From the Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, The New Jersey Medical School, Newark, New Jersey 07103
Received for publication, July 31, 2000, and in revised form, October 9, 2000
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ABSTRACT |
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Na+/Ca2+ exchange
activity in Chinese hamster ovary cells expressing the bovine cardiac
Na+/Ca2+ exchanger was inhibited by the short
chain ceramide analogs N-acetylsphingosine and
N-hexanoylsphingosine (5-15 µM). The
sphingolipids reduced exchange-mediated Ba2+ influx by
50-70% and also inhibited the Ca2+ efflux mode of
exchange activity. The biologically inactive ceramide analog
N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the The Na+/Ca2+ exchanger is the principal
Ca2+ efflux mechanism in cardiac myocytes and plays a
critical role in regulating the force of cardiac muscle contraction
(1). Its stoichiometry is thought to be 3 Na+/Ca2+ (2), although a recent report suggests
that it may have a higher, or variable, stoichiometry (3). Exchange
activity is regulated by Ca2+-dependent and
Na+-dependent processes; cytosolic
Ca2+ activates exchange activity by binding to high
affinity regulatory sites located within a large (546 residues)
hydrophilic domain situated between the fifth and sixth transmembrane
segments of the exchanger (4, 5). Cytosolic Na+ is thought
to induce the time-dependent formation of an inactive state
(Na+-dependent inactivation) after it binds to
the Na+ translocation sites on the exchanger (6).
Na+-dependent inactivation can be antagonized
by ATP-dependent synthesis of phosphatidylinositol
4,5-bisphosphate, by elevated concentrations of cytosolic
Ca2+ and by certain mutations within the "regulatory"
hydrophilic domain of the exchanger (6-8).
The physiological significance of these regulatory mechanisms is
uncertain (9, 10). In intact cells, the Kd for
Ca2+-dependent activation of the exchanger is
~50 nM (11-13), suggesting that the exchanger may be
nearly fully activated under "resting" conditions. The low
cytosolic Na+ concentration and the high levels of ATP and
phosphatidyl 4,5-bisphosphate in healthy cells preclude a major role
for Na+-dependent inactivation in regulating
exchange activity under physiological conditions. Indeed, there is no
direct evidence that Na+/Ca2+ exchange activity
is in fact regulated in functioning cardiac myocytes. The present
report addresses the possibility that sphingolipids such as ceramide
and sphingosine serve as physiological regulators of
Na+/Ca2+ exchange activity.
Ceramide is a central component of the sphingomyelin cycle, a
stress-activated signaling pathway that participates in the induction
of apoptosis and growth arrest. The activating or inhibiting effects of
ceramide on a host of intracellular signaling pathways have been
described in several recent reviews (14-16). Ceramide can also be
converted to sphingosine and sphingosine phosphate, two other signaling
lipids with important regulatory effects of their own.
Here we show that short chain ceramide analogs and sphingosine inhibit
Na+/Ca2+ exchange activity in transfected
Chinese hamster ovary (CHO)1
cells expressing the bovine or canine cardiac
Na+/Ca2+ exchangers. Similar effects were noted
when the cells were treated for 60 min with an inhibitor of endogenous
ceramide metabolism. The differential effects of ceramide on exchanger
mutants defective in Na+- or
Ca2+-dependent regulation suggest that ceramide
blocks the conformational transitions associated with
Ca2+-dependent activation of exchange activity.
We propose that in functioning cardiac myocytes, sphingolipids and
diastolic Ca2+ levels interact to control the distribution
of exchangers between the Ca2+-activated and nonactivated forms.
Cells--
CHO cells expressing Na+/Ca2+
exchange activity (CK1.4 cells) were prepared by transfecting the cells
(CCL 61; American Type Culture Collection) with the expression vector
pcDNA I/Neo (Invitrogen Corp., Carlsbad, CA) containing a cDNA
insert coding for the bovine cardiac Na+/Ca2+
exchanger (17). The Materials and Solutions--
Na-PSS contained 140 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 10 mM glucose, and 20 mM
Mops adjusted to pH 7.4 (37 °C) with Tris. K-PSS had the same
composition as Na-PSS except that NaCl was replaced with KCl (total
concentration, 140 mM). Na-PSS was diluted 7-fold with
K-PSS to yield 20/120 Na/K-PSS. Fura-2/acetoxymethylester was
purchased from Molecular Probes, Inc. (Eugene, OR). Ceramide analogs
and sphingosine were obtained from Biomol Research Laboratories (Plymouth Meeting, PA). All other biochemicals were purchased from
either Sigma or Calbiochem (La Jolla, CA).
Fura-2-based Assays of Ba2+ Influx--
The
Ba2+ influx assay for Na+/Ca2+
exchange activity is fully described in Ref. 20. Cells were grown to
confluence in 75-cm2 culture flasks, washed 3 times with
Na-PSS and incubated for 1 min at 37 °C with Na-PSS containing 5 mM EDTA to detach cells from the flask. The suspended cells
were centrifuged at 700 × g for 1 min, resuspended in
Na-PSS + 1 mM CaCl2, centrifuged again, and
resuspended in 4-5 ml of Na-PSS + 1 mM CaCl2
containing 1% bovine serum albumin. The cells were divided into
300-µl aliquots and incubated for 30 min with 3 µM
fura-2/acetoxymethylester and 0.25 mM sulfinpyrazone (to
retard transport of fura-2 out of the cells). The fura-2 and
sulfinpyrazone were added as 1000-fold concentrated stock solutions in
dimethyl sulfoxide.
After the 30-min loading period, the cells were centrifuged for several
seconds in an Eppendorf Mini-centrifuge, washed, and preincubated for 5 min in 100 µl of Na-PSS + 1 mM CaCl2 or with other additions and conditions as indicated in individual experiments. These cells were then added directly to fluorescence cuvettes containing 3 ml of 20/120 Na/K-PSS + 0.3 mM EGTA.
Gramicidin (1 µg/ml) was added to the cuvettes to equilibrate
transmembrane Na+ and K+ gradients.
Sphingolipids were then added to the cuvette as 1000-fold concentrated
stock solutions in dimethyl sulfoxide. After monitoring fura-2
fluorescence for 60 s, 1 mM BaCl2 (30 µl
of 0.1 M stock solution) was added to the cuvette, and
fura-2 fluorescence was followed for an additional 240 s.
Fura-2 fluorescence was measured at 510 nm with alternate excitation at
350 and 390 nm using a Photon Technology International RF-M 2001 fluorometer (South Brunswick, NJ); data points were obtained at 1.8-s
intervals. All fluorescence values were corrected for autofluorescence
using cells that had not been loaded with fura-2. Data are presented as
the ratio of fluorescence for excitation at 350/390 nm and represent
the mean values (± S.E.; error bars shown in figures) for
the number of experiments (n) indicated in the figure
legends. Statistical testing utilized Student's t test for
unpaired samples, unless indicated otherwise.
Assay for Ca2+ Efflux--
To measure
Ca2+ efflux activity, the cells were placed in cuvettes
containing either Na- or K-PSS + 0.3 mM EGTA.
Ca2+ release from internal stores was initiated by adding
thapsigargin (3 µl; 1 mM in dimethyl sulfoxide), and
fura-2 fluorescence was monitored at the excitation wavelengths
appropriate for Ca2+ (340 and 380 nm). Dimethyl sulfoxide
alone at the concentrations used had no effect on Ca2+
release. The difference in the amplitude and duration of the ionomycin-induced [Ca2+]i transient in K-PSS
versus Na-PSS was taken as an indication of
exchange-mediated Ca2+ efflux activity (21).
Treatment with PPMP--
Cells were distributed in 300-µl
aliquots as described above for the fura-2-based assay of
Ba2+ influx, except that 0.1% bovine serum albumin was
present in the medium instead of 1% bovine serum albumin. PPMP (30 µM) was added from a 1000-fold concentrated stock
solution in dimethyl sulfoxide; for the control cells, an equal volume
of dimethyl sulfoxide alone was added. After 30 min of incubation, 3 µM fura-2/acetoxymethylester was added, and after an
additional 30 min of incubation, the cells were centrifuged, washed,
and preincubated for 2 min in Na-PSS with the additions specified in
individual experiments. PPMP was not present in the wash or the 2-min
preincubation solutions.
Ceramide Analogs Inhibit Exchange-mediated Ba2+
Influx--
The data in Fig. 1 show that
15 µM N-acetylsphingosine (C2-ceramide), a
short chain fatty acid analog of ceramide, inhibited Na+/Ca2+ exchange activity in transfected CHO
cells expressing the bovine cardiac Na+/Ca2+
exchanger (CK1.4 cells). Exchange activity was assayed as the rate of
Ba2+ influx in cells treated with gramicidin in a medium
containing 20 mM NaCl and 120 mM KCl; the
gramicidin was added to equalize the Na+ concentrations
across the plasma membrane (20). As shown in Fig. 1, the initial rate
of Ba2+ influx was inhibited by 38% (p < 0.02) when 15 µM C2-ceramide was included in the assay
medium. The degree of inhibition increased with time, as shown by the
downward curvature of the trace in Fig. 1. By 200-300 s, the rate of
Ba2+ influx in the presence of C2-ceramide was 72% less
(p < 0.001) than that of control cells over the same
interval (Fig. 1B).
The inhibition of Ba2+ influx by C2-ceramide was mimicked
by C6-ceramide, another short chain ceramide analog (Fig.
2A, trace c). In
this experiment, the cells were preincubated with thapsigargin prior to
assaying exchange activity. Thapsigargin inhibits the Ca2+-ATPase in the endoplasmic reticulum (22), leading to
the release of Ca2+ from internal stores. This treatment
accelerates the development of inhibition by ceramide and yields a
greater initial inhibition of Ba2+ influx (cf.
"Discussion"). Under these conditions, C2- and C6-ceramide inhibited Ba2+ influx by 55 and 51%, respectively
(p < 0.005). As shown in trace b in Fig.
2A, C2-dihydroceramide, a biologically inactive analog (14),
had only a small effect on exchange activity. The initial rate of
Ba2+ influx was reduced 25% by C2-dihydroceramide
(p < 0.05), but at later times, the rate of
Ba2+ influx in the presence of C2-dihydroceramide was
nearly identical to that of the control cells.
Ceramide and Ca2+ Efflux--
The traces in Fig.
3 depict the effects of C2-ceramide on
Nao+-dependent Ca2+ efflux. In this
experiment, cells were placed in either Na- or K-PSS, and thapsigargin
(1 µM) was added 60 s later. Following the addition
of thapsigargin, an increase in [Ca2+]i was
observed because of the release of Ca2+ from internal
stores. The traces in panel A for control cells show that the rise in
[Ca2+]i was reduced in Na-PSS compared with
K-PSS; as discussed in detail elsewhere (21), this difference is due to
Ca2+ efflux by the Na+/Ca2+
exchanger. When 15 µM C2-ceramide was included in the
assay medium, the difference between Na- and K-PSS was substantially
reduced, indicating that exchange-mediated Ca2+ efflux was
inhibited by the presence of ceramide. The peak rise in
[Ca2+]i in K-PSS was reduced in the
ceramide-treated cells compared with untreated cells, and a small,
gradual rise in [Ca2+]i was observed during the
30 s prior to the addition of thapsigargin (Fig. 3B).
The rise in [Ca2+]i evoked by ceramide was
clearly evident during a 3-min incubation period (data not shown),
suggesting that ceramide itself induced a slow release of
Ca2+ from the stores.
Ceramide and Mutant Exchangers--
The exchange protein consists
of multiple transmembrane segments with a large hydrophilic domain of
546 amino acids on the cytosolic membrane surface between the fifth and
sixth transmembrane segments (4, 5). The hydrophilic domain is thought
to be essential for the regulation of exchange activity because its elimination by proteolysis or mutagenesis eliminates normal regulatory behavior (23). The data in Figs. 4 and
5 show the effects of ceramide in cells
expressing several different mutations involving the hydrophilic
domain. The traces in Fig. 4A show that C2-ceramide had only
a modest effect on exchange activity in cells expressing a mutant,
A 20-residue region at the N-terminal end of the hydrophilic domain
plays an important role in exchanger regulation (24-26). This region
contains positively charged and hydrophobic residues in a pattern that
suggests an amphipathic helix. A peptide corresponding to this region
inhibits Na+/Ca2+ exchange activity when
applied to the cytosolic membrane surface, and this has therefore been
designated the XIP region for exchange inhibitory peptide (25). Site-directed
mutagenesis studies have shown that substitution of key positively
charged residues in this region with uncharged amino acids abrogates
sodium-dependent inactivation, suggesting an important
contribution of this region to regulatory behavior. In Fig. 4, the
traces in Panel B show the effect of ceramide on
cells expressing a mutant exchanger, designated XIPA, in which all the
positively charged residues in the XIP region have been substituted
with alanines. The data show that C2-ceramide strongly inhibits
exchange activity in this mutant. Traces a and b
in Fig. 4B show Ba2+ influx in a
Na+-free assay medium; under these conditions, exchange
activity is absent, and Ba2+ enters cells through passive
leak pathways. After correcting for passive Ba2+ entry, the
initial rate of Ba2+ influx was found to be inhibited by
68% (p < 0.001) in the presence of C2-ceramide. The
biologically inactive C2-dihydroceramide (15 µM) had no
effect on exchange activity in this mutant (data not shown).
The behavior of cells expressing a small deletion, Ceramide and [Ca2+]i--
Cytosolic
Ca2+ activates Na+/Ca2+ exchange
activity by binding to regulatory sites located in the exchanger's
central hydrophilic domain (reviewed in Refs. 1, 9). To determine
whether the effects of ceramide could be overcome by increasing
cytosolic Ca2+, we preincubated cells with ionomycin in
medium containing either 0.3 mM Ca2+ or 0.3 mM EGTA, so that the initial values of
[Ca2+]i prior to adding Ba2+ were
110 ± 11 and 44 ± 5 nM, respectively. As shown
in Fig. 6, the initial rate of
Ba2+ influx (in the absence of ceramide) was 3.7-fold
greater at the higher value of [Ca2+]i (compare
traces in panels A and B and rates in panel C). C2-ceramide (15 µM) inhibited Ba2+
influx in both cases but seemed to be less effective for the cells
preincubated in EGTA (35% inhibition) compared with cells preincubated
with 0.3 mM CaCl2 (59% inhibition) (Fig.
6C). However, this difference disappeared when the data were
corrected for the rate of passive Ba2+ leakage into the
cell in the absence of Na+/Ca2+ exchange.
Traces a and b in Fig. 6A show passive
Ba2+ influx when the cells were preincubated and assayed in
medium lacking Na+. The Ba2+ leak was very low
under control conditions (trace b) but increased 2.3-fold
(p
Two other features of the data in Fig. 6 deserve mention. First, the
slope of the control trace in Fig. 6A increases with time
following Ba2+ addition; the rate of Ba2+
influx at 150-200 s is 84% greater than at 65-95 s (p
Sphingosine Inhibits Exchange Activity--
The ceramide analogs
used in this study are short chain acyl derivatives of sphingosine. The
data in Fig. 7 demonstrate that sphingosine itself (2.5 µM) inhibited exchange-mediated
Ba2+ influx in cells expressing the wild-type exchanger but
not in cells expressing the Endogenous Sphingolipids and Exchange
Activity--
DL-Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol
(PPMP) and its shorter chain analog PDMP inhibit glucosylceramide synthase and sphingomyelin synthase in CHO cells (28) and other cell
types (reviewed in Ref. 29). Because they block important pathways of
ceramide utilization, they have been used to elevate endogenous
ceramide levels in various cell types (30-33). To determine whether an
elevation of endogenous sphingolipids would mimic the effects of the
exogenous short chain ceramide analogs, we incubated CK1.4 cells with
30 µM PPMP for 60 min prior to assaying
Na+/Ca2+ exchange activity. The results are
shown in Fig. 8 and Table I.
The initial rate of Ba2+ influx was inhibited by 49% in
CK1.4 cells following the PPMP treatment (Fig. 8A and Table
I). In this experiment, the cells were treated with thapsigargin
following the preincubation with PPMP and then assayed for
Ba2+ influx. When the experiment was repeated with cells
expressing the
To examine this issue, we pre-incubated the PPMP-treated and control
cells for 2 min with ionomycin in Na-PSS containing either 0.3 mM EGTA or 0.3 mM CaCl2, exactly as
described above for Fig. 6. As shown in Fig. 8C
(traces a) and in Table I, the initial values for
[Ca2+]i were identical for the PPMP-treated and
control cells after exposure to ionomycin + 0.3 mM
CaCl2. Importantly, the rate of Ba2+ influx
under these conditions was inhibited by 42% in the PPMP-treated cells,
indicating that the PPMP treatment inhibited exchange activity by a
mechanism that was independent of changes in
[Ca2+]i.
This experiment also shows that the initial rates of Ba2+
influx for the control and PPMP-treated cells were stimulated 3.6- and
4.1-fold, respectively, in the cells incubated with 0.3 mM CaCl2 compared with those incubated with EGTA (Fig.
8D and Table I). Thus, PPMP treatment did not block the
activation of the exchanger by Ca2+, despite the lower
overall exchange activity. As in the experiments with C2-ceramide (Fig.
6), the rate of Ba2+ influx for the PPMP-treated cells
incubated with 0.3 mM CaCl2 gradually declined
following the addition of Ba2+ and by 200-300 s became
identical to that for the EGTA-treated cells (110%, p = 0.17). (Note that with this batch of cells, the initial values of
[Ca2+]i following treatment with ionomycin + 0.3 mM CaCl2 were lower than for the experiment in
Fig. 6; the reasons for this variability are not known.)
In two of the experiments shown in Fig. 8 (A and trace
b. Cont in C), the slopes of the traces for
Ba2+ influx in control cells increased following the
addition of Ba2+. In both cases, this upward curvature was
not observed in the PPMP-treated cells. This behavior is essentially
identical to that observed with C2-ceramide, as discussed in connection
with Fig. 6.
Some additional observations with PPMP-treated cells are listed below,
although detailed data will not be presented in most instances:
(a) The inhibitory effects of PPMP required time to develop.
For example, after 30 min of incubation with 30 µM PPMP, inhibition of exchange activity was poor compared with cells incubated for 60 min. This behavior is consistent with a
time-dependent accumulation of endogenous ceramide and is
not compatible with a direct pharmacological effect of PPMP on exchange
activity. Indeed, 30 µM PPMP added directly to the
cuvette, without preincubation, did not inhibit exchange activity.
(b) The data shown in Fig. 8 (A and B)
were obtained with cells that had been treated with thapsigargin before
assaying for Na+/Ca2+ exchange activity.
Without thapsigargin, Ba2+ influx in the PPMP-treated cells
was inhibited by 64% relative to controls (Table I). Thus,
thapsigargin treatment did not enhance or accelerate inhibition of
exchange activity, in contrast to its effects with C2-ceramide (Figs. 1
and 2). For the cells expressing the The results presented here demonstrate that short chain ceramide
analogs inhibited both the Ca2+ influx and Ca2+
efflux modes of Na+/Ca2+ exchange activity
(Figs. 1-3). A biologically inactive ceramide analog, in which the
essential double bond in the sphingosine moiety is hydrogenated, had
only minor effects on exchange activity (Fig. 2). Moreover, certain
mutant exchangers that are defective in their regulatory behavior were
not inhibited by ceramide (Figs. 4 and 5). These data suggest that the
effects of ceramide are not simply due to a generalized membrane
perturbation but that ceramide acts in a biologically relevant manner
and specifically targets the mechanisms that regulate exchange
activity. Recent results indicate that 15 µM C2-ceramide
also inhibits Na+/Ca2+ exchange currents in
cardiac myocytes but does not inhibit either Na+ or
Ca2+ channel
activity.2
Ceramide is a central component of the sphingomyelin signaling pathway
and plays a critical role in the activation of the caspase cascade that
leads to apoptosis (14-16). In cardiac myocytes, ceramide and
sphingosine levels increase following ischemia/reperfusion (36, 37) and
after exposure of the cells to tumor necrosis factor Short chain ceramide analogs sometimes induce effects that are not
mimicked by increases in endogenous ceramide (reviewed in Ref. 39).
Moreover, the short chain analogs may themselves bring about
alterations in endogenous ceramide and/or sphingosine levels (35, 40,
41). To address these issues, we preincubated the cells for 60 min with
PPMP, an agent that blocks conversion of ceramide to glucosylceramide
and has been used to elevate endogenous ceramide levels in several
different cell types (30-33). As shown in Fig. 8, treatment with PPMP
mimicked the effects of the short chain ceramide analogs on
Na+/Ca2+ exchange activity. We conclude that
exchange activity is inhibited by increased endogenous ceramide and/or
sphingosine as well as by the exogenous analogs.
Ceramide and sphingosine did not inhibit exchange activity in certain
regulatory-deficient mutants, and we therefore conclude that the
sphingolipids target one of the mechanisms that regulate exchange
activity. The best characterized regulatory mechanisms involve two
time-dependent processes that promote inactive states of
the exchanger (6, 7). The first process is called
"Na+-dependent inactivation" and is
observed in excised patches as an exponential decay of current to a
steady-state value following application of cytosolic Na+.
Na+-dependent inactivation is counteracted by
the presence of phosphatidylinositol 4,5-bisphosphate (42), by high
concentrations of cytosolic Ca2+ (6), and by mutations
involving key basic residues or tyrosines in the XIP region (24, 43).
The second regulatory process involves the interaction of
Ca2+ with high affinity regulatory sites within the central
hydrophilic domain of the exchanger (7, 44); the binding of
Ca2+ to these regulatory sites appears to be required for
all modes of exchanger operation (1, 9).
Na+-dependent inactivation does not appear to
be involved in the effects of sphingolipids, because the XIPA mutant
was as sensitive to inhibition by ceramide as the wild type (Fig.
4B); this mutant does not display
Na+-dependent inactivation because of the
alteration of critical basic residues in the XIP region. Moreover,
ceramide inhibited wild-type exchange activity equally well at high (95 mM) and low (9 mM) Na+
concentrations (data not shown). Because
Na+-dependent inactivation requires high
concentrations of cytosolic Na+ (6), these results provide
another indication that ceramide does not promote this inactivation process.
The findings with the various exchanger mutants and the effects of
alterations in Ca2+ homeostasis suggest that sphingolipids
interfere with the regulatory activation of the exchanger by
Ca2+. C2-ceramide did not inhibit the activities of the
deletion mutants Experimental conditions that altered Ca2+ homeostatic
processes also affected the response of exchange activity to ceramide. For example, the effects of ceramide developed slowly in cells with
filled Ca2+ stores, as shown by the
time-dependent decline in the rate of Ba2+
influx in the presence of C2-ceramide (Fig. 1A). When
internal Ca2+ stores were depleted by prior treatment of
the cells with thapsigargin or ionomycin, the development of the
inhibition of ceramide was accelerated, and the traces for
Ba2+ influx no longer displayed a downward curvature (Figs.
2 and 6A). The results suggest that there is a link between
the filling state of intracellular Ca2+ stores and the
susceptibility of the exchanger to ceramide inhibition. The basis for
this observation is not known; perhaps filled Ca2+ stores
generate local gradients of elevated [Ca2+]i in
the vicinity of the exchanger, and this antagonizes ceramide
inhibition. For PPMP-treated cells, inhibition of exchange activity in
the absence of thapsigargin was immediate (Table I) and did not
increase with time following Ba2+ addition (data not
shown). This observation is entirely consistent with the observations
described above, because internal Ca2+ stores in the
PPMP-treated cells were already depleted, presumably because of an
inhibition of Ca2+ influx (see discussion under
"Results").
In several experiments, control cells showed a
time-dependent increase in the rate of Ba2+
influx, as shown by the upward curvature of the traces (Figs. 4B, 6A, 7A, and 8, A and
C, trace b. Cont). This behavior was not seen
with the These considerations suggest that ceramide/sphingosine impairs the
regulatory activation of exchange activity by Ca2+. Its
precise mechanism of action is not known, however. It does not seem
likely that these hydrophobic lipids would interact directly with high
affinity Ca2+ regulatory sites, because these are located
in the hydrophilic domain of the exchanger. A more plausible
possibility is that they interact with the transmembrane segments of
the exchanger and stabilize the inactive conformation that is attained
upon dissociation of Ca2+ from its regulatory binding sites
(Fig. 9). In this way, the sphingolipids
might increase the Kd for Ca2+
activation, slow the conformational transitions involved, or block
Ca2+ activation of the exchanger altogether in a
subpopulation of exchangers. In any event, it is clear that ceramide
does not completely block Ca2+ activation, because exchange
activity was stimulated by increasing [Ca2+]i in
the ionomycin experiments, both in the presence of C2-ceramide (Fig.
6C) and in the PPMP-treated cells (Fig. 8D and
Table I).
(241-680) and
(680-685) deletion mutants of the Na+/Ca2+ exchanger were not
inhibited by ceramide; these mutants show defects in both
Na+-dependent and
Ca2+-dependent regulatory behavior. Another
mutant, which was defective only in
Na+-dependent regulation, was as sensitive to
ceramide inhibition as the wild-type exchanger. Inhibition of exchange
activity by ceramide was time-dependent and was accelerated
by depletion of internal Ca2+ stores. Sphingosine (2.5 µM) also inhibited the Ca2+ influx and efflux
modes of exchange activity in cells expressing the wild-type exchanger;
sphingosine did not affect Ba2+ influx in the
(241-680)
mutant. The effects of the exogenous sphingolipids were reproduced by
blocking cellular ceramide utilization pathways, suggesting that
exchange activity is inhibited by increased levels of endogenous
ceramide and/or sphingosine. We propose that sphingolipids impair
Ca2+-dependent activation of the exchanger and
that in cardiac myocytes, this process serves as a feedback mechanism
that links exchange activity to the diastolic concentration of
cytosolic Ca2+.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(241-680) deletion mutant was prepared and
expressed in CHO cells (CK138 cells) as described (18). For XIPA cells,
the basic residues in the XIP region of the exchanger (219RRLLFYKYVYKRYRAGKQR) were replaced by alanines
(219AALLFYAYVYAAYAAGAQA). For this purpose,
RsrII and BclI restriction sites were introduced
into the cDNA for the bovine exchanger (p17; Ref. 19) by
site-directed mutagenesis at positions 1016 and 1079, respectively; an
oligonucleotide cassette with the desired sequence was ligated into the
cDNA after cleavage with RsrII and BclI to
create the XIPA mutant. cDNAs for the
(680-685) mutant of the
canine exchanger, as well as the wild-type canine exchanger, was kindly
provided by Drs. Kenneth D. Philipson and Debora A. Nicoll, UCLA School
of Medicine; the full-length cDNAs were ligated into the pcDNA3
expression vector (Invitrogen) and used for transfection of CHO cells
following the procedures described previously (17). The cells were
grown in Iscove's modified Dulbecco's medium containing 10% fetal
calf serum and antibiotics as described (17).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Inhibition of
Na+/Ca2+ exchange by C2-ceramide.
A, CK1.4 cells were loaded with fura-2 and preincubated for
5 min in Na-PSS + 1 mM CaCl2. The cells were
assayed for Na+/Ca2+ exchange activity in
20/120 Na/K-PSS + 0.3 mM EGTA containing 1 µg/ml
gramicidin, with or without 15 µM C2-ceramide, as
indicated. BaCl2 (1 mM) was added
(arrow) to initiate exchange mediated Ba2+
influx. B, rates of Ba2+ influx, given by the
slopes of the traces in A between 65-95 and
200-300 s, as indicated (n = 5). RU/s,
ratio units/second.
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Fig. 2.
Na+/Ca2+ exchange
activity in the presence of different ceramide analogs.
A, fura-2-loaded cells were preincubated for 2 min in Na-PSS + 0.3 mM EGTA with 1 µM thapsigargin and then
assayed for exchange activity as described in the legend to Fig. 1
alone (trace a) or with 15 µM C2-ceramide
(trace d), C6-ceramide (trace c), or
C2-dihydroceramide (trace b) present. B, initial
rates of Ba2+ influx given by the slopes of the traces
between 65-95 s (n = 5-8).
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Fig. 3.
C2-ceramide inhibits Ca2+ efflux
by the exchanger. A, cells were preincubated for 5 min.
in Na-PSS + 1 mM CaCl2 and placed in cuvettes
containing either Na-PSS or K-PSS as indicated. Thapsigargin (1 µM) was added after 60 s. Vertical arrow,
Tg. B, conditions were identical to A, except
that 15 µM C2-ceramide was added to the cuvettes
(n = 6-10).
(241-680), in which 440 amino acids were deleted from the
exchanger's hydrophilic domain. The initial rate of Ba2+
influx was reduced by 28% in the presence of C2-ceramide
(p = 0.05, paired t test), a value similar
to that seen with the wild-type exchanger when treated with the
biologically inactive C2-dihydroceramide (Fig. 2). As with
C2-dihydroceramide, little or no inhibition was observed during the
later portions of the time course of Ba2+ uptake.
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Fig. 4.
Effect of C2-ceramide in cells
expressing (241-680) and XIPA mutations of
the exchanger. A, cells expressing the
(241-680)
exchanger mutant were pretreated with thapsigargin and assayed for
Ba2+ influx with or without 15 µM C2-ceramide
as described in Fig. 2 (n = 6). B, as in
A, but with XIPA cells (see text; n = 9-10). For traces a and b, cells were pretreated
with 1 µM thapsigargin in K-PSS/EGTA and assayed for
Ba2+ influx in Na-free assay medium (K-PSS/EGTA + 1 µg/ml
gramicidin) (n = 3).
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Fig. 5.
Effect of C2-ceramide in cells
expressing (680-685) mutation of the
exchanger (A) and the canine wild-type exchanger
(B). A, cells expressing the canine
(680-685) exchanger mutant were pretreated with thapsigargin and
assayed for Ba2+ influx with (n = 10) or
without (n = 18) 15 µM C2-ceramide;
conditions were as described in Fig. 2. except that the
Ba2+ concentration was 5 mM. B, As
in A., but with cells expressing the canine wild-type
exchanger (n = 10-11). For traces a and
b in both panels, the cells were pretreated with 1 µM thapsigargin in K-PSS and assayed for Ba2+
influx (5 mM Ba2+) in sodium-free assay medium
(K-PSS + 1 µg/ml gramicidin; n = 9-13).
C, initial rates of Ba2+ influx, given as the
slopes of the traces in panels A and B
between 64 and 82 s.
(680-685),
within the hydrophilic domain of the canine cardiac exchanger is
presented in Fig. 5A. The activity of this mutant is not
regulated by cytosolic Ca2+ and shows little or no
Na+-dependent inactivation (27). The extent of
Ba2+ influx in these cells was reduced compared with
wild-type cells (see below), and so 5 mM BaCl2
was used in these experiments to increase the signal obtained. As shown
in Fig. 5 (A and C), exchange activity was not
significantly inhibited by 15 µM C2-ceramide (11%
inhibition; p > 0.3). Traces a and
b in Fig. 5A show the rate of passive
Ba2+ influx in the absence of Na+ and
demonstrate that exchange activity can readily be distinguished from
passive Ba2+ entry. The data in Fig. 5B show the
results of experiments carried out under identical conditions with
cells expressing the wild-type canine cardiac exchanger. In these
cells, the rate of Ba2+ influx, after subtracting the rates
of passive Ba2+ entry (traces a and
b), was inhibited by 64% (p
0.001) in the presence of 15 µM C2-ceramide. The time course of
Ba2+ entry for the canine exchanger under these conditions
was similar to that for cells expressing the bovine exchanger (Fig. 2).
However, for cells expressing the
(680-685) deletion mutant, the
rate of Ba2+ influx declined markedly within 40 s
following the addition of Ba2+. Thereafter, the traces were
nearly parallel to the traces for passive Ba2+ influx
(traces a and b in Fig. 5A). The
explanation for this curious behavior is not known.
0.01) following the addition of C2-ceramide
(trace a), suggesting that ceramide had increased the
passive permeability of the cell membrane to Ba2+. This
increase in passive Ba2+ entry was not observed in the
experiments described earlier (Figs. 4 and 5) and may reflect a
leakiness induced by the combination of ceramide and ionomycin. Even in
the presence of C2-ceramide, however, passive Ba2+ influx
was less than the rate of Ba2+ influx because of
Na+/Ca2+ exchange. When the passive rates of
Ba2+ influx were subtracted from the data obtained under
the standard assay conditions for Na+/Ca2+
exchange, the inhibition by C2-ceramide was essentially the same for
cells preincubated in either 0.3 mM CaCl2 or
0.3 mM EGTA (67% versus 62% inhibition,
respectively). We conclude that within this range of
[Ca2+]i values, increased activation of exchange
activity by cytosolic Ca2+ did not antagonize the
inhibitory effects of ceramide.
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Fig. 6.
Exchange activity in cells at different
[Ca2+]i and the effect of C2-ceramide. CK1.4 cells
were pretreated for 2 min with 10 µM ionomycin in Na-PSS + 0.3 mM EGTA (A) or Na-PSS + 0.3 mM
CaCl2 (B). Ba2+ influx was assayed
in 20/120 Na/K-PSS/EGTA with (C2) or without
(Cont) 15 µM C2-ceramide, as indicated
(n = 6-7). For traces a and b in
panel A, the cells were preincubated for 2 min with 10 µM ionomycin in K-PSS + 0.3 mM EGTA, and
assayed for Ba2+ influx in K-PSS + 0.3 mM EGTA
with (a) or without (b) 15 µM
C2-ceramide (n = 3). C, initial rates of
Ba2+ influx (65-95 s) for cells preincubated in 0.3 mM Ca, 0.3 mM EGTA or assayed in the absence of
Na+ (K-PSS).
0.001). This pattern was also observed for the XIPA cells
(Control trace, Fig. 4B). We have previously
suggested that this behavior reflects the auto-activation of exchange
activity by Ba2+, acting at the Ca2+ regulatory
sites to accelerate exchange activity as it accumulates within the
cytosol. In the ceramide-treated cells, the traces remain linear
throughout the entire time course, indicating that ceramide blocks this
effect. Second, for the cells that were preincubated with 0.3 mM Ca2+, the slope of the trace for
Ba2+ influx showed a time-dependent decline in
the presence of C2-ceramide (trace labeled C2 in
Fig. 6B). By 200-300 s, the rate of Ba2+ influx
became identical (104%) to that of cells preincubated in EGTA
(p > 0.3). This was not simply due to saturation of
the fura-2 or to Ba2+ uptake achieving a steady state,
because other traces with C2-ceramide (e.g. in Fig. 2) did
not display a downward curvature over a similar range of fura-2 ratios.
We suggest that [Ca2+]i probably continued to
decline following the addition of Ba2+ in these
experiments, leading to gradual decline in the
Ca2+-dependent activation of exchange activity
in the ceramide-treated cells.
(241-680) mutant. At a concentration of 2.5 µM, sphingosine did not make the cells leaky to
Ba2+, as shown in Fig. 7A (trace a).
At higher concentrations, however, a substantial Ba2+ leak
was induced, and so we were restricted to sphingosine concentrations
2.5 µM. At this concentration, sphingosine inhibited
the initial rate of exchange-mediated Ba2+ influx by 68%,
after correcting for the passive rates of Ba2+ influx. The
data in Fig. 7D show that sphingosine partially inhibited the Ca2+ efflux mode of exchange activity. The difference
between the traces for Tg-induced Ca2+ release in K-PSS
versus Na-PSS was reduced compared with the control cells
(Fig. 7C), indicating a reduction in
Na+-dependent Ca2+ efflux. Note
that sphingosine also induced a substantial leak of Ca2+
from internal stores, as shown by the increase in the fura-2 signal
prior to the addition of Tg; this probably accounts for the more rapid
rise and shorter duration of the Tg-evoked
[Ca2+]i transient in the presence of sphingosine
compared with controls.
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Fig. 7.
Sphingosine inhibits
Na+/Ca2+ exchange activity. A,
fura-2 loaded CK1.4 cells were preincubated for 2 min in Na-PSS + 0.3 mM EGTA with 1 µM thapsigargin and then
assayed for exchange activity as described in the legend to Fig. 1,
with (SPH) or without 2.5 µM sphingosine
present (n = 11-12). For traces a and
b, the cells were preincubated in K-PSS with thapsigargin
and assayed for Ba2+ influx in K-PSS + gramicidin with
(a) or without (b) sphingosine.
(n = 5-6). B, as in A, except
that cells expressing the (241-680) deletion mutant were used
(n = 4-6). C, cells were preincubated for 5 min. in Na-PSS + 1 mM CaCl2 and placed in
cuvettes containing either Na-PSS or K-PSS as indicated. Thapsigargin
(1 µM) was added after 60 s (n = 6-7). Vertical arrow, Tg. D, conditions were
identical to C, except that 2.5 µM sphingosine
was included in the medium in the cuvettes (n = 8).
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Fig. 8.
Endogenous sphingolipids inhibit exchange
activity. A, CK1.4 cells were preincubated for 60 min
with 30 µM PPMP and loaded with fura-2 as described under
"Experimental Procedures." The cells were then preincubated for an
additional 2 min in Na-PSS + 0.3 mM EGTA with 1 µM thapsigargin and assayed for exchange activity as
described in the legend to Fig. 1 (n = 7).
B, as in A, except that cells expressing the
(241-680) mutant were used (n = 9-10).
C, CK1.4 cells were preincubated with 30 µM
PPMP and loaded with fura-2 as described under "Experimental
Procedures." The cells were then incubated for 2 min in Na-PSS + 10 µM ionomycin containing either 0.3 mM
CaCl2 (traces a) or 0.3 mM EGTA
(traces b) and assayed for exchange activity as described in
the legend to Fig. 1 (n = 7). D, initial
rates of Ba2+ influx (34-64 s) for the data in
A-C, as indicated.
Effect of PPMP treatment on Na+/Ca2+ exchange activity
(241-680) mutant (Fig. 8B), only 21%
inhibition was observed, a value that was not statistically significant
(p = 0.09). With the CK1.4 cells (Fig. 8A)
but, curiously, not the
(241-680) cells, the initial value of
[Ca2+]i (prior to adding Ba2+) was
lower following PPMP treatment than for the control cells (Table I).
The lower value for [Ca2+]i probably reflects the
reduced Ca2+ entry and the greatly decreased size of
internal Ca2+ stores observed in the PPMP-treated cells
(see below). Because exchange activity is highly dependent upon
[Ca2+]i in this concentration range (11), it was
important to determine whether or not the inhibition induced by PPMP
was merely a secondary consequence of the reduced
[Ca2+]i.
(241-680) mutant, a 35%
inhibition (p < 0.02) of exchange activity by PPMP was
observed when the thapsigargin treatment was omitted (Table I). Thus,
the exchange activity of this mutant can be inhibited by PPMP, but less
effectively than the wild type, under some experimental conditions.
(c) PDMP produced similar effects to PPMP. However, higher
concentrations and/or longer incubation times were required for PDMP.
This finding is consistent with reports that PPMP is considerably more
effective than PDMP in inhibiting glucosylceramide synthesis in intact
cells (34). (d) Elevation of cellular ceramide levels has
been shown to inhibit store-dependent Ca2+
entry in T lymphocytes (35), and we have found that exogenous C2-ceramide inhibits store-dependent Ca2+ entry
in CHO cells. As a positive control for the studies conducted above, we
observed that the influx of Mn2+, a Ca2+
surrogate often used to provide an index of store-dependent
Ca2+ entry, was strongly inhibited in the PPMP-treated
CK1.4 cells. Internal Ca2+ stores (measured as ionomycin-
or thapsigargin-releasable Ca2+) were markedly reduced in
the PPMP-treated cells, presumably because of the reduced
Ca2+ entry. It should be noted that store-operated channels
do not contribute to Ba2+ influx during the assay for
Na+/Ca2+ exchange; the depolarized membrane
potential resulting from the gramicidin treatment (see "Experimental
Procedures") blocks any measurable Ba2+ entry by this
route (20). (e) Finally, we note that the PPMP treatment did not induce
a passive leak for Ba2+, as measured in gramicidin-treated
cells in the absence of Na+.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(38). Ceramide
induces a multitude of cellular responses, including induction of the
stress-activated protein kinase pathway, activation or inhibition of
various individual protein kinases, activation of protein phosphatase
2A, inhibition of mitochondrial respiration, activation of cytochrome
c release from mitochondria, and inhibition of phospholipase
D (reviewed in Refs. 14-16). Moreover, ceramide can be rapidly
converted to ceramide-1-phosphate, sphingosine, and
sphingosine-1-phosphate, signaling lipids that have multiple effects of
their own. The rapid time course of sphingolipid-induced inhibition of
exchange activity in the present study suggests that these agents exert
their effects directly on the exchanger rather than through one of the
above signal transduction pathways. This conclusion is strongly
supported by the recent finding that both sphingosine and C2-ceramide
inhibit exchange activity when applied to excised patches from cardiac
myocytes.3
(680-685) and
(241-680) (Figs. 4 and 5), which
are defective in both Ca2+-dependent activation
and Na+-dependent inactivation. As mentioned
above, C2-ceramide did inhibit the activity of the XIPA mutant (Fig.
4B), in which regulatory Ca2+ activation is
intact, but Na+-dependent inactivation does not
occur (24, 45). Thus, ceramide inhibited the activity of exchangers
that display Ca2+-dependent activation but not
in exchanger mutants in which this regulatory mechanism was defective.
(241-680) mutant (Figs. 4B, 7B, and
8B), but it was observed with the XIPA mutant (Fig.
4B). We have previously suggested that the acceleration in
Ba2+ influx is due to the auto-activation of the exchanger
by cytosolic Ba2+ through its interaction with the
Ca2+ regulatory sites (11). In the presence of ceramide or
sphingosine or in PPMP-treated cells, the corresponding traces for
Ba2+ influx remained linear throughout the entire time
course, indicating that the sphingolipids blocked this process.
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Fig. 9.
Proposed mechanism of ceramide
inhibition. When [Ca2+]i dissociates from
regulatory binding sites in the exchanger's hydrophilic domain, a
conformational transition from an active state to an inactive state
(I2) occurs. The designation I2 is to
distinguish this inactive state from the one produced by
Na+-dependent inactivation (I1;
Refs. 6 and 7). Elevation of [Ca2+]i (left
arrow) leads to refilling of the regulatory binding sites and a
rapid reactivation of exchange activity because of re-establishment of
the exchanger's active state. We propose that ceramide stabilizes the
I2 state through an interaction with the transmembrane
regions of the exchanger and thereby interferes with the I2
A transition. In beating cardiac myocytes, dissociation of
Ca2+ from regulatory sites during diastole would act in
conjunction with endogenous ceramide to govern the distribution of
exchangers between the A and I2 states.
In the experiments with ionomycin-treated cells (Figs. 6 and 8C), we sought to determine whether an increase in [Ca2+]i would antagonize the inhibitory effects of ceramide or PPMP treatment, as expected if a competitive effect were involved. In both cases, however, the degree of inhibition was similar at high and low [Ca2+]i. The highest value of [Ca2+]i attained in these experiments was only ~110 nM, however, and it will be important to re-examine this issue in an experimental system (e.g. excised patches) that permits a broader range of [Ca2+]i values to be studied. These experiments also showed that for the ceramide- or PPMP-treated cells at the elevated [Ca2+]i, the rate of Ba2+ influx declined gradually following Ba2+ addition and eventually became equal to that seen at the lower [Ca2+]i (Figs. 6B and 8C). This behavior probably reflects the gradual reduction in regulatory activation of exchange activity because the initially high level of [Ca2+]i declined following Ba2+ addition. In the corresponding experiments with control cells, the rate of Ba2+ influx did not decline in this manner, consistent with our hypothesis that the ability of Ca2+ and/or Ba2+ to activate exchange activity is impaired by the sphingolipids.
What are the physiological implications of our results? Ceramide and sphingosine are elevated in cardiac myocytes during stress. The resulting inhibition of exchange activity could be viewed as a protective measure to preserve Ca2+ stores and maintain contractile strength under stressful conditions. Alternatively, when cytosolic [Na+] is elevated, as in ischemia, inhibition of exchange activity could be a means of protecting the cell against Ca2+ overload by reducing exchange-mediated Ca2+ influx. A more interesting possibility, however, is that under nonpathological conditions endogenous sphingolipids and the exchanger work coordinately as a Ca2+-dependent feedback mechanism to control the distribution of exchangers between active and inactive states, in the manner described below.
The high affinity of the exchanger for regulatory Ca2+ (Kd ~50 nM) (11-13) would seem to provide no opportunity for meaningful regulation of exchange activity within a physiological range of [Ca2+]i values. In a functioning cardiac myocyte, the small fraction of exchangers that become inactive because of dissociation of regulatory Ca2+ during diastole ([Ca2+]i ~100 nM) would be rapidly reactivated by the ensuing rise in [Ca2+]i during the next systole. However, if endogenous ceramide/sphingosine were to interfere with Ca2+-dependent activation of the exchanger by any of the mechanisms suggested above (Fig. 9), a fraction of the inactive exchangers would be retained in the inactive state (I2 in Fig. 9) despite the rise in [Ca2+]i. Over multiple contraction/relaxation cycles, the distribution of exchangers between active and inactive states would be determined by two principal factors: diastolic [Ca2+]i and endogenous levels of ceramide/sphingosine. A fall in diastolic [Ca2+]i would increase the population of inactive exchangers, thereby reducing exchange-mediated Ca2+ efflux and eventually restoring diastolic [Ca2+]i to its normal level. An increase in endogenous ceramide or sphingosine, e.g. during stress, would have the same effect and establish a new steady-state relation between diastolic [Ca2+]i and exchange activity.
This hypothesis, although speculative, provides a welcome framework for
understanding the physiological role of
Ca2+-dependent activation of exchange activity
in light of the high affinity of the exchanger for regulatory
Ca2+ (11-13). In considering the physiological role of
sphingolipid-exchanger interactions, it will be essential to define
more precisely its effects on Ca2+-dependent
activation and the influence of internal Ca2+ stores on
this process. Work toward this end is currently in progress.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Kenneth D. Philipson
and Debora A. Nicoll (Departments of Physiology and Medicine, UCLA
School of Medicine) for generously providing the cDNAs for the
canine exchanger and the (680-685) mutant. We thank Drs. Donald W. Hilgemann (Department of Physiology, Southwestern Medical Center) and
Neal Shepherd (Duke University and VA Medical Center) for sharing
unpublished results on the effects of ceramide in excised patches and
cardiac myocytes.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL49932.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.
To whom correspondence should be addressed: Dept. of
Pharmacology and Physiology, UMDNJ-NJ Medical School, 185 South
Orange Ave., Newark, NJ 07103. Tel.: 973-972-3890; E-mail:
reeves@umdnj.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M006862200
2 N. Shepherd, personal communication.
3 D. Hilgemann, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CHO, Chinese hamster ovary; C2-ceramide, N-acetylsphingosine; C6-ceramide, N-hexanoylsphingosine; C2-dihydroceramide, N-acetylsphinganine; PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol; PPMP, DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol; Na-PSS, sodium physiological salts solution; K-PSS, potassium physiological salts solution; Mops, 4-morpholinepropanesulfonic acid.
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