C6-pyridinium-ceramide large ampli-tude swelling was suppressed by FCCP (Fig. 9B, trace 4).

As shown in Fig. 11B, incubation of mitochondria with C6-pyridinium-ceramide resulted in progressive large amplitudeswelling. After 20 min of incubation with C6-pyridinium-ceram-ide, �40% of the cytochrome c was released from mitochondria(Fig. 11A). When C6-pyridinium-ceramide-induced mitochondrialswelling was suppressed by the addition of FCCP plus antimycinA (Fig. 11B), an �3-fold decrease in cytochrome c release wasobserved (Fig. 11A), which was comparable with the controlvalue. C6-ceramide exerted no effect on cytochrome c release inboth the absence and presence of uncouplers of oxidative phos-phorylation compared with the control (Fig. 11A). Under thesame conditions, C6-ceramide failed to increase large amplitudeswelling (Fig. 11B). The addition of the pore-forming peptidealamethicin provided a 100% response in the parameters of in-terest that can be observed under the conditions employed. Theseresults indicate that the preferential mechanism of cytochrome crelease by C6-pyridinium-ceramide is permeabilization of theinner membrane as an initial step, with subsequent swelling andrupture of the outer membrane.

DISCUSSION

In this study, we have shown that positively charged C6-pyridinium-ceramide readily permeates the lipid bilayer andspecifically targets the inner mitochondrial membrane and ma-trix space. Because of the large mitochondrial inner membranepotential (negative inside), these molecules accumulate insideisolated mitochondria and within mitochondria in culturedcells. Moreover, accumulation of these molecules is reversibleand can be prevented by discharge of ��. In addition, theaccumulation of these ceramides in the mitochondrial matrixspace increases permeability of mitochondrial membranes byactivating putative ion porters of the inner mitochondrial mem-brane: PTP and the electrogenic H� channel.

Several observations are in favor of this conclusion. First,C6-pyridinium-ceramide induced a light-scattering response(indicative of change in mitochondrial ultrastructure) that wassimilar in magnitude to that observed upon conventional Ca2�

treatment (data not shown) or in the presence of the pore-forming peptide alamethicin (Fig. 5B, trace 3). This suggeststhat the light-scattering response observed in the presence ofC6-pyridinium-ceramide reflects mitochondrial large ampli-tude swelling, which is colloid/osmotic in nature as opposed tononspecific amphiphilic compound-mediated solubilization ofmitochondrial membranes. Additional support for the relativespecificity of the permeability defect created by C6-pyridinium-ceramide in the inner membrane comes from examination ofmitochondrial ultrastructure by electron microscopy (Fig. 6, Aand B). Comparison of mitochondrial ultrastructure before andafter ceramide treatment revealed all the features of classicalpermeability transition: increased mitochondrial volume, un-folded cristae, ruptured outer membranes, and apparent in-tactness of the inner membrane.

A second observation in favor of PTP opening came from theuse of the PTP inhibitors CSA and DCCD. These inhibitorssuppressed or delayed mitochondrial large amplitude swellingand discharge of �� by 60 and 90%, respectively. This indicatesthat the permeability transition observed in the presence of

FIG. 9. CSA and FCCP suppress C6-pyridinium-ceramide-in-duced decreases in �� (A) and large amplitude mitochondrialswelling (B). Mitochondria were incubated under the conditions de-scribed under “Experimental Procedures,” except that 2 �M TPP� waspresent from the beginning of the experiment. Traces 2 (A and B), noaddition; traces 3 (A and B), 1 �M CSA present from the beginning of theexperiment; trace 4 (B), 1 �M FCCP present from the beginning of theexperiment. For traces 1, 3, and 4, C6-pyridinium-ceramide (C-6 pyr; 40�M) was present from the beginning of the experiment. Alamethicin(ALA; 7 �g/mg of protein), a pore-forming peptide, was added as indi-cated to induce permeabilization and to determine the full extent ofpotential changes in the parameters of interest. RLM, rat livermitochondria.

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C6-pyridinium-ceramide is likely attributed to the activation ofprotein transporters of the inner mitochondrial membranerather than the formation of lipid channels created by segre-gation of ceramides in a special domain, as was proposed pre-viously for the outer membrane (16).

Our data also provide evidence that C6-pyridinium-ceramideactivates additional ion transport pathways distinct from PTP.Indeed, the shrinkage phase observed during the first minutesafter ceramide addition and the accompanying discharge of ��indicate selective loss of cations from the mitochondrial matrixand activation of electrogenic ion fluxes without a simultane-ous increase in permeability to sucrose, which is usually ob-served in classical models of permeability transition. Perhapsthese relatively specific cation fluxes reflect operation of PTP ina low conductance (impermeable to sucrose) state, as has been

demonstrated previously (31, 32). However, the lack of sensi-tivity of these fluxes to CSA does not support this notion. It alsoshould be kept in mind that, although DCCD suppresses boththese selective fluxes and the nonspecific permeability in-crease, this does not unequivocally indicate operation of PTPbecause, in contrast to CSA, this compound can modify othermitochondrial proteins such as the K�/H� exchanger (33) of theinner mitochondrial membrane and the F0 channel of mito-chondrial ATPase (34, 35).

The best explanation for the initial mitochondrial responseto ceramide treatment seems to be simultaneous activation ofselective electrogenic K� and H� fluxes. K� is known to be themost abundant ion in the mitochondrial matrix, playing amajor role in regulation of mitochondrial volume (36). In thismodel, increased H� permeability across the inner membrane

FIG. 10. DCCD suppresses C6-pyridinium-ceramide-induced large amplitude swelling (A) and electrogenic ion fluxes (B) inisolated rat liver mitochondria. A, mitochondria were incubated under the conditions described under “Experimental Procedures.” Traces 1 and2, C6-pyridinium-ceramide (C-6 pyr; 40 �M) was added were indicated. For trace 2, DCCD (100 �M) was added at the beginning of the experiment.Alamethicin (ALA) was added as indicated to determine the full degree of permeabilization. The inset shows the dose-response curve of the DCCDeffect on C6-pyridinium-ceramide-induced permeabilization. The curve was generated from experiments similar to those depicted in traces 1 and2 with the indicated concentration of DCCD present from the beginning of the experiment. The degree of mitochondrial swelling was assessed 30min after the addition of C6-pyridinium-ceramide. B, respiration of rat liver mitochondria at state 4 was measured as described under“Experimental Procedures.” DCCD (100 �M) was present from the beginning of the experiments. C6-pyridinium-ceramide (40 �M) was added 2 minafter the addition of mitochondria. For bars 1–4, the data show the respiratory rate 1 min after the addition of C6-pyridinium-ceramide. It shouldbe noted that the respiratory rate in the presence of DCCD was linear for at least 8 min. For bar 5, FCCP (1 �M) was added 8 min after the additionof C6-pyridinium-ceramide. RLM, rat liver mitochondria.

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dissipates ��, which allows K� to be lost from the matrixaccording to its electrochemical potential, which, in turn, re-sults in mitochondrial shrinkage.

One of the interesting questions is the structural specificityof C6-pyridinium-ceramide action in the induction of mitochon-drial permeabilization. Some of the cellular responses and en-zymes (e.g. apoptosis (37) and ceramide-activated protein phos-phatase (38)) demonstrate a high degree of specificity forceramide versus dihydroceramide. At the same time, genera-tion of reactive oxygen species by mitochondria appears to lackthis specificity (12). Also, a report from Richter and co-workers(17) demonstrates a �3-fold increase in cytochrome c releasefrom isolated mitochondria under the effect of C2-dihydrocer-amide compared with the control. These considerations areimportant with regard to ceramide interactions with PTP,which has been implicated by this and previous studies to

mediate the ceramide effect on mitochondria. Previous work byGudz et al. (39) and Walter et al. (40) postulated the presenceof low and high affinity binding sites that can nonspecificallyinteract with a variety of hydrophobic compounds, resulting inPTP opening or closure. The natural effectors of these sites areunknown, but ubiquinones of the respiratory chain (40) orceramides (18, 19) may be good candidates for this role. Thiscould explain our observation that the selectivity for C6-pyri-dinium-ceramide versus C6-pyridinium-dihydroceramide is notabsolute.

Our observation that suppression of C6-pyridinium-ceram-ide-induced mitochondrial swelling by FCCP also resulted insuppression of cytochrome c release indicates that mitochon-drial swelling is a prerequisite for the outer membrane perme-ability alterations. Even at 40 nmol/mg of protein, a concentra-tion twice that used by Siskind et al. (16), neutral C6-ceramidefailed to induce considerable cytochrome c release comparedwith the control. It has been reported that loss of cytochrome cby mitochondria under the effect of C2-ceramide is highly de-pendent on the redox state of this protein, with the oxidizedstate favoring the release (17). However, we found no substan-tial release (compared with the control) of cytochrome c byC6-ceramide and C6-pyridinium-ceramide under conditions inwhich the respiratory chain downstream of complex III is com-pletely oxidized by the presence of oxidative phosphorylationuncouplers. On the contrary, suppression of cytochrome c re-lease was observed. This provides evidence that, in our exper-iments, the limiting step in cytochrome c release is not a redoxstate value, but the formation of a permeability pathway forcytochrome c across the outer membrane. This conclusion fitswell with the observation of Kristal and Brown (41), who sug-gested that, under conditions in which C2-ceramide (100nmol/mg of protein) is unable to induce the permeability tran-sition of the inner membrane, no cytochrome c release is ob-served. In agreement with these data, in the experiments ofSzalai et al. (19), conditions that resulted in C2-dependentincrease in permeability of the inner membrane were alsofound to trigger cytochrome c release from mitochondria in aCSA-sensitive manner.

Notably, previous studies suggested that either Ca2� at 100–150 �M or Bax is required in addition to ceramide to causepermeability change in the outer and inner membranes (18,19). In contrast, in our experiments, C6-pyridinium-ceramideby itself induced permeabilization of the mitochondria, or therequirement for Ca2� was extremely low. (The estimated en-dogenous Ca2� concentration is �10 nmol/mg of protein.) Thiseffectiveness of C6-pyridinium-ceramide is best explained by itsgreater accumulation in the mitochondrial matrix. In addition,the low potency of C2-pyridinium compared with C6-pyridini-um-ceramide likely excludes the possibility of a nonspecificeffect of the pyridinium group on mitochondrial membranesand underscores the importance of the length of the N-fattyacylsphingosine moiety in mitochondrial permeabilization.

Our results obtained by in vitro experiments indicate thatmitochondria are the primary targets for C6-pyridinium-ceram-ide in cell death and that the mechanism of cell death involvesdisruption of mitochondrial function. Indeed, by confocal mi-croscopy, we observed preferential accumulation of C6-pyri-dinium-ceramide in the mitochondrial compartment, and therelative potency of C6-pyridinium-ceramide to induce perme-abilization of isolated mitochondria corresponds well with itsability to kill cells. One of the factors that should be kept inmind while considering the effect of ceramide treatment on cellviability is the concentration of ceramide in the vicinity of itstarget. Electroneutral ceramides redistribute preferentially inthe Golgi apparatus (Fig. 2, A and B), which decreases their

FIG. 11. C6-pyridinium-ceramide-induced large amplitudeswelling (B) is accompanied by cytochrome c release (A). Mito-chondria were incubated under the conditions described under “Exper-imental Procedures.” C6-pyridinium-ceramide (C-6 pyr) or C6-ceramide(C-6) at 40 �M was added at 2 min, and mitochondria were incubated foran additional 20 min, followed by the addition of CSA (1 �M) and EGTA(1 mM) to prevent further permeabilization. Two minutes after theaddition of CSA and EGTA, samples were collected and treated forcytochrome c analysis as described under “Experimental Procedures.”Alamethicin (7 �g/mg of protein) was added as indicated to determinethe full degree of permeabilization and maximum cytochrome c release.Where indicated, FCCP (1 �M) and antimycin A (0.5 �g/mg of protein)were present from the beginning of the experiment. The total amount ofcytochrome c is 1.95 �g/mg of protein. Data are expressed as themeans � S.E. (n 3). *, p 0.05 versus the control.

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effective concentration in mitochondria. In contrast, positivelycharged ceramides are specifically concentrated within theirimmediate target, the inner mitochondrial membrane, whereasredistribution to other compartments is relatively small (Fig.2C). This specific redistribution of positively charged ceramidecorrelates well with its higher potency in cell killing comparedwith its neutral counterpart. In this way, our results supportthe hypothesis that the mechanism by which ceramides inducecell killing is permeabilization of the inner mitochondrial mem-brane with subsequent release of cytochrome c. With respect tothe mechanism of pyridinium-ceramide-induced cell death, itshould be noted that the permeability alterations of the innermembrane and the subsequent release of cytochrome c ob-served in isolated mitochondria under the effect of pyridinium-ceramide are compatible with both apoptotic and necrotic path-ways. Also, the results from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of cell viability based onmeasurement of mitochondrial dehydrogenase activities reflectboth necrotic and late apoptotic cell death. Therefore, experi-ments in our laboratory are ongoing to determine the mecha-nism of pyridinium-ceramide-induced cell death and the par-ticular steps involved in ceramide signal propagation.

Irrespective of the mechanism of cell death, our data suggestthat positively charged ceramides could be effective in selectivekilling of cancer cells. The basis for this selectivity is a subst-antial difference in �� between normal and tumor cells(42–44). The difference in �� between carcinoma and controlepithelial cells can be �60 mV higher in carcinoma cells (43,44), a difference that may allow for 10-fold greater accumula-tion of positively charged ceramides in tumor mitochondria.Thus, future studies are aimed at a better understanding of thenature of molecular targets for ceramide in mitochondria andtoward optimization of the molecular structure of positivelycharged ceramides to increase their accumulation in the mito-chondrial matrix. Overall, our results indicate the presence ofspecific ceramide targets in the mitochondrial matrix, the occ-upation of which alters permeability of the inner and outermembranes; these findings support a possible novel therape-utic role for positively charged ceramides.

Acknowledgments—We thank George O. Washington for technicalassistance with tissue cultures and Kathy Wiita-Fisk for administrativeassistance.

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in press

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Bielawski, Alicja Bielawska, Yusuf A. Hannun and Lina M. ObeidSergei A. Novgorodov, Zdzislaw M. Szulc, Chiara Luberto, Jeffrey A. Jones, Jacek

Positively Charged Ceramide Is a Potent Inducer of Mitochondrial Permeabilization

doi: 10.1074/jbc.M411707200 originally published online February 18, 20052005, 280:16096-16105.J. Biol. Chem. 

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