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J. Biol. Chem., Vol. 277, Issue 14, 11788-11794, April 5, 2002
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From the Unidad de Biofísica (Centro Mixto Consejo Superior
de Investigaciones Científicas/Universidad del
País Vasco/Euskal Herriko Unibertsitatea) and
the Departamento de Bioquímica, Universidad del País
Vasco, Aptdo. 644, 48080 Bilbao, Spain
Received for publication, December 5, 2001, and in revised form, January 14, 2002
The capacity of ceramides to modify the
permeability barrier of cell membranes has been explored. Membrane
efflux induced either by in situ generated ceramides
(through enzymatic cleavage of sphingomyelin) or by addition of
ceramides to preformed membranes has been studied. Large unilamellar
vesicles composed of different phospholipids and cholesterol, and
containing entrapped fluorescent molecules, have been used as a system
to assay ceramide-dependent efflux. Small proportions of
ceramide (10 mol % of total lipid) that may exist under physiological
conditions of ceramide-dependent signaling have been used
in most experiments. When long chain (egg-derived) ceramides are used,
both externally added or enzymatically produced ceramides induce
release of vesicle contents. However, the same proportion of ceramides
generated by sphingomyelinase induce faster and more extensive efflux
than when added in organic solution to the preformed vesicles. Under
our conditions 10 mol % of N-acetylsphingosine
(C2-ceramide) did not induce any efflux. On the other hand,
sphingomyelinase treatment of bilayers containing 50 mol % sphingomyelin gave rise to release of fluorescein-derivatised dextrans
of molecular mass Ceramides have emerged in the last decade as important
messengers in cell signaling involved among others, in processes of cell differentiation, growth suppression, and apoptosis. Mechanisms of ceramide-mediated signal transduction are now starting to be understood (see Refs. 1 and 2, for reviews). One striking property of
ceramides, that may be linked to their physiological effect, is their
capacity to restructure the permeability barrier of model and cell
membranes, thus giving rise to vesicle or cell efflux. Ceramide-induced
release of aqueous contents from liposomes and resealed erythrocyte
ghosts was first observed by Ruiz-Argüello et al. (3),
who induced in situ generation of ceramide by treating the
sphingomyelin-containing model or cell membranes with bacterial sphingomyelinase. Ceramide-induced membrane efflux may be important physiologically, e.g. in generating local ion fluxes, or
even in the release of large molecules, like cytochrome c,
whose efflux from mitochondria is crucial for the activation of apoptosis.
In more recent years, a number of studies have dealt with the issue of
membrane rearrangement by ceramides. Simon and Gear (4) found that
N-acetylsphingosine
(C2-ceramide),1 a
short chain ceramide, caused release of [3H]adenine from
platelets at a ceramide:lipid ratio of 0.2. Ghafourifar et
al. (5) showed that C2- and C6-ceramide
induced cytochrome c release from isolated mitochondria. Di
Paola et al. (6) showed cytochrome c release from
isolated mitochondria induced by C2-and C16-ceramide. However, Di Paola et al. (6)
observed that C2-ceramide, but not
N-palmitoylsphingosine (C16-ceramide, an
abundant natural ceramide) was able to induce efflux from the inner
mitochondrial membrane in isolated mitochondrial suspensions as
evidenced from its ability to dissipate the inner mitochondrial
membrane potential. Also recently, Siskind and Colombini (7) used
electrophysiological methods to demonstrate the formation of stable
pores by short and long chain ceramides in planar lipid bilayers. In
their studies ceramides in organic solvent were added to preformed
bilayers or admixtured with the component lipids at total
ceramide:lipid ratios of 0.05. Pore formation was observed after a few minutes.
In the present study, we have attempted to answer a number of questions
that remain open after publication of the above data, in particular the
possibility of inducing the release of large molecules (the size of
cytochrome c) by ceramide, the existence of common aspects
in the efflux induced by in situ enzyme-generated and by
externally added ceramide, and the correlation between electrophysiological obervations of channel formation and
biochemical/biophysical detection of release of vesicle or cell
contents. For that purpose we have prepared large unilamellar vesicles
with varying lipid compositions, sphingomyelin ranging between 0 and 50 mol % of the total lipid and loaded them with water-soluble
fluorescent molecules. Ceramides have been generated in situ
by sphingomyelinase, or added to the preformed vesicles, or mixed with
the other lipids in the process of liposome preparation. Our results
show that the presence of ceramides can indeed allow the efflux of
large molecules (molecular mass Sphingomyelinase (EC 3.1.4.12) from Bacillus cereus
was supplied by Sigma. Egg PC and egg PE were purchased from Lipid
Products (South Nutfield, UK). Egg SM, egg ceramide, lyso-PC, and plant asolectin were from Avanti Polar Lipids (Alabaster, AL). Plant asolectin (soya bean phospholipids) contained 46% phosphatidylcholine, 22% phosphatidylethanolamine, 18% phosphatidylinositol, 7%
phosphatidic acid, 7% others, according to the manufacturer. ANTS and
DPX were supplied by Molecular Probes, Inc. (Eugene, OR). Ch was from
Sigma, and FITC-dextran was from Serva (Frankfurt, Germany).
Large unilamellar vesicles (LUV) of diameters 100-150 nm were prepared
by the extrusion method (8) using Nuclepore filters of 0.1 µm pore
diameter at room temperature, in 10 mM HEPES, 200 mM NaCl, 10 mM CaCl2, 2 mM MgCl2, pH 7.0. Quantitative analysis of the
LUV preparations, as described by Ruiz-Argüello et al. (3), showed that their composition did not differ significantly from
the initial lipid mixture. All experiments were performed at 37 °C,
except when asolectin vesicles were involved, in which case temperature
was 20 °C, because of the large spontaneous permeability of
asolectin vesicles at 37 °C. Lipid concentration was 0.3 mM, and sphingomyelinase was used at 1.6 units/ml.
Sphingomyelinase activity was assayed by determining phosphorus
contents in the aqueous phase of an extraction mixture
(chloroform:methanol, 2:1) after addition of aliquots from the reaction
mixture at different times. Because of the 1:1 ceramide:phosphate
stoichiometry of the enzyme products, enzyme activities could be given
either as ceramide production or as phosphate production. Since
commercial sphingomyelinase preparations may contain phospholipase C
impurities, 2 mM o-phenanthroline (9) was
routinely added in all our enzyme assays. In the presence of this
specific inhibitor of phospholipase C, thin layer chromatography
experiments demonstrated that only SM, and not PC or PE, were degraded
by the enzyme.
Vesicle efflux was usually assayed with the ANTS:DPX fluorescent system
(10). Alternatively, FITC-derived dextran of molecular mass 20,000 was
entrapped in the vesicles. Details on the use of these fluorescent
probes, including assay calibration, have been given elsewhere (11,
12). Fluorescence measurements were performed in an Aminco Bowman
Series 2 luminescence spectrometer.
Ceramide was introduced in the vesicle membranes by one of these four
procedures: (i) sphingomyelinase (0.8 unit) was added to 0.5 ml of
vesicle suspension, 0.3 mM in lipid (3); (ii) ceramide was
dissolved (3.75 mM) in dodecane/ethanol (2:98 by volume)
(13) and a small volume (2-4 µl) added to 0.5 ml of vesicle
suspension (0.3 mM in lipid) in the cuvette; (iii) ceramide was dissolved (3.75 mM) in ethanol at 40 °C and a small
volume (2-4 µl) added to 0.5 ml of vesicle suspension; and (iv)
ceramide was mixed in chloroform-methanol with the other lipids at the beginning of the process of vesicle preparation, then solvent was
evaporated and the lipid mixture hydrated in buffer.
In Situ Generated Ceramides--
When large unilamellar vesicles
composed of SM:PE:Ch (2:1:1 mole ratio) were treated with
sphingomyelinase under the conditions described under "Materials and
Methods," ceramide was generated within the lipid bilayers as a
result of sphingomyelin cleavage (Fig.
1A). When the vesicles were
loaded, under isotonic conditions, with water-soluble fluorescent
probes, efflux could be observed concomitantly with ceramide production
(Fig. 1B). In a previous study (3) we had described the
release of low molecular weight markers, i.e. ANTS, DPX,
from the vesicles. We have now extended these observations to include
the release of larger molecules, of molecular masses up to 20 kDa. As seen in Fig. 1B, fluorescent dextrans the size of
small proteins, e.g. cytochrome c, could be
released through the activity of sphingomyelinase on SM-containing bilayers.
In another series of experiments, the proportion of sphingomyelin in
the bilayer, thus of ceramide formed by enzyme action, was varied. For
this purpose a number of LUV preparations were made, of general
composition SM:PC:PE:Ch (X:Y:25:25, mole ratio), such that X + Y was always 50 mol %, and
X varied from 0 (control) to 50. Fig.
2 shows the kinetics of ceramide
production and ANTS release from LUV composed of SM:PC:PE:Ch
(10:40:25:25, mole ratio). Under these conditions release was slow,
note the time scale in the abscissa, as compared with Fig.
1. Virtually no release was observed in the first 10 min after enzyme
addition. When the proportion of SM in the bilayer was increased, at
least in the 0-50% range, both the initial rates and extents of probe
liberation increased accordingly (Fig.
3). This result shows that efflux is very
sensitive to the concentration of ceramide in the bilayer.
Externally Added Ceramides--
The above results, and those
published by Siskind and Colombini (7) (see Introduction), prompted us
to study in our system the effect of adding ceramides to lipid
bilayers. This was done in either of two forms, (i) co-dissolving
ceramide with phospholipids in organic solvent, then drying and
preparing the liposomes in the usual way or (ii) adding a small volume
of ceramide in organic solvent to a suspension of vesicles. In the
latter case, ceramides were dissolved either in dodecane:ethanol (2:98,
by volume) at room temperature or in absolute ethanol at ca. 40 °C.
The bilayer lipid composition was either SM:PE:Ch (2:1:1, mole ratio),
or egg PC:Ch (5:1, mole ratio), or plant asolectin. The first
composition was also used by Ruiz-Argüello et al. (3),
and the last two were used by Siskind and Colombini (7). With asolectin
vesicles measurements were performed at 20 °C, because the liposomes
had a very high spontaneous permeability at 37 °C, the temperature at which our experiments were routinely conducted. In all cases, a
control experiment ("0% ceramide") was performed in which only the
organic solvent was added.
The results of ceramide-induced efflux are summarized in Table
I, and some selected observations are
shown in Fig. 4. Ceramides added to
bilayers, the latter either in the process of preparation or already
formed in water, did elicit a certain degree of release of ANTS,
although at a slower rate and to smaller extent than enzymatically
generated ceramides (see, e.g. Figs. 2B and
4A). Bilayer lipid composition was important in the process,
PC:Ch being more stable than SM:PE:Ch or asolectin (see Table I) (more on this subject below). Ceramide solvent was also significant when
ceramides were added to preformed liposomes, dodecane:ethanol being
consistently more efficient than warm ethanol (Table I).
The effect of short chain ceramides (i.e.
N-acylsphingosine with C2 or C6 acyl
chains) on membrane restructuring was also tested in a similar way.
Only ethanol was used as solvent, since both C2- and
C6-ceramides were highly soluble in it. The results are
also summarized in Table I (bottom lines).
C6-ceramide induced some efflux on SM:PE:Ch but not on
asolectin bilayers, while C2-ceramide was inactive on both systems.
The Role of Lipid Geometry--
We have suggested elsewhere (2, 3,
14) that the tendency of ceramides to induce a "negative" curvature
in the bilayer could be related to its membrane-restructuring
properties (for the convention of negative and "positive"
curvatures see (15)). To test this hypothesis, we prepared LUV whose
bilayers contained lysophosphatidylcholine, a lipid with the same
headgroup as SM, but inducing a positive curvature because of its
peculiar geometry. Fig. 5A
shows the effect of lyso-PC on the passive efflux of ANTS induced by
5% ceramide (added in dodecane/ethanol) in SM:PE:Ch (2:1:1) bilayers.
Lyso-PC caused a clear inhibition of ceramide-induced release that was
particularly evident at longer incubation times (after 6 h). The
effect when ceramide was enzymatically generated in the bilayer is
interesting (Fig. 5B). Lyso-PC shifted the efflux curve
toward longer times, i.e. a lag time of ~200 s was
observed. The simplest explanation is that while the number of ceramide molecules was smaller than that of lyso-PC molecules the geometric effects of each other were compensated, release starting only when the
ceramide molecules "outnumbered" or overcame the opposite influence
of lyso-PC.
The main results in this paper concur in demonstrating that
ceramides induce the rearrangement of lipid bilayers, irrespective of
the method followed to bring them into the membrane. However, important
differences in the kinetics and extent of efflux exist, according to
the experimental procedures used. Thus there are two main topics for
discussion in this context, the mechanism of bilayer restructuring,
that may allow even the passage of macromolecules, and the concordance
of the different experimental methods.
The Mechanism of Bilayer Restructuring and Efflux--
In our
previous studies (2, 3, 14) we have suggested that two properties of
ceramides may be related to the release effect, namely the ceramide
geometry, that promotes the negative curvature of a lipid monolayer and
its tendency to form ceramide-rich domains segregated in the plane of
the membrane. As a result of its geometrical constraints ceramide
facilitates the lamellar-to-hexagonal transitions in lipid bilayers (3,
14), and such transitions, even if localized at certain points in the
membrane, are likely to allow extensive communication between the inner
and outer compartments. Perhaps not even the actual transition is
required to take place, but the "propensity" (16) of the bilayer to
adopt the hexagonal form is enough to destabilize transiently the
bilayer. This would explain our observations (3) of efflux in the
absence of nonlamellar signals in 31P NMR spectra.
The role of ceramide geometry in promoting efflux is clearly supported
by the data in Fig. 5, in which the presence of lyso-PC in the bilayers
counteracts the effects of ceramide. In fact, the geometry of lyso-PC
opposes that of ceramide, the former favoring positive curvature of
monolayers, or, in other words, micelle formation (15, 17). PE, a lipid
that favors negative curvature, has the opposite effect than lyso-PC,
i.e. it enhances the effect of ceramide. This is
demonstrated by the higher efflux from SM:PE:Ch bilayers as compared
with PC:Ch bilayers (other conditions being the same) (Table I). Note
that SM forms more stable bilayers than PC, despite which SM:PE:Ch
membranes are more easily reorganized by ceramide than PC:Ch ones.
The lateral segregation of ceramide-rich domains in the plane of the
membrane is another important mechanism that explains efflux. The
phenomenon was first observed by Huang et al. (18) and then
reported, on the basis of different techniques, by Holopainen et
al. (19, 20), Carrer and Maggio (21), and Veiga et al. (14). Ceramides have much higher melting points than SM (
The fact that enzyme-derived ceramide induces very fast efflux is also
in agreement with the hypothesis that lateral separation of
ceramide-rich domains is at the origin of release (see below). In
summary, the data in this paper are in agreement with the idea that
ceramide lateral segregation and ceramide tendency to induce nonlamellar lipid phases can jointly explain bilayer restructuring by
ceramides. Note that, in our view, the most likely mode for solute
efflux from vesicles is through transient, irregular interfaces between
ceramide-rich and -poor domains or may be through local destabilization
points adopting short-lived nonlamellar structures, rather than through
well structured channels.
A Comparison of Methods and Results--
Of the various reports
describing ceramide-induced restructuring of membranes (see
Introduction), those involving C2-ceramide, notably the
work by Di Paola et al. (6) and by Simon and Gear (4),
should be analyzed separately. Di Paola et al. (6) showed
that C2-ceramide can dissipate the inner mitochondrial membrane potential. Simon and Gear (4) observed actual lysis of platelets at ceramide:lipid ratios of 0.5. This, together with the
structure of C2-ceramide, very similar to lyso-PC or to
palmitoylcarnitine (24), suggests that C2 may have
detergent properties, so that its efflux-inducing and lytic properties
could occur, particularly at high ceramide:lipid ratios, through a
mechanism different from that of the long chain ceramides. It is
noteworthy that, under our conditions, low proportions (10 mol %) of
C2 did not induce efflux, while the longer chain
C6-ceramide was more active in this respect. Work from this
and other laboratories (25-27) has shown that the transmembrane
asymmetry of ceramide distribution is very important in the
induction of membrane reorganization in liposomes. Bai and Pagano (28)
have estimated the t1/2 for the transbilayer
movement of a fluorescent ceramide derivative at
Release induced by long chain ceramides has been
explicitely shown by Ruiz-Argüello et al. (3) and by
Siskind and Colombini (7). In the former case ceramide was generated
in situ by enzymatic cleavage of SM, in the latter case it
was mixed in organic solvent with the other bilayer components prior to
membrane formation. Ruiz-Argüello et al. (3) used LUV
with entrapped fluorescent probes to detect efflux, while Siskind and
Colombini (7) detected pore formation in planar bilayers through
electrophysiological methods. The "large, stable channels" observed
by Siskind and Colombini (7) in the presence of 5%
C16-ceramide pose a problem. Theoretical calculations
predict (29) that a vesicle of our size (~100 nm) containing one of
those channels would become empty in less than 10
Other methodological aspects are also relevant when
measuring release, e.g. dodecane-ethanol leads to more
efflux than warm ethanol, for reasons that are probably related to
ceramide solubility. Also significant is the fact that 10% ceramide
generated by sphingomyelinase leads to a faster and larger efflux than
the same amount introduced in organic solvent (cf. Figs.
2B and 4A and Table I). This, together with the
very fast and extensive release at higher ceramide proportions (Fig.
3), suggests that enzyme generation of ceramide is a localized process, occurring whenever a sphingomyelinase molecule binds the
membrane and goes beyond the lag period (30). The ensuing rapid
hydrolysis generates a ceramide-rich microdomain, thus an interdomain
interface through which efflux is facilitated (see above). On the
contrary, addition of ceramide in organic solvent may give rise to a
more even distribution of that lipid in the bilayer, release becoming
secondary to the process of ceramide lateral segregation and domain formation.
Physiological Relevance--
The above observations in model
membranes may be relevant in understanding ceramide-mediated processes
at the cellular level. However, a degree of caution must be exerted
when comparing experimental data from cell and model membranes. One
important issue is the actual concentration of ceramide in living
mammalian cells and the change in ceramide concentrations upon
activation. The overall concentration of ceramide in cells
is smaller, by 2-3 orders of magnitude, than the one used in our
experiments, and activation of ceramide-dependent pathways
leads only to a modest increase in overall concentration
(31), but average figures of lipid composition may hide the localized
existence of ceramide-rich domains. In fact, ceramide signaling is
believed to occur in small well defined regions of the cell membranes
(rafts, caveolae) (2, 32). Our experimental system is not intended to
mimic the whole plasma or outer mitochondrial membrane, but only those
sphingolipid-rich domains where ceramide signaling is localized.
Another interesting point concerns the miscibility of ceramide in
the biologically relevant liquid-crystalline phospholipid bilayers
present in mammalian cells at 37 °C. Differential scanning calorimetry studies using DPPC or DEPE bilayers have shown
lateral separation of ceramide-rich domains with as little as 1-5 mol % ceramide (14, 21). However, will these domains also form in the more
complex and fluid cell membrane environment? Holopainen et
al. (19, 33) found ceramide-rich domain formation both in
L-
Finally, we note that in our experiments efflux occurs in a
protein-free system, while in the living cell, e.g. in the
apoptotic process, proteins such as Bax and tcBcl-xL (the C-terminal
fragment of Bcl-xL generated during apoptosis) permeabilize the outer
mitochondrial membrane to cytochrome c (36, 37). However,
the nature of the apoptotic pore is unclear at present, and there are
suggestions that the apoptotic proteins would induce formation of
pores, composed at least partly by lipid, through changes in membrane
curvature (38). Our view of this matter is that the lipids alone, as
shown in the present paper, may restructure to allow efflux of large molecules, but that, under physiological conditions, these processes will be catalyzed and modulated, both positively and negatively, by proteins.
In conclusion, from the above results and discussion, it can be
established that (i) ceramide induces the restructuring of the lipid
bilayer allowing the passage of even large molecular weight compounds,
(ii) both in situ generated and externally added ceramide
induce an increased efflux in membranes, and (iii) in situ
generation of ceramides by enzymatic cleavage of sphingomyelin leads to
a faster and more extensive efflux than addition of the same amount of
ceramide to a pre-existing membrane.
We are indebted to Dr. R. N. Kolesnick for helpful comments and critical reading of the manuscript.
*
This work was supported in part by grants from
Dirección General de Investigación Científica y
Técnica (Grant PB 96/0171), the Basque Government (Grant PI
99/7), and the University of the Basque Country (Grant UPV 13552/2001).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by a grant from the Spanish Ministerio de Educación
y Ciencia.
¶
To whom correspondence should be addressed. Tel.:
34-94-601-26-25; Fax: 34-94-464-85-00; E-mail:
gbpaliza@lg.ehu.es.
Published, JBC Papers in Press, January 16, 2002, DOI 10.1074/jbc.M111568200
The abbreviations used are:
C2-ceramide, N-acetylsphingosine;
C6-ceramide, N-hexanoylsphingosine;
LUV, large
unilamellar vesicle(s);
ANTS, 8-aminonaphthalene-1,3,6-trisulfonic
acid;
DPX, p-xylenebis (pyridinium bromide);
FITC, fluorescein isothiocyanate;
PC, phosphatidylcholine;
PE, phosphatidylethanolamine;
SM, sphingomyelin;
Ch, cholesterol;
DPPC, dipalmitoylphosphatidylcholine;
DEPE, dielaido-
ylphosphatidylethanolamine.
Membrane Restructuring via Ceramide Results in Enhanced
Solute Efflux*
,
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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20 kDa, i.e. larger than
cytochrome c. These results have been discussed in the
light of our own previous data (Ruiz-Argüello, M. B.,
Basañez, G., Goñi, F. M., and Alonso, A. (1996)
J. Biol. Chem. 271, 26616-26621) and of the
observations by Siskind and Colombini (Siskind, L. J., and
Colombini, M. (2000) J. Biol. Chem. 275, 38640-38644). Our spectroscopic observations appear to be in good
agreement with the electrophysiological studies of the latter authors.
Furthermore, some experiments in this paper have been designed to
explore the mechanism of ceramide-induced efflux. Two properties of
ceramide, namely its capacity to induce negative monolayer curvature
and its tendency to segregate into ceramide-rich domains, appear to be
important in the membrane restructuring process.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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20 kDa) through membranes,
that both in situ generation and external addition of
ceramides can induce release, and that electrophysiological detection
of pore formation correlates with release of vesicular contents
(although with significant differences in the time scale of detection
of the phenomena). Moreover, the role of the molecular geometry of
ceramides, and of their immiscibility with other lipids in the
mechanism of membrane permeabilization are supported by the
experimental data.
MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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DISCUSSION
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Fig. 1.
Ceramide-induced efflux of FITC-dextran
20000. Ceramide was generated by sphingomyelinase action on
SM:PE:Ch (2:1:1) LUV. Lipid concentration: 0.3 mM. Enzyme
concentration: 1.6 units/ml. A, ceramide production,
expressed as mol % of total lipid. B, release of entrapped
FITC-dextran 20000. 100% release was obtained after addition of 5 mM Triton X-100.
View larger version (14K):
[in a new window]
Fig. 2.
Ceramide-induced efflux of ANTS-DPX.
Ceramide was generated by sphingomyelinase action on SM:PC:PE:Ch
(10:40:25:25) LUV. A, ceramide production, expressed as mol
% of total lipid. B, release of entrapped ANTS-DPX. 
,
control, in the absence of enzyme; 
, in the presence of
sphingomyelinase, 1.6 units/ml.
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Fig. 3.
Effect of bilayer composition on
ceramide-induced efflux of ANTS-DPX. Ceramide was generated by
sphingomyelinase action on LUV. A, Time course of ANTS-DPX
release. , SM:PC:PE:Ch (10:40:25:25); , SM:PC:PE:Ch
(20:30:25:25); 
, SM:PC:PE:Ch (30:20:25:25); 
, SM:PC:PE:Ch
(40:10:25:25). B, changes in rate () and extent () of
ANTS-DPX release as function of SM contents in the bilayer; data were
taken from experiments as shown in A.
Ceramide-induced release of vesicle aqueous contents
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Fig. 4.
Time course of ceramide-induced efflux of
ANTS-DPX. Long chain ceramide added to either the lipid mixture or
to preformed vesicles. A, LUV composed of SM:PC:Ch (2:1:1).
, control containing ethanol, but no ceramide; , 10 mol % ceramide in ethanol added to vesicles in suspension; , 10 mol % ceramide in dodecane-ethanol added to vesicles in suspension; , 10 mol % ceramide added to lipids in organic solvent prior to vesicle
formation. B, other bilayer compositions. , bilayers
composed of plant asolectin, control containing dodecane-ethanol, but
no ceramide; , bilayers composed of PC:Ch (5:1), 10 mol % ceramide
in dodecane-ethanol added to vesicles in suspension; , bilayers
composed of plant asolectin. 10 mol % ceramide in dodecane-ethanol was
added to vesicles in suspension.
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Fig. 5.
Opposite effects of lyso-PC and ceramide on
release of vesicle contents. A, ANTS-DPX efflux from
bilayers composed of SM:PE:Ch (2:1:1) ( ) or SM:PE:lysoPC:Ch
(2:0.5:0.5:1) (). 5 mol % ceramide in dodecane-ethanol was added to
vesicles in suspension. B, ANTS-DPX leakage from bilayers
composed of SM:PE:Ch (2:1:1) (continuous line) or
SM:PE:lysoPC:Ch (2:0.5:0.5:1) (dotted line).
Sphingomyelinase (1.6 units/ml) was added to the vesicle
suspension.
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80 °C
versus 40 °C) (22). Consequently, at 37 °C the
ceramide-rich domains constitute rigid islands in a sea of fluid lipid.
Co-existing ordered and disordered domains is a well known condition
that allows efflux to occur (23). The interfaces between ceramide-rich and -poor regions could give rise to the observed release of vesicle contents. It is interesting in this context that C2
ceramides did not elicit efflux under our conditions (Table I, bottom
lines). Huang et al. (18) observed that
C16-ceramide, but not C2-ceramide, gave rise to
in-plane phase separations according to their NMR measurements.
22 min, and
additional data support the idea that natural ceramides should
have flip-flop t1/2 values of the same order of
magnitude. However, short chain (e.g. C2)
ceramides are expected to have significantly faster transbilayer movements. In our experiments, ceramides are added to, or generated in,
the outer monolayer of the liposomal membrane, thus a clearly asymmetric distribution of the natural ceramides may be expected at
least for the initial stages of our observations, when efflux is
faster. With C2 ceramide, transbilayer equilibration may be much faster, and distribution consequently symmetric, contributing to
the observed lack of effect. Nevertheless, a specific investigation of
the surfactant properties of C2-ceramide and of its dehydro derivative is required to clarify this matter.
2 s. But
the fact is that, as just mentioned, efflux takes place slowly for many
hours. The answer to the paradox must rely on intrinsic aspects of the
respective techniques. We may suggest, among others, that electric
measurements detect individual events, while fluorescence is reporting
on the overall effect. The surface area of a typical planar membrane
compared with that of one liposome shows a difference of roughly 6 orders of magnitude. The observation of one permeability pathway in a
planar membrane experiment would then correspond to one permeability
pathway in 106 liposomes. The formation of one permeability
pathway in 106 liposomes would not be detected by the
fluorescence method, but one pathway in one planar membrane would
elicit an electric signal. It should also be considered that the
precise electric signal given by a channel whose size will allow
passage of ANTS/DPX cannot be accurately predicted. Moreover the
results from planar lipid membranes may be influenced by residual
solvent. Finally, LUV may display curvature effects that are lost in
the planar membranes.
-dimyristoylphosphatidylcholine and in
the much more fluid 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.
Moreover cell plasma membranes and outer mitochondrial membranes
contain cholesterol, and hydrogen bonding between ceramide and
cholesterol is important in stabilizing sphingolipid-rich domains (34,
35). Thus we conclude that the lateral phase separation of
ceramide-enriched domains observed in DPPC or DEPE
bilayers may well occur in our model membranes, containing fluid
phospholipids and cholesterol, and in cell membranes.
ACKNOWLEDGEMENT
FOOTNOTES
Supported by a predoctoral grant from the Basque Government.
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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