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INTRODUCTION |
Ceramide is a sphingosine-based lipid involved in the regulation
of several cellular processes, including differentiation, growth
suppression, cell senescence, and apoptosis (1-5). Ceramide can be
generated in cells via de novo synthesis or sphingomyelin hydrolysis. Several inducers of cellular stress leading to apoptosis have been shown to cause a net increase in cellular ceramide levels (2-4). Increases in cellular ceramide levels often precede the mitochondrial phase of apoptosis (6-13). Mitochondria are believed to
be the targets in ceramide-mediated apoptosis and mitochondria are
known to play a major regulatory role in cell death via apoptosis (14-18). Thus, ceramides may act on mitochondria to induce apoptosis.
In most cell types, a key event in apoptosis is the release of proteins
from the intermembrane space of mitochondria to the cytoplasm,
including apoptosis-inducing factor, cytochrome c, procaspases, and heat shock proteins (14-17, 19). In general, it is
the release of these intermembrane space proteins that activates caspases and DNase that are responsible for the execution of apoptosis. Short-chain cell permeable ceramide analogues such as
N-acetyl-D-erythro-sphingosine (C2-ceramide)1
and N-hexanoyl-D-erythro-sphingosine
(C6-ceramide) have been shown to induce cytochrome
c release when added to whole cell cultures (20-26) and
isolated mitochondria (27-29); this cytochrome c release
was preventable by preincubation with or overexpression of the
anti-death protein, Bcl-2 (29-31), or transfection of cells with
Bcl-xL (32, 33). In addition, Di Paola et al.
(28) reported the release of cytochrome c from isolated
mitochondrial suspensions by solubilized, long-chain, naturally
occurring
N-hexadecyl-D-erythro-sphingosine (C16- ceramide). We hypothesize that ceramide channels
forming in the outer mitochondrial membrane are responsible for the
ceramide-induced cytochrome c release. We have reported that
both short- and long-chain ceramides form large channels in planar
membranes (34) and that some of these are predicted to be large enough
to allow cytochrome c to permeate. Others have reported
ceramide-induced increases in the permeability of liposomes (35-37).
For example, Montes et al. (37) showed that long-chain
ceramides, both externally added or enzymatically produced, can induce
release of vesicle contents. They showed that sphingomyelinase
treatment of large unilamellar vesicles containing sphingomyelin
gives rise to release of fluorescein-derivatized dextrans of
molecular weight of about 20,000, i.e. larger than cytochrome c (37). Dihydroceramide, despite the fact that it differs from ceramide only by the reduction of one double bond, does
not induce apoptosis nor form channels (34). The ceramide channels are
composed of many ceramide monomers and thus their formation is
exceedingly sensitive to the free ceramide concentration. Altering the
activity of local enzymes that synthesize and catabolize ceramide would
cause channels to assemble or disassemble thus regulating the
permeability of the outer membrane to small proteins.
Ceramides have been reported to have other effects on mitochondria
including enhanced generation of reactive oxygen species (20, 28,
38-40), alteration of calcium homeostasis of mitochondria and
endoplasmic recticulum (20, 38, 40-42), ATP depletion (27), collapse
of the inner mitochondrial membrane potential (
) (20, 27-29),
and inhibition and/or activation of the activities of various components of the mitochondrial electron transport chain (28, 43). It
is therefore unclear whether ceramide acts directly or indirectly on
cytochrome c release.
Here we show that treatment of rat liver mitochondria with either
C2- or C16-ceramide causes the outer membrane
to be freely permeable to cytochrome c, not just cytochrome
c release, and allows the release of proteins of up to about
60 kDa from the intermembrane space. The permeability increase induced
by C2-ceramide is largely reversed by treatment with fatty
acid-depleted bovine serum albumin (BSA). These results bolster the
hypothesis that ceramide-induced cytochrome c release from
mitochondria is via the formation of ceramide channels in dynamic
equilibrium with ceramide in other structural states.
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EXPERIMENTAL PROCEDURES |
Reagents--
The following reagents were purchased from Avanti
Polar Lipids (Alabaster, AL): C2-ceramide,
C2-dihydroceramide, C16-ceramide, C18-dihydroceramide, and asolectin (polar extract of
soybean phospholipids). Antimycin A, 2,4-dinitrophenol (DNP), fatty
acid-depleted BSA, horse heart cytochrome c, and sodium
ascorbate were purchased from Sigma.
Electrophysiological Recordings--
Planar membranes were
formed by the monolayer method (44), as modified (45), across a
100-µm diameter hole in a Saran partition using 1% (w/v) asolectin
(soybean phospholipids), 0.2% (w/v) cholesterol in hexane solution.
The aqueous solution contained 1.0 M KCl, 1 mM
MgCl2, and 5 mM HEPES or 5 mM PIPES
(pH 7.0). The voltage was clamped (trans-side was ground)
and the current was recorded. C2-ceramide was stirred into
the water phase from a Me2SO solution, whereas
C16-ceramide was first dissolved in ethanol at 37 °C
prior to addition. In both cases, the final concentration of the
vehicle was no more than 0.5%. Fatty acid-depleted BSA was prepared as
a stock solution (0.5 mM) in the same aqueous solution
bathing the membrane with 0.5% azide (for storage purposes).
Preparation of Mitochondria--
Rat liver mitochondria were
isolated by differential centrifugation of tissue homogenate as
previously described (46). Briefly, livers from male Sprague-Dawley
rats (fasted overnight with water ad labitum) were quickly
excised, cut, washed repeatedly in cold isolation medium (70 mM sucrose, 210 mM mannitol, 0.1 mM
EGTA, 1.0 mM Tris-Cl, pH 7.4), and minced. Homogenization
and differential centrifugation were performed with isolation medium
supplemented with 0.5% (w/v) BSA. BSA was removed by a final 9000 × g centrifugation and subsequently mitochondria were
suspended in BSA-free medium.
Preparation of Reduced Cytochrome c--
1 mM horse
heart cytochrome c was reduced by an excess of ascorbate (20 mM), 0.2 M Tris-Cl (pH 7.5). The reduced
cytochrome c was separated from the ascorbate using a
Sephadex G-10 gel filtration column. The concentration of reduced
cytochrome c was determined spectrophotometrically
(
550 nm(Red.-Ox.) = 18.5 mM
1
cm1).
Detection of Cytochrome c Permeation through the Outer
Membrane--
Mitochondria were diluted 20-40-fold in isolation
buffer to achieve an appropriate rate of cytochrome c
oxidation after hypotonic shock and kept on ice. 20 µl of diluted
mitochondria were added to 750 µl of isolation buffer supplemented
with 0.5 mM DNP and 5 µM antimycin A (final
concentration of 0.3-1.2 mg of mitochondrial protein/ml) and allowed
to reach room temperature. Ceramide was added in such a way that the
vehicle was no more than 1% of the total volume. Me2SO was
the vehicle for C2-ceramide and
C2-dihydroceramide. C16-ceramide and
C18-ceramide were dissolved in 100% ethanol at 37 °C as
described in Ref. 28 and added to mitochondria at room temperature.
After the indicated incubation period, 20 µl of reduced cytochrome
c (25-35 µM final concentration in the
cuvette) was added and the initial rate of oxidation was assayed
spectrophotometrically as a decrease in absorbance at 550 nm
(Red.-Ox. = 18.5 mM1
cm1) or the difference in absorbance at 550 and 536 nm
(550 nm = 27.7 mM1
cm1; 536 nm = 7.7 mM1 cm1). All initial rates
were expressed as nanomoles of cytochrome c oxidized per
s/mg of mitochondrial protein. KCN was used to inhibit cytochrome
c oxidase and hence stop the reaction. Equal volumes of
vehicle and/or dihydroceramide were used as controls.
Assessing the Ability of Ceramides to Release Adenylate Kinase
and Fumarase--
The intermembrane space enzyme adenylate kinase (47)
and the matrix enzyme fumarase (48) were assayed by standard methods. To increase the concentration of any released enzyme in the medium (for
detection purposes) following exposure to ceramide, it was necessary to
use higher mitochondrial concentrations than those used to measure
cytochrome c permeation through the outer membrane. To
achieve comparable conditions in both types of experiments, the
ceramide to mitochondria ratio was kept constant rather than the
total concentration of added ceramide. This can be justified by
recalling that ceramide exerts its effect on the membranes and that the
critical concentration should be the concentration of ceramide in the
membrane; a constant ratio of ceramide to mitochondrial protein should
result in a constant level of ceramide in the mitochondrial outer
membrane. The literature supports this notion. Muriel et al.
(42) found that the concentration of C2-ceramide that
induced the death of 50% of the PC12 cells after 24 h depended on
the cell density used. Similarly, Simon and Gear (35) found that C2-ceramide inhibition of platelet aggregation and its
ability to induce 6-carboxyfluorescein release from vesicles was
dependent on the ratio of ceramide to total lipid, as opposed to the
absolute ceramide concentration. Thus, we scaled the amount of ceramide to the amount of mitochondria used (i.e. moles of ceramide
to milligrams of mitochondrial protein).
The mitochondrial preparation was diluted 5-fold in isolation buffer
without antimycin A or DNP. A 250-µl aliquot (5-10 mg of protein/ml)
was incubated for 10 min with C2- or
C16-ceramide at either a high or low molar ratio, 5 and 20 µM equivalent ratios of ceramide to milligrams of
mitochondrial protein as in the accompanying cytochrome c
oxidation experiments. The mitochondria were then pelleted at 12,000 rpm for 5 min and 200 µl of supernatant was then added to 700 µl of
either adenylate kinase reaction mixture (50 mM Tris-HCl,
pH 7.5, 5 mM MgSO4, 10 mM glucose,
5 mM ADP, 0.2 mM NADP, 10 units of hexokinase,
and 10 units of glucose-6-phosphate dehydrogenase) or fumarase reaction
mixture (50 mM sodium phosphate and 50 mM
L-malate, pH 7.3). Adenylate kinase was detected as an
increase in absorbance at 340 nm. Fumarase was detected as an increase
in absorbance at 250 nm. Untreated mitochondria and mitochondria with
lysed outer (adenylate kinase) or lysed outer and inner (fumarase)
membranes served as negative and positive controls, respectively. In
the case of the adenylate kinase assay, the outer mitochondrial
membrane was lysed hypotonically. In the fumarase assay, the outer and
inner mitochondrial membranes were lysed via sonication under severe
hypo-osmotic conditions in the presence of 1 mM EDTA as
described in Ref. 49. Equal volumes of vehicle and dihydroceramide were
used as controls.
Assessment of Ceramide-induced Protein Release--
Five
milliliters of mitochondria that was diluted 5-fold (5-10 mg/ml) with
isolation buffer (without antimycin A or DNP) was allowed to reach room
temperature and then incubated for 10 min with C2- or
C16-ceramide at 20 µM equivalent ratios of
moles of ceramide to mitochondrial protein. Untreated mitochondria,
dihydroceramide, and vehicles served as controls. The mitochondria were
then spun at 35,000 rpm (50Ti rotor) for 30 min at 4 °C. The
supernatant was removed and treated with 10% trichloroacetic acid
overnight at 4 °C to pellet the proteins. The pellet was washed
repeatedly with 1:1 ethanol:ether (v/v) to remove the trichloroacetic
acid. The pellets were redissolved by adding Tris-OH,
-mercaptoethanol, and SDS in amounts equivalent to those used in the
SDS-PAGE sample buffer and heated to near boiling for 10 min. After
returning to room temperature, bromphenol blue was added and then HCl
was added just until the color changed. 8 M urea was added
to help stabilize the solution and provide for the added density
(instead of glycerol). Proteins from the hypotonically lysed
mitochondria were diluted 4-, 8-, and 16-fold. Samples were separated
on a 15% acrylamide SDS-PAGE supplemented with 4 M urea
and the bands stained with GelCode Blue stain (Pierce).
Determination of Protein Concentration--
Mitochondrial
protein was measured using the BCA method (Pierce). Bovine serum
albumin was the standard.
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RESULTS |
Ceramide Increases the Permeability of the Mitochondrial Outer
Membrane to Cytochrome c--
It has already been reported that
addition of either C2- or C16-ceramide to
isolated mitochondria results in cytochrome c release
(27-29). However, from the literature it is not clear how this release
might come about. We previously demonstrated that ceramides form large
channels in phospholipid membranes (34), whereas the biologically
inactive C2- and C18-dihydroceramides do not
(34). The addition of either C16- or
C2-ceramide to the aqueous phase on either one or both
sides of a planar phospholipid membrane results in pore formation as
indicated by discrete stepwise current increases (Fig.
1). Discontinuous changes in current are diagnostic of channel formation.
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Fig. 1.
Sample traces of C2- and
C16-ceramide channels in planar membranes. Continuous
current recordings induced by the addition of ceramide to the aqueous
solution as described under "Experimental Procedures." The applied
voltage was clamped at 10 mV. a, 1 µM
C16-ceramide was added only to one side (the cis
side) of the membrane. b, 5 µM
C2-ceramide was added to both sides of the membrane.
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Ceramide channels have a wide distribution of discrete conductance
increases (34) (Fig. 1), ranging from 1 nanoSiemens to more than 200 nanosiemens. Small channels are seen early on when the total membrane
conductance is low. With time, larger discrete conductance increases
are evident reflecting the formation of larger structures. Eventually
the conductance grows with fluctuations of current overlapping into
current noise. Depicted in Fig. 1 are representative examples of some
of these small and large channels observed with C2- and
C16-ceramide. According to the bulk properties of water,
pores with conductance ranging from 1 to 200 nanosiemens would have
estimated diameters ranging from 0.8 to 11 nm, respectively. We
hypothesize that similar channels form in the outer mitochondrial membrane, channels large enough to allow cytochrome c to
exit. In this hypothesis, ceramide does not trigger a cytochrome
c secretion or release mechanism, but simply raises the
permeability of the outer mitochondrial membrane, via ceramide channel
formation, to include small proteins. The hypothesis predicts the
following: 1) cytochrome c should freely permeate and thus
both entry to and exit from the intermembrane space should be
facilitated; 2) the permeability pathways should be eliminated by
removal of ceramide; 3) the release should not be specific to
cytochrome c.
To test the first prediction, we did not measure cytochrome
c release (already reported in Refs. 20-29), but rather the
ability of cytochrome c to permeate through the outer
membrane. We determined the rate at which cytochrome oxidase of the
mitochondrial inner membrane would oxidize exogenously added reduced
cytochrome c. Because of the exceedingly small volume of the
mitochondrial intermembrane space, cytochrome c would need
to cross the outer membrane twice for appreciable oxidation to take
place and be detected spectrophotometrically.
Both C2- and C16-ceramide caused a rapid
increase in the rate of cytochrome c oxidation (Fig.
2a). The ceramide-induced
permeability increase of the mitochondrial outer membrane occurred in a
dose-dependent manner in the concentration range 0.5 to 40 µM. Dihydroceramide and vehicle controls were essentially
identical to untreated mitochondria controls (Fig. 2b). To
ensure that the decrease in absorbance at 550 nm in mitochondria
incubated with ceramide reflected the oxidation of exogenously added
reduced cytochrome c and not mitochondrial volume changes
(swelling), mitochondria were exposed to C2- and C16-ceramide without adding exogenously reduced cytochrome
c and the absorbance was recorded at 540 and 550 nm. We
detected no changes in absorbance at 540 or 550 nm (data not shown).
Therefore, the decrease in absorbance at 550 nm in mitochondria
incubated with ceramide reflects the oxidation of exogenously added
reduced cytochrome c and not volume changes. This does not
rule out the possibility that ceramides can cause mitochondrial volume
changes as previously reported (50). However, it does exclude the
possibility that absorbance changes associated with volume changes were
causing part of the recorded change in absorbance.
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Fig. 2.
C2- and C16-ceramide
increase the permeability of the mitochondrial outer membrane to
cytochrome c in a dose- and time-dependent
manner. Mitochondria were incubated for the indicated time periods
and with the indicated concentrations of C2-
(circles) or C16-ceramide (triangles)
as described under "Experimental Procedures." Reduced cytochrome
c was then added and the absorbance was monitored at 550 nm.
The initial rates plotted were in nanomoles of cytochrome c
oxidized per s/mg of mitochondrial protein. Results are representative
experiment of at least 3 performed on separate mitochondrial
preparations. a, mitochondria incubated for the indicated
time periods with 20 µM ceramides. b,
mitochondria incubated with the following treatments: mitochondrial
controls (M; untreated mitochondria, vehicle controls, 83 µM BSA for 15 min, with overlapping lines); 20 µM C2-dihydroceramide for 10 min
(DH); 20 µM C2-ceramide for 10 min
(C2); 20 µM C16-ceramide for 10 min (C16); lysed mitochondria were incubated for 10 min with
20 µM C2-dihydroceramide (L-DH);
lysed mitochondrial controls (L; untreated lysed
mitochondria, lysed mitochondria incubated for 10 min with 20 µM C2-ceramide, C16-ceramide, or
vehicles or 15 min with 83 µM BSA, with overlapping
lines).
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The measured rates of cytochrome c oxidation were not
influenced by direct effects of ceramide on mitochondrial enzymes. Di Paola et al. (28) reported that C2- and
C16-ceramide had stimulatory and inhibitory effects on
cytochrome c oxidase, respectively, measured as
ascorbate/TMPD oxidase activity, in isolated rat heart mitochondria or
isolated respiratory complexes from bovine heart. However, we found
that when mitochondria with lysed outer membranes were incubated with
C2- or C16-ceramide, the rate of cytochrome c oxidation was identical to the control (Fig.
2b). Similarly, Gudz et al. (43) found no effect
of C2-ceramide on cytochrome c oxidase activity
in isolated rat heart mitochondria, using the ascorbate/TMPD oxidase
system in the same range of C2-ceramide concentrations
as utilized here.
In addition, ceramides have been reported to affect complex III (43),
other components of the electron transport system (28), and the inner
mitochondrial membrane potential () (20, 27-29). Although
important, these effects were not relevant as the medium was
supplemented with both the complex III inhibitor antimycin A and the
uncoupler DNP. Antimycin A prevents re-reduction of cytochrome
c and DNP eliminates the protonmotive force that could slow
down cytochrome oxidase activity. Contrary to the findings of
Ghafourifar et al. (29) that ceramide-induced cytochrome c release was blocked in the absence of (achieved
when they blocked respiration and uncoupled mitochondria), we detect
cytochrome c permeation through the outer mitochondrial
membrane in both uncoupled (Fig. 2, a and b) and
coupled (data not shown) mitochondria incubated with C2-
and C16-ceramide.
The Permeability Increase Induced by C2-ceramide Can Be
Reversed--
If the C2-ceramide-induced increase in
permeability of the mitochondrial outer membrane to cytochrome
c is because of ceramide channels in dynamic equilibrium
with monomers then the normal permeability should be restored when
ceramide monomers are removed from solution, resulting in channel
disassembly (prediction number 2). Experiments with planar membranes
showed that fatty acid-depleted BSA could be used to remove added
ceramide. In the representative experiment in Fig.
3a, 10 µM fatty
acid-depleted BSA (a 2:1 mol ratio of BSA to C2-ceramide)
was added to the aqueous phase on both sides of a phospholipid membrane
containing C2-ceramide channels. The membrane conductance
decreased in a stepwise manner until the total membrane conductance
returned to baseline. The time required for a complete disassembly of
ceramide channels by BSA varied greatly between experiments. Although
the conductance decreased soon after BSA addition, it took between 5 min and 2 h before all the channels disassembled. This
may indicate that the structures formed have a varying degree of
structural stability. In any case, BSA can be used as a tool to
disassemble C2-ceramide channels.
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Fig. 3.
The permeability increase induced by
C2-ceramide can be reversed with BSA. a,
BSA was added (10 µM final) to the aqueous phase on both
sides of a planar phospholipid membrane containing
C2-ceramide channels (5 µM
C2-ceramide on both sides). The applied voltage was clamped
at 10 mV. b, mitochondria were incubated with 20 µM C2-ceramide for the indicated time periods
and where indicated with 83 µM BSA for the indicated time
periods as described under "Experimental Procedures." Reduced
cytochrome c was then added and the absorbance was monitored
at 550 nm. The initial rates were plotted as nanomoles of cytochrome
c oxidized per s/mg of mitochondrial protein. Error
bars represent standard deviations. Results are a representative
experiment of at least 3 performed on separate mitochondrial
preparations.
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This approach was applied to intact mitochondria. A 5-min preincubation
of isolated mitochondria with 20 µM
C2-ceramide was followed by a 15-min incubation with excess
BSA (83 µM; 5:1, BSA:C2-ceramide mole ratio).
This resulted in a marked drop in the cytochrome c oxidation
rate indicating an almost complete restoration of outer membrane
permeability characteristics (Fig. 3b). If the BSA was
simply preventing any further effects of the short-chain ceramide on
the outer membrane permeability instead of actually reversing the
ceramide channels then the rate of oxidation following the 5-min 20 µM C2-ceramide and then the 15-min 83 µM BSA incubation would only be lower than a 20-min
incubation with 20 µM C2-ceramide. Instead, the rate of oxidation was substantially lower than the rate
seen with a 5-min exposure of the mitochondria to 20 µM
C2-ceramide and was almost as low as the untreated
mitochondria. Incubation of the mitochondria with BSA prior to
C2-ceramide addition or mixing the BSA and
C2-ceramide prior to their addition also had very low rates
of oxidation, showing that BSA can both reverse and inhibit
C2-ceramide channel formation (Fig. 3b).
Complete reversal was not achieved, in contrast with the observations
made in the planar membrane experiments, and this was likely because of
the inability to incubate the mitochondria at room temperature for a
sufficient amount of time to allow for 100% reversal without their
degradation. The time required for complete reversal of the
C2-ceramide channels in the planar membrane experiments was
highly variable and for the most part required a longer time period
than 15 min. Mitochondria with lysed outer membranes exposed to BSA had
essentially identical rates of oxidation of the reduced cytochrome
c as lysed mitochondria not exposed to BSA, ruling out an
effect of the BSA on cytochrome oxidase activity. An inert molecule
impermeable to the outer membrane, polyvinyl pyrrolidine (40 kDa), was
used as a control for osmotic changes and the possibility that a
reduction in the volume of the intermembrane space might somehow affect
the rate of cytochrome c oxidation. Addition of 83 µM polyvinyl pyrrolidine had no effect.
Only C2-ceramide was used for the BSA reversal experiments
because of the inability of BSA to reverse or prevent the permeability induced by C16-ceramide, which could be because of a
greater affinity of C16-ceramide for membranes as opposed
to BSA. C16-ceramide has two long fatty acyl chains like
phospholipids, whereas C2-ceramide has only one as in the
free fatty acids that bind to BSA. By doubling the non-polar portion of
ceramide one would expect that the hydrophobic component of the
interaction energy between ceramide and membranes would also double.
This change could not only make ceramide-membrane interactions stronger
than ceramide-BSA interactions, but also greatly reduce the free
ceramide concentration in solution, favoring the dissociation of
ceramide from BSA. In addition, BSA is known to bind associated forms
of organic ligands poorly relative to unassociated forms (monomers)
(51).
C2- and C16-ceramides Allow the Release of
Low Molecular Weight Proteins from the Intermembrane Space--
It has
been proposed that cytochrome c release is the prime
mitochondrial target of ceramide (29). However, if C2- and
C16-ceramide-induced cytochrome c release was
because of ceramide channel formation in the outer mitochondrial
membrane then these channels should not be specific to the release of
cytochrome c, but should also allow the release of other
proteins from the mitochondrial intermembrane space (prediction number
3). We therefore assayed for adenylate kinase (26 kDa) (52) release
from the intermembrane space of isolated mitochondria by
C2- and C16-ceramide addition. To obtain a
sufficient amount of adenylate kinase activity, a higher amount of
mitochondria was used and therefore 5 and 20 µM
equivalent ceramide to mitochondria ratios (labeled low and high) were
used to achieve comparable conditions as in the cytochrome c
permeability assays. Mitochondria incubated for 10 min with
C2- and C16-ceramide, but not their
dihydroceramide counterparts, released adenylate kinase from the
intermembrane space; an increased amount of adenylate kinase was
released at the higher ceramide dose (Table
I). Vehicles and dihydroceramides were
essentially identical to the untreated mitochondrial control.
Therefore, the ceramide channels allow the passage of adenylate kinase
as well as cytochrome c.
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Table I
Effects of ceramides on the release of the intermembrane space enzyme
adenylate kinase and the matrix enzyme fumerase
Enzymatic activity (absorbance change/min ± S.D.) released from
mitochondria (2.4 (adenylate kinase) or 1.8 (fumerase) mg of protein)
after they were untreated (M) or treated with C2-ceramide
(C2), C16-ceramide (C16), or
C18-dihydroceramide (DH) or hypotonically shocked to damage
either the outer membrane (L, adenylate kinase) or both membranes (L,
fumarase). Treatments utilized low or high levels of ceramide (4 or 16 for adenylate kinase and 5 or 20 for fumarase in nanomoles/mg of
protein). The results are representative of three different experiments
using separate mitochondrial preparations.
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To assess whether the inner membrane was also being permeabilized to
proteins, we tested for the release of fumarase (50 kDa) (53), a
soluble matrix enzyme, in mitochondrial supernatants. This is a typical
test for damage/disruption of the mitochondrial inner membrane (53). No
increase in fumarase activity was detected in supernatants from
mitochondria incubated with C2- or C16-ceramide at either low or high ceramide to mitochondrial protein ratios (Table
I).
If ceramide channels were forming in the mitochondrial outer membrane
that allow the release of relatively small soluble proteins from the
intermembrane space, then other proteins of similar molecular weight
should also be released. SDS-PAGE results show that C2- and
C16-ceramides favor the release of low molecular weight
proteins from mitochondria over high molecular weight ones (Fig.
4, a-c). The bands from the
representative gel in Fig. 4a were quantified using
densitometry and the individual gel background for each lane in the gel
was subtracted out. To more accurately reflect the differences between
the ceramides, dihydroceramide, and positive control (mitochondria with
hypo-osmotically lysed outer membranes), the data from the untreated
mitochondrial control (the background) was also subtracted out. Fig.
4b quantitatively represents the differences in the proteins
released by the ceramides and those that were present in the
intermembrane space. C18-dihydroceramide was essentially
identical to the untreated mitochondria, as its intensity was basically
zero when the data from the mitochondrial control was subtracted out
(Fig. 4b). Mitochondria with hypo-osmotically lysed outer
membranes show proteins both above and below the RF value of 0.2 (Mr of 60,000). However,
mitochondria incubated with C2- and
C16-ceramides show proteins only above the
RF value of 0.2 (Fig. 4b). Therefore,
there was a sharp molecular weight cut-off of the proteins released by
C2- and C16-ceramides (Fig. 4b).
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Fig. 4.
C2- and C16-ceramide
increase the permeability of the mitochondrial outer membrane to
intermembrane space proteins with a cut-off of about 60 kDa.
Mitochondria were incubated with or without ceramides as described
under "Experimental Procedures" and the released proteins were run
on a 15% acrylamide SDS-PAGE. The ceramide level was 18 nmol of
ceramide/mg of mitochondrial protein. Results are a representative
experiment of 3 performed on separate mitochondrial preparations. The
following abbreviations are used: STD, standard proteins;
M, mitochondrial control; C2,
C2-ceramide; C16, C16-ceramide;
DH or DH-C18, C18-dihydroceramide;
L8, lysed mitochondria diluted 8-fold; L4, lysed
mitochondria diluted 4-fold; L16, lysed mitochondria diluted
16-fold. a, SDS-PAGE was stained with GelCode Blue stain
(Pierce). b, densitometry was performed on the gel from
a with individual backgrounds for each lane and the
mitochondrial control was subtracted out. Results were expressed as
intensity of staining versus the RF
value. c, results were expressed as the % of lysed
(undiluted) versus the RF value.
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Upon initial examination of Fig. 4b, it appears that more of
the high molecular weight proteins (less than 60,000, but greater than
about 20,000) were released by the ceramides than the lower molecular
weight ones (less than about 20,000). However, when the data was
expressed as the percent of total proteins in the intermembrane space
(released by osmotic shock), on average the same percentage of proteins
less than the cut-off of 60,000 was released by the ceramides (Fig.
4c). It appears that at a ceramide dose of 18 (nanomoles of
ceramide/mg of mitochondrial protein) of either C2- or
C16-ceramide increases the permeability of the outer
membranes of about 10-15% of mitochondria to proteins of molecular
weight 
60,000 (Table II).
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Table II
Effects of ceramides on the release of intermembrane space proteins
Individual bands from polyacrylamide gels such as the one illustrated
in Fig. 4 were quantitated by densitometry. The intensity was corrected
for gel background and for the intensity of the corresponding band in
the untreated mitochondrial control. The results were expressed as a
percentage of the intensity of the corresponding band for protein
released after selective damage of the outer membrane by hypotonic
shock. Errors represent the S.E. of the mean for three separate
mitochondrial preparations.
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DISCUSSION |
The results of this paper extend our previous observations that
C2- and C16-ceramide form large stable channels
in planar membranes to isolated mitochondria. Here, several
predictions/criteria for channel formation were utilized to test our
hypothesis that ceramide channels, similar to the ones that we observe
in planar phospholipid membranes, form in the mitochondrial outer
membrane, raising its permeability to intermembrane space proteins. The results of this paper show that by fulfilling all of the criteria for
channel formation, the ceramide-induced increases in the permeability of isolated rat liver mitochondrial outer membranes were in fact because of ceramide channels.
First, as expected for a channel, the ceramides do not simply allow the
release of cytochrome c or trigger a release mechanism, but
allow its bidirectional flux across the outer mitochondrial membrane
(Fig. 2). Our results strongly argue for specific pathways, channels of
a size capable of allowing the passage of proteins. They are
inconsistent with a specific pathway for cytochrome c. Ghafourifar et al. (29) showed an increase in the release of mitochondrial proteins apart from cytochrome c upon
treatment of isolated rat liver mitochondria with C2- and
C6-ceramide. However, it should be noted that in their
experiments they only found release of cytochrome c and
other mitochondrial proteins by ceramide when cytochrome c
was mainly oxidized, achieved after blocking complex III (29). In
contrast, we found release of adenylate kinase (Table I) and other
proteins of similar size from mitochondria (Fig. 4, a and
b) when complex III was not blocked. Similarly, Di Paola
et al. (28) reported C2- and
C16-ceramide-induced cytochrome c release from
isolated mitochondria without blocking complex III. In addition, we
found that exogenously added reduced cytochrome c was able
to cross the outer mitochondrial membrane of mitochondria incubated
with either C2- or C16-ceramide; hence, we
found no correlation between the redox state of cytochrome c
and its ability to transverse the outer mitochondrial membrane via
ceramide channels.
Second, as would be expected for a channel, the permeability pathways
in the mitochondrial outer membranes were eliminated by removal of
ceramide. The permeability barrier of the mitochondrial outer membranes
was almost completely restored when BSA removed C2-ceramide
and this mirrors exactly the results obtained with C2-ceramide channels reconstituted into planar membranes.
The simplest conclusion is that C2-ceramide forms the same
channels in both mitochondrial and phospholipid membranes and these
channels are in dynamic equilibrium with monomers in solution. As BSA
binds monomers reducing the monomer concentration, the channels
disassemble. The fact that the integrity of planar membranes and the
outer membranes of isolated mitochondrial suspensions can be restored upon removal of ceramide monomers argues against an indirect action by
ceramide in our system. Ceramides might act indirectly by triggering the permeability transition or catalyzing the insertion and channel formation by Bax or by triggering lipid reorganization in a
detergent-like manner. Whereas not specifically excluding any of these
possibilities for the in vivo situation, clearly our results
in isolated mitochondria can be easily explained by ceramide channel
formation without the need for ancillary factors. In fact, Polster
et al. (54) found no Bax associated with mitochondria
isolated from rat liver. In addition, in the planar membrane
experiments no proteins were used except for BSA.
Third, the ceramide-induced increase in the permeability of the outer
mitochondrial membranes was not specific to cytochrome c.
The ceramide channels allow the release of other intermembrane space
proteins and, as expected for a channel, there was a distinct molecular
weight cut-off for the proteins released at about 60,000. This cut-off
is in line with the sizes of proteins that were released from the
mitochondrial intermembrane space during apoptosis (55-57). The
observed cut-off is a lower limit because the proteins have been
reduced to individual polypeptides by treatment with SDS under reducing
conditions. In addition, no effects were observed when the biologically
inactive C18-dihydroceramide was used and this was in
harmony with the inability of this molecule to form channels in
phospholipid membranes (34).
It has been suggested that C2-ceramide can cause unspecific
increases in membrane permeability via perturbation of the membrane structure (1, 28, 35, 58). In addition, there has been recent
controversy over the type of ceramide used in apoptosis research. The
majority of experiments with cells and isolated mitochondria have used
short-chain cell-permeable ceramides (such as C2- or
C6-ceramide) because of their ease in handling. However, these short-chain ceramides are not the major forms found in cells and
their use has been criticized because they were thought to cause an
unspecific increase in membrane permeability (28, 58). For example, Di
Paola et al. (28) showed that only C2-ceramide, but not C16-ceramide, was able to dissipate the inner
mitochondrial membrane potential and the potential in complex
III-reconstituted liposomal vesicles. However, we argue that the
differences between short- and long-chain ceramides lies in their
ability to insert and exchange between membranes. The virtually
non-existent second acyl chain leads to a much higher water solubility
and thus short-chain ceramides would be expected to be able to
partition promiscuously into different populations of membranes,
whereas long-chain ceramides would be expected to exert their effects
only locally. Indeed, Simon et al. (59) found that the rate
of exchange of C16-ceramide between populations of lipid
vesicles was on the order of days.
Short-chain ceramides have also been proposed to have detergent-like
effects on membranes. Amphipathic molecules are labeled as detergents
based on a variety of properties such as the ability to dissolve
relatively insoluble molecules into an aqueous environment, ability to
disrupt membranes by solubilizing components and favoring the formation
of micelles, possessing a relatively high critical micellar
concentration, and the like. Often, molecules that act as channels or
carriers display detergent-like behavior by destabilizing membranes.
However, the fact that ceramides do not destabilize solvent-free planar
phospholipid membranes and form channels in said membranes demonstrates
that they are not significantly detergent-like. We argue that
C2-ceramide does not cause cytochrome c release by simply rupturing or dissolving the outer membrane in a
detergent-like manner because its effects are reversible,
i.e. the low permeability of the membrane can be restored.
In addition, whereas some might argue that a detergent-like effect may
also be reversible with BSA, disruption of the outer membrane with
digitonin was not reversed by washing away the digitonin. In addition,
detergents would not have a molecular weight cut-off for the proteins
released from the mitochondrial intermembrane space as was the case for
ceramides. Finally, erythrocytes are sensitive to detergent-induced
lysis, but we found no such effect with C2-ceramide added
at a ratio of ceramide to surface membrane lipids that was 4-fold
higher than those used in the experiments reported here (data not
shown). Most importantly, it was the combined results of testing all
three predictions of channel activity that allows us to conclude that ceramide-induced increases in the permeability of outer membranes isolated from rat liver mitochondria was through ceramide channel formation.
To apply our results obtained with isolated mitochondria to the
in vivo situation, one must first consider the levels of
ceramide used and whether or not they were physiologically relevant. At a ratio of 18 nmol of ceramide/mg of mitochondrial proteins,
if all of the added ceramide inserted into the mitochondrial
outer membrane then the ceramide would constitute about 14 mol % of the outer membrane phospholipids. The ceramide content of isolated healthy rat liver mitochondria has been measured at 1.7 nmol/mg of
protein (60). If all of the ceramide was inserted into the mitochondria
then there would be about a 10-fold increase in ceramide above the
level of ceramide in healthy rat liver mitochondria. Apoptotic agents
can cause up to 20-fold increases in cellular ceramide levels (1) and
these represent average increases for the entire cell. Local increases
in ceramide may very well be of a much higher magnitude. (Ongoing
research in our laboratory indicates that only about 10% of the
C2-ceramide added actually gets into the mitochondria.)
Another consideration was the way ceramide levels could be raised
locally. Enzymes capable of both de novo synthesis
(ceramide synthase) and hydrolysis (ceramidase) have been found in
mitochondria (61, 62). (The de novo synthetic pathway was
distinct from ceramide generation in the plasma membrane through the
breakdown of sphingomyelin.) Altering the activity of one or both of
these mitochondrial enzymes would change the local steady-state level of ceramide thus increasing or decreasing the propensity for channel formation. Garcia-Ruiz et al. (40) reported that
mitochondria isolated from cells treated with tumor necrosis factor
showed a 2-3-fold increase in mitochondrial ceramide concentrations as compared with control cells. Their failure to increase mitochondrial ceramide levels by adding sphingomyelinases (40) indicates that the
increase was most likely through de novo synthesis. Ionizing radiation has been shown to induce both a prolonged ceramide generation exclusively within mitochondrial membranes and an increase in mitochondrial ceramide synthase
activity.2 Other reports show
that many chemotherapy drugs and radiation that induce ceramide
generation do so via the de novo synthesis route (9,
64-70). For example, Charles et al. (10) found that exposure of human breast cancer cells to Taxol resulted in an enhanced
formation of ceramide that was inhibited by both fumonisin B1 (a ceramide synthase inhibitor) and
L-cycloserine (a serine palmitoyltransferase inhibitor).
Additionally, Perry et al. (8) reported ceramide
accumulation following etoposide treatment of Molt-4 human leukemia
cells as a result of activation of the de novo synthesis
enzyme serine palmitoyltransferase. Kroesen et al. (12)
reported early generation of de novo derived
C16-ceramide in response to B-cell receptor cross-linking
that was linked to a loss of mitochondrial function and subsequent
activation of the apoptotic program. They showed that fumonisin
B1 completely prevented not only ceramide production, but
also a drop in , mitochondrial swelling and disruption of
mitochondrial membranes, poly(ADP-ribose) polymerase cleavage,
and DNA fragmentation (12). Kawatani et al. (63) reported a
decrease in cytochrome c release upon treatment of cells
with fumonisin B1. These studies implicate a role for
de novo generated ceramide in cytochrome c
release and subsequent activation of downstream effectors that lead to the execution of apoptosis.
In conclusion, ceramide channels have the right biophysical properties
to be responsible for the release of pro-apoptotic factors from
mitochondria. When added to isolated mitochondria they increase the
permeability of the outer membrane in a way that is consistent with the
formation of dynamic channels. The appropriate enzymes capable of
regulating ceramide levels are located in mitochondria. There is
evidence that the activity of synthetic enzymes is elevated early in
apoptosis. Taken together, these findings make a compelling case that
ceramide channels are good candidates for the pathway in the outer
membrane that is responsible for the release of pro-apoptotic factors
leading to irreversible apoptosis.
§,
TOP
ABSTRACT
INTRODUCTION
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addressed. Tel.: 301-405-6925; Fax: 301-314-9358; E-mail:
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