Inversion of the Intracellular Na+/K+
Ratio Blocks Apoptosis in Vascular Smooth Muscle at a Site Upstream
of Caspase-3*
Sergei N.
Orlov
§,
Nathalie
Thorin-Trescases
,
Sergei V.
Kotelevtsev§,
Johanne
Tremblay
, and
Pavel
Hamet
¶
From the
Centre de Recherche du Centre Hospitalier de
l'Université de Montréal, Campus Hôtel-Dieu,
University of Montreal, Montreal, Quebec, Canada and
the § Laboratory of Biomembranes, Faculty of
Biology, M. V. Lomonosov Moscow State University, Moscow,
Russia
 |
ABSTRACT |
Long term elevation of the intracellular
Na+/K+ ratio inhibits macromolecule
synthesis and proliferation in the majority of cell types studied so
far, including vascular smooth muscle cells (VSMC). We report here that
inhibition of the Na+,K+ pump in VSMC by
ouabain or a 1-h preincubation in K+-depleted medium
attenuated apoptosis triggered by serum withdrawal, staurosporine, or
okadaic acid. In the absence of ouabain, both DNA degradation and
Caspase-3 activation in VSMC undergoing apoptosis were insensitive to
modification of the extracellular Na+/K+ ratio
as well as to hyperosmotic cell shrinkage. In contrast, protection of
VSMC from apoptosis by ouabain was abolished under equimolar
substitution of Na+o with K+o,
showing that the antiapoptotic action of
Na+,K+ pump inhibition was caused by inversion
of the intracellular Na+/K+ ratio. Unlike VSMC,
the same level of increment of the
[Na+]i/[K+]i ratio caused
by a 2-h preincubation of Jurkat cells with ouabain did not affect
chromatin cleavage and Caspase-3 activity triggered by treatment with
Fas ligand, staurosporine, or hyperosmotic shrinkage. Thus, our results
show for the first time that similar to cell proliferation, maintenance
of a physiologically low intracellular Na+/K+
ratio is required for progression of VSMC apoptosis.
 |
INTRODUCTION |
The maintenance of the transmembrane gradient of monovalent cation
(high [K+]i and low [Na+]i)
is a universal property of all nucleated cells, and its dissipation is
viewed as a hallmark of necrotic-type cell death (1, 2). It was shown
that a transient and moderate rise of intracellular Na+
concentration in mitogen-treated cells is involved in rejoining DNA
strand breaks preceding DNA synthesis (3), whereas long term inversion
of the intracellular Na+/K+ ratio blocks
macromolecular synthesis and cell cycle progression in the majority of
eukaryotic cells studied so far (4-7), including vascular smooth
muscle cells (VSMC)1 (8, 9).
Much less is known about the role of the transmembrane gradient of
monovalent ions in the triggering and progression of programmed cell
death (apoptosis).
Cell shrinkage is one of the initial morphological markers of apoptosis
in all types of cells, particularly in VSMC (10). In immune system
cells, apoptotic shrinkage is so impressive that the term
"shrinkage-mediated necrosis" was originally proposed to describe
this type of cell death (11), and the striking increase in the density
of shrunken cells was used to separate intact from apoptotic cells (12,
13). In lymphocytes, the apoptotic cell volume decrease is caused
by the loss of potassium chloride (14) and a major organic osmolyte,
taurine (15), because of the CD95 receptor-mediated activation of
Cl
and K+ channels and the taurine outward
transporter (for a recent review, see Ref. 16). However, the
involvement of perturbation of intracellular ion composition and the
ionic strength of the cytoplasm in the triggering and development of
the apoptotic machinery remains unclear. Recently, it was shown that
equimolar substitution of extracellular Na+ by
K+ protects Jurkat cells from apoptosis induced by Fas
ligand receptors (14), suggesting that dissipation of K+
gradients plays a role in the triggering of apoptosis in immune system
cells. Here, we report that in contrast to Jurkat cells, inversion of
the [Na+]i/[K+]i ratio
blocks apoptosis of VSMC at a site upstream of Caspase-3, independently
of the transmembrane gradient of monovalent cations and cell volume.
 |
EXPERIMENTAL PROCEDURES |
Cells--
VSMC were obtained by explant methods from the aortas
of 10-13-week-old male rats as described previously (17), cultured in
DMEM with 10% calf serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin, and used between 10 and 16 passages. Cells transfected
with c-myc (VSMC-MYC) and E1A adenovirus (VSMC-E1A) were
obtained in accordance with a previously described protocol (18, 19)
and were cultured in the same medium with the addition of 500 µg/ml
Geneticin. Jurkat cells were obtained from the American Type
Culture Collection and cultured in RPMI 1640 medium supplied with 10%
calf serum, antibiotics, sodium pyruvate, glutamate, and
-mercaptoethanol.
Chromatin Cleavage Assay--
Chromatin cleavage in VSMC was
estimated by a technique described previously in detail (10). Briefly,
VSMC grown in 24-well dishes were labeled for 24 h in
serum-supplied DMEM with [3H]thymidine (1 µCi/ml),
washed with 2 × 2 ml of DMEM, and incubated in serum-supplied
DMEM. For 48 h, the cells were pretreated with ouabain or
K+-depleted medium as indicated in Fig. 2 and Table III,
washed twice with serum-containing medium, and incubated with 0.5 ml of
medium with or without ouabain and containing different inducers of
apoptosis. To measure the content of chromatin fragments, the cells
were transferred on ice, and 1 ml of ice-cold lysis buffer (10 mM EDTA, 10 mM Tris-HCl, 0.5% Triton X-100 (pH
8.0)) was added. In 15 min, the cell lysate was transferred to
Eppendorf tubes and sedimented (12,000 rpm, 10 min), and 1 ml of
supernatant was transferred for the measurement of radioactivity
(A1t). The remaining radioactivity from
pellets and wells was extracted with 0.5 ml of a mixture of 1% SDS and
4 mM EDTA, combined, and counted
(A2). Chromatin cleavage was quantified as the
content of chromatin fragments normalized by total content of
3H-labeled DNA in accordance with the equation (1.5 × (A1t
A1o)/(A2 + A1t
1.5A1o)) × 100%, where
A1o is the value of
A1t before induction of apoptosis. To
measure chromatin fragmentation in [3H]thymidine-labeled
Jurkat cells, the cells were resuspended at a density of
106 cells/ml in medium containing 10% calf serum; the
additions are indicated in Fig. 4 and Table IV. At the time intervals
shown in Fig. 4, 100 µl of cell suspension was mixed with 100 µl of phosphate-buffered saline containing 0.5% Triton X-100, and the content of chromatin fragments was measured as described previously in
detail (20).
3'-End DNA Labeling--
VSMC-E1A seeded in 75-cm2
flasks were treated with 4 ml of lysis buffer containing 50 mM Tris-HCl, 20 mM EDTA (pH 8.0), 0.5% SDS,
and 500 µg/ml proteinase K. The lysate was incubated at 50 °C for
3 h, mixed with 250 µg/ml RNase A, and incubated for 1 h at
37 °C. After phenol-chloroform extraction, DNA was precipitated by
the addition of a mixture containing 500 mM potassium
acetate and ethanol (1:2, v:v). The precipitate was resuspended in
water, and 1 µg of DNA was mixed with a solution containing 2 mM CoCl2, 0.2 mM dithiothreitol,
100 mM potassium cacodilate, 0.5 mM
[32P]dCTP (3000 Ci/mmol), and 15 units/µl terminal
deoxynucleotidyl transferase (final volume, 20 µl). After a 1-h
incubation at 37 °C, the aliquots were loaded on a 1.5% agarose
gel, run at 100 V for 3-4 h, transferred onto a nylon membrane (Hybond
N+, Amersham Pharmacia Biotech), and analyzed with a PhosphorImager
(for more details, see Ref. 21).
Caspase Activity--
VSMC-E1A seeded in 20-cm2
flasks and treated as indicated in Fig. 5 were scratched, transferred
to centrifuge tubes, washed twice with phosphate-buffered saline, and
lysed in 0.5 ml of Buffer A containing 50 mM Tris-HCl (pH
7.4), 5 mM MgCl2, 1 mM EGTA, and 0.1% CHAPS. Then, 50-100 µl of cell lysate was mixed with 600 µl
of Buffer A containing 1 mM dithiothreitol and 40 µM YVAD-AMC or DEVD-AMC, with or without 1 µM Caspase-1 and Caspase-3 inhibitors (Ac-YVAD-CHO and
Ac-DEVD-CHO, respectively), incubated for 2 h at 37 °C, and
diluted 15-fold with 80 mM glycine-NaOH buffer (pH 10).
Fluorescence of the samples was measured at
ex = 365 nm and
em = 465 nm and calibrated with AMC in the range of
10 to 100 nM. To measure Caspase activity in control and
ouabain-treated Jurkat cells, 5 ml of suspension containing ~2 × 107 cells was transferred onto ice and sedimented. The
pellet of cells was washed and treated with 0.3 ml of Buffer A in the
same way as VSMC. Protein content was estimated by the Bradford method.
Intracellular Na+ and K+
Content--
The intracellular content of exchangeable Na+
and K+ was measured as steady-state distribution of extra-
and intracellular 22Na and 86Rb, as described
previously in detail (22). Briefly, to establish the steady-state
distribution of isotopes, VSMC growing in 12 (22Na)- or 24 (86Rb)-well plates or ~106 Jurkat cells in
suspension were preincubated for 24 h in DMEM containing 10% calf
serum and 0.5 µCi/ml 86RbCl or 2 µCi/ml
22NaCl and then treated in medium with the compositions
shown in Fig. 1 and Tables III and IV and with the same specific
radioactivity. At the end of the incubation time, the VSMC were
transferred on ice, washed four times with 2 ml of ice-cold medium W,
containing 100 mM MgCl2 and 10 mM
HEPES-Tris buffer (pH 7.4), and lysed with an SDS/EDTA mixture. One ml
of Jurkat cell suspension was applied on a Whatman type C filter with
the cells being washed with 3 × 5-ml aliquots of ice-cold medium
W under negative pressure at 20-30 mm Hg. The level of radioactivity
of the incubation medium and the cell lysate was measured with a liquid
scintillation analyzer. Intracellular cation content was determined as
A/am, where A was the radioactivity of the
samples (cpm), a was the specific radioactivity of
Na+ and K+ (86Rb) in the medium
(cpm/nmol), and m was the protein content determined by the
method of Lowry.
 |
RESULTS |
Inhibitors of the Na+,K+ Pump Block
Apoptosis in VSMC--
Previously, it was shown that the activity of
apoptotic pathways triggered by serum deprivation can be heightened by
transfection of VSMC with c-myc or with its functional
analogue E1A adenoviral protein (VSMC-MYC and VSMC-E1A cells,
respectively) (18, 19). We used these cells to study the involvement of
the [Na+]i/[K+]i ratio in
the regulation of VSMC apoptosis. To increase the
[Na+]i/[K+]i ratio, we
inhibited Na+,K+ pump activity with ouabain or
by depleting the incubation medium of K+. In VSMC-E1A,
2 h after the addition of ouabain, intracellular Na+
content was augmented from 38 ± 8 to 283 ± 30 nmol/mg
protein, whereas K+i dropped from 405 ± 41 to
131 ± 19 nmol/mg protein (Fig. 1,
curves 1 and 3). Prolongation of the incubation
with ouabain for up to 5 h did not significantly modify
Na+i and K+i content in
VSMC-E1A. The same kinetics of modulation of Na+i
and K+i content were observed in ouabain-treated
Jurkat cells (Fig. 1, curves 2 and 4) and in VSMC
incubated in K+-depleted medium (data not shown). A 10-fold
increase in the content of intracellular Na+ in ouabain
treated Jurkat lymphocytes (Fig. 1, curve 2) is consistent with the same level of elevation of free Na+i
concentration in human peripheral lymphocytes after inhibition of the
Na+,K+ pump in K+-depleted medium
and measured with a fluorescent Na+-indicator, sodium
binding benzofuran isophthalate (23). Considering these results, the
effect of the [Na+]i/[K+]i
ratio on apoptosis was studied after 1-2 h of
Na+,K+ pump inhibition.

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Fig. 1.
Kinetics of modulation of intracellular
Na+ (1, 2) and K+ (3, 4) content by ouabain in
VSMC-E1A (1, 3) and Jurkat cells (2, 4). Cells were preincubated
for 24 h in the presence of 10% calf serum in control medium
(consisting of 91 mM NaCl, 5 mM KCl, 0.9 mM NaH2PO4, 44 mM
NaHCO3, 1.8 mM CaCl2, 0.8 mM MgCl2, 33 mM HEPES, and 5 mM D-glucose (pH 7.4) with 2 µCi/ml of
22Na or 0.5 µCi/ml 86Rb), and 1 mM ouabain was added at the time interval indicated by the
arrow. At specific time points, the intracellular
Na+ and K+ contents were measured
as described under "Experimental Procedures." Mean values ± S.E. obtained in experiments performed in quadruplicate (VSMC-E1A) or
triplicate (Jurkat cells) are given. prot, protein.
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As seen in Fig. 2a, serum
deprivation drastically potentiated chromatin cleavage in VSMC-E1A, and
after 6 h the content of chromatin fragments was 4.5 ± 0.4 and 26.3 ± 3.0% in control and serum-deprived media,
respectively. Treatment with ouabain blocked base-line as well as serum
withdrawal-induced chromatin cleavage. Similar to VSMC-E1A, the
antiapoptotic effect of ouabain was also seen in serum-deprived
VSMC-MYC as well as in nontransfected VSMC (Table
I). In these experiments,
Na+,K+ pump activity was also blocked by
incubation of cells in K+-depleted medium. As with ouabain,
this procedure sharply inhibited VSMC apoptosis (Table I).

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Fig. 2.
Effect of ouabain on chromatin cleavage in
VSMC-E1A. a, kinetics of the accumulation of chromatin
fragments in control (curves 1 and 3) and
ouabain-treated (curves 2 and 4) VSMC-E1A in the
presence of 10% calf serum (CS, curves 1 and
2) or in CS-free medium (curves 3 and
4). Mean values ± S.E. obtained in experiments
performed in quadruplicate are given. b, content of
chromatin fragments in VSMC-E1A subjected to 6 h of incubation in
serum-supplied medium with or without 1 mM ouabain, 0.25 µM staurosporine, 1 µM okadaic acid, and
350 mM mannitol. Mean values ± S.E. obtained in three
experiments performed in quadruplicate are given. * and **,
p < 0.05 and 0.001, respectively, compared with
ouabain-untreated cells. In both experiments, ouabain was added 1 h before the triggering of apoptosis.
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Table I
Effect of ouabain and K+-depleted medium on chromatin cleavage
in nontransfected VSMC and in VSMC transfected with c-myc and E1A
adenovirus
Cells were incubated in K+-containing medium consisting of 91 mM NaCl, 5 mM KCl, 0.9 mM
NaH2PO4, 44 mM NaHCO3, 1.8 mM CaCl2, 0.8 mM MgCl2, 33 mM HEPES, and 5 mM D-glucose (pH 7.4) (lines 1 and 2) or in K+-depleted medium (equimolar substitution of
potassium chloride by sodium chloride, line 3) with or without 10%
calf serum (CS) and 1 mM ouabain as indicated. In
experiments with VSMC-MYC and VSMC-E1A, cells were preincubated for
1 h with ouabain or with K+-depleted medium in the
presence of CS and then subjected to treatment with serum-free medium.
After 24 h (VSMC) or 6 h (VSMC-MYC and VSMC-E1A), the content
of chromatin fragments was measured as described under "Experimental
Procedures." Mean values ± S.E. obtained in three experiments
performed in quadruplicate (VSMC) or triplicate (VSMC-MYC, VSMC-E1A)
are given.
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Fig. 2b shows that apart from serum withdrawal, VSMC
apoptosis can be triggered by inhibitors of protein kinase C and
serine-threonine phosphatase. After 6 h of incubation of VSMC-E1A
with staurosporine and okadaic acid, the content of chromatin fragments
was increased up to 30.1 ± 4.7 and 28.3 ± 4.2%,
respectively. Similar to serum-deprived cells (Fig. 2a),
apoptosis triggered by these compounds was blocked by treatment with
ouabain (Fig. 2b). Chromatin cleavage in control and
ouabain-treated VSMC-E1A was insensitive to hyperosmotic shrinkage caused by the addition of 350 mM mannitol (Fig.
2b). This is consistent with previous results obtained in
nontransfected VSMC (10).
The antiapoptotic action of ouabain in VSMC was further confirmed by
phase contrast microscopy and by analysis of DNA laddering. Fig.
3A shows that pretreatment
with ouabain sharply decreased the number of apoptotic cells after
6 h of incubation of VSMC-E1A in serum-deprived medium. Treatment
of VSMC-E1A with ouabain prevented DNA laddering and reduced the
accumulation of low molecular weight 3'-end-labeled DNA
oligonucleosomal fragments triggered by serum deprivation (Fig.
3B). Similar results were obtained with cells that underwent
apoptosis in the presence of staurosporine and okadaic acid (data not
shown).

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Fig. 3.
Effect of ouabain on morphology
(A) and DNA laddering (B) in
VSMC-E1A. In experiments with ouabain, cells were pretreated with
this compound (1 mM) 1 h before a 6-h exposure to
serum-free medium. A, cell morphology was evaluated by phase
contrast microscopy without preliminary fixation: a and
c, DMEM containing 10% calf serum; b and
d, serum-free DMEM; a and b, without
ouabain; c and d, in the presence of ouabain.
Micrographs were prepared using a Nikon phase contrast microscope at
×100 magnification. Apoptotic cells are shown by arrows.
B, DNA laddering (left side) was analyzed with a
PhosphorImager, and the relative content of low molecular weight DNA
fragments (1500-125 base pairs, right side) in cells
incubated with 10% calf serum (CS) in the absence of
ouabain was taken as 100%. Means ± S.E. obtained in two
experiments performed in triplicate are shown.
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Recently, we reported that heat stress triggers necrosis but does not
affect apoptosis in VSMC (24). Indeed, as can be seen from Table
II, in contrast to serum-deprived cells,
heat stress (46 °C, 30 min) did not modulate chromatin cleavage in
VSMC but led to a 5-fold increase of lactate dehydrogenase release, a
marker of necrosis. In contrast to apoptosis, we did not observe
modulation by ouabain of heat stress-induced lactate dehydrogenase
release. Viewed collectively, these data demonstrate that inhibition of the Na+,K+ pump protects VSMC against apoptosis
but does not affect the necrotic type of cell death.
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Table II
Effect of ouabain on chromatin cleavage and lactate dehydrogenase
release in serum-deprived and heat-treated VSMC
VSMC growing in six-well (lactate dehydrogenase (LDH) release) or
24-well (chromatin cleavage assay) plates were preincubated for 3 h with or without 1 mM ouabain. In part of the experiments,
the cells were subjected to a 30-min incubation at 46 °C
(heat-treated cells, line 3). They were then incubated in the absence
(line 2) or presence (lines 1 and 3) of 10% calf serum during an
additional 8 h, and chromatin cleavage and LDH release were
measured as indicated under "Experimental Procedures" and with a
spectrophotometric kit (Sigma), respectively. Means ± S.E. from
experiments performed in triplicate (LDH release) or quadruplicate
(chromatin cleavage) are given.
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Ouabain Does Not Affect the Induction of Apoptosis in Jurkat
Cells--
Fig. 4a shows that
during a 7-h incubation of Jurkat cells in control medium, the content
of chromatin fragments was increased monotonously up to ~5%. After a
1.5-h incubation in the presence of Fas ligand (anti-human Fas, mouse
monoclonal IgM), the content of chromatin fragments was increased by
10-fold and did not change much during the next 5.5 h of
incubation (Fig. 4a, curve 3). These kinetics are
consistent with previously reported data (20, 25). Apart from Fas
receptor-induced apoptosis playing a key role in the functioning of
activated T-lymphocytes, the apoptotic machinery in immune system cells
can be triggered by other stimuli, including inhibitors of protein
kinase C (14, 26) and hyperosmotic shrinkage (27). After a 3-h
incubation of Jurkat cells in the presence of 0.25 µM
staurosporine or in hyperosmotic medium (addition of 350 mM
mannitol), the content of chromatin fragments was increased from
2.6 ± 1.2 to 19.7 ± 4.3 and 15.6 ± 2.9%,
respectively (Fig. 4b).

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Fig. 4.
Effect of ouabain on chromatin cleavage in
Jurkat cells. a, kinetics of accumulation of chromatin
fragments in control (curves 1 and 3) and
ouabain-treated (curves 2 and 4) cells in the
absence (curves 1 and 2) or presence (curves
3 and 4) of 100 ng/ml anti-human Fas (mouse
monoclonal IgM, Upstate Biotechnology, Lake Placid, NY). b,
content of chromatin fragments in Jurkat cells subjected to 3 h of
incubation with or without 1 mM ouabain, 0.25 µM staurosporine, and 350 mM mannitol. In
both experiments, ouabain was added 2 h before the triggering of
apoptosis. Mean values ± S.E. obtained in experiments performed
in triplicate are given.
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In the absence of triggers of apoptosis, a 2-h pretreatment of Jurkat
cells with ouabain did not significantly modify chromatin cleavage
after a subsequent 3 h of incubation. Prolongation of incubation
with ouabain for up to 7 h increased the content of chromatin
fragments from 4.9 ± 2.1 to 16.0 ± 4.6% (Fig.
4a, curve 2 versus 1). We
did not observe any effect of ouabain on the increment of chromatin
cleavage triggered by a 3-h treatment of Jurkat cells with Fas ligand,
staurosporine, or mannitol (Fig. 4b). After a 7-h incubation
in the presence of Fas ligand, chromatin cleavage was significantly
higher in ouabain-treated cells (Fig. 4a, curve 4 versus 3). However, increment of the content of
chromatin fragments triggered by Fas ligand in control and
ouabain-treated Jurkat cells was not different (23.6 ± 3.9 and
26.2 ± 4.7%, respectively).
Effect of Equimolar Substitution of Extracellular Na+
by K+--
To examine whether or not protection of VSMC
cells against apoptosis by inhibition of
Na+,K+-ATPase is mediated by the alteration of
the intracellular Na+/K+ ratio, we compared the
modulation of apoptosis and the intracellular content of
Na+ and K+ by ouabain in control
([Na+] = 137 mM; [K+] = 5 mM) and K+-enriched, Na+-depleted
medium ([Na+] = 14 mM; [K+] = 128 mM). Table III shows that
in contrast to control medium, neither the intracellular content of
Na+ and K+ in VSMC-E1A nor base-line apoptosis
or apoptosis triggered by serum withdrawal was affected by ouabain in
K+-enriched, Na+-depleted medium. Similar to
VSMC-E1A, ouabain did not significantly affect the
[Na+]i/[K+]i ratio in
Jurkat cells incubated in K+-enriched,
Na+-depleted medium (Table
IV). In Jurkat cells, substitution of Na+ by K+ in incubation medium attenuated
apoptosis induced by a 7-h treatment with Fas ligand (20.0 ± 2.2 versus 28.5 ± 3.3% in K+-enriched and
control medium, respectively) and completely blocked the effect of long
term ouabain administration on base-line chromatin cleavage (5.7 ± 2.0 versus 16.0 ± 4.6% in K+-enriched
and control medium, respectively; Table IV).
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Table III
Effect of equimolar substitution of extracellular Na+ by
K+ on intracellular monovalent cation content and chromatin
cleavage in control and ouabain-treated VSMC-E1A
To estimate chromatin cleavage, cells were incubated for 1 h in
the presence of 10% calf serum (CS) in the control solution (DMEM,
[Na+] = 137 mM; [K+] = 5 mM; line 1) or under equimolar substitution of NaCl by KCl
([Na+] = 14 mM; [K+] = 128 mM; line 2) with or without 1 mM ouabain and
then for an additional 6 h in the same media with or without CS.
Intracellular Na+ and K+ content was measured as
steady-state distribution of isotopes after a 24-h preincubation of
cells in control Na+-enriched solution containing 0.5 µCi/ml
86Rb or 2 µCi/ml 22Na and an additional 6-h
incubation in Na+ (line 1)- or K+ (line 2)-enriched
medium, containing 10% CS and isotopes with the same specific activity
with or without 1 mM ouabain. For more details, see
"Experimental Procedures." Mean values ± S.E. obtained in
three experiments performed in triplicate (ionic content) or
quadruplicate (chromatin cleavage) are given.
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Table IV
Effect of equimolar substitution of extracellular Na+ by
K+ on intracellular monovalent cation content and chromatin
cleavage in control and ouabain-treated Jurkat cells
To estimate chromatin cleavage, cells were incubated for 2 h in
the presence of 10% calf serum in the control solution (DMEM,
[Na+] = 137 mM; [K+] = 5 mM; line 1) or under equimolar substitution of NaCl with
KCl ([Na+] = 14 mM; [K+] = 128 mM; line 2) with or without 1 mM ouabain and
then for an additional 7 h in the same media with or without 100 ng/ml Fas ligand (Fas-L). Intracellular Na+ and K+
content was measured as steady-state distribution of isotopes after a
24-h preincubation of cells in control Na+-enriched solution
containing 0.5 µCi/ml 86Rb or 2 µCi/ml 22Na and an
additional 6-h incubation in Na+ (line 1)- or K+ (line
2)-enriched medium containing 10% calf serum and isotopes with the
same specific activity with or without 1 mM ouabain. For
more details, see "Experimental Procedures." Mean values ± S.E. obtained in experiments performed in triplicate are given.
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Modulation of Caspase Activity--
Activation of the Caspase
superfamily protease cascade is involved in apoptotic DNA degradation
in the majority of cells studied so far (28, 29). However, to the best
of our knowledge, there are no data on the measurement of Caspase
activity in VSMC undergoing apoptosis. Using YVAD-AMC and DEVD-AMC
as substrates for the Caspase-1 and Caspase-3 subfamilies (30), we
observed that base-line activities of YVAD-ase and DEVD-ase in VSMC-E1A
were 1571 ± 256 and 673 ± 235 pmol (mg
protein)
1 h
1. In these cells, activity of
the Caspase-1 and Caspase-3 measured as YVAD-CHO and DEVD-CHO-sensitive
components of YVAD-ase and DEVD-ase, respectively, was 56 ± 47 and 547 ± 111 pmol (mg protein)
1 h
1
(n = 9). In Jurkat cells, total YVAD-ase and DEVD-ase
activity was 1830 ± 201 and 831 ± 95 pmol (mg
protein)
1 h
1, whereas Caspase-1 and
Caspase-3 activity levels were in the range of 121 ± 67 and
305 ± 41 pmol (mg protein)
1 h
1,
respectively (n = 12), which is in accordance with
previously reported data (31).
Neither Jurkat cells nor VSMC-E1A undergoing apoptosis showed any
modulation of Caspase-1 activity (data not presented). In contrast to
Caspase-1, Caspase-3 activity in VSMC-E1A was increased after a 6-h
incubation in serum-deprived medium or in the presence of staurosporine
and okadaic acid by 6-, 8-, and 10-fold, respectively, but was
insensitive to mannitol-induced cell shrinkage (Fig.
5A). Pretreatment of VSMC-E1A
for 1 h with ouabain decreased the base-line activity of Caspase-3
from 560 ± 41 to 101 ± 39 pmol (mg protein)
1
h
1 (p < 0.001) and abolished the
increment of Caspase-3 activity under induction of apoptosis (Fig.
5A). We did not observe any effect of ouabain on Caspase-3
activity in VSMC-E1A undergoing apoptosis triggered by okadaic acid in
K+-enriched, Na+-depleted medium
([Na+]o/[K+]o = 14/128)
(Fig. 5A). Similar negative results were obtained with
serum-deprived VSMC-E1A and VSMC-E1A treated with staurosporine in
K+-enriched, Na+-depleted medium (data not
shown).

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Fig. 5.
Effect of ouabain on Caspase-3 activity in
VSMC-E1A (A) and Jurkat cells
(B). VSMC-E1A and Jurkat cells were pretreated
with 1 mM ouabain for 1 and 2 h, respectively, and the
additions indicated below the bars were delivered in media
with 10% calf serum (CS) and with the base-line
(137/5) or inverse (14/128)
[Na+]o/[K+]o ratio for 6 (VSMC-E1A) or 3 h (Jurkat cells). In experiments with VSMC-E1A,
calf serum was omitted where indicated ( ). staur.,
staurosporine (0.25 µM); ok. ac., okadaic acid
(1 µM); mann., mannitol (350 mM);
Fas-L, Fas ligand (100 ng/ml). Mean values ± S.E.
obtained in experiments performed in quadruplicate (A) or
triplicate (B) are given. *, p < 0.001 compared with ouabain-untreated cells.
|
|
In Jurkat cells, Caspase-3 activity was increased by ~30-, 20-, and
10-fold after 3 h of treatment with Fas ligand, staurosporine, and
mannitol, respectively. Pretreatment of Jurkat cells for 2 h with
ouabain increased base-line Caspase-3 activity from 296 ± 38 to
497 ± 67 pmol (mg protein)
1 h
1.
Neither ouabain nor equimolar substitution of Na+ by
K+ in the incubation medium affected the increment of
Caspase-3 activity in Jurkat cells triggered by Fas ligand,
staurosporine, and hyperosmotic shrinkage (Fig. 5B).
 |
DISCUSSION |
The data obtained in the present study show for the first time
that independently of the origin of apoptotic signals and transfection with c-myc or its functional analogue E1A-adenoviral
protein, inhibition of the Na+,K+ pump blocks
the development of apoptosis in VSMC (Figs. 2 and 3) without any
modulation of VSMC necrosis triggered by severe heat stress (Table II).
In contrast to VSMC, a 2-h preincubation with ouabain in the absence of
triggers of apoptosis following a 3-h incubation in the presence of
three different apoptotic stimuli (Fas ligand, staurosporine, and cell
shrinkage with mannitol) did not modulate apoptosis in Jurkat cells
(Fig. 4). Prolongation of incubation with ouabain for up to 7 h
resulted in the activation of base-line chromatin cleavage in Jurkat
cells but did not affect the increment of accumulation of chromatin
fragments triggered by activation of the Fas receptor (Fig.
4a). Our results on the activation of base-line apoptosis
under a 7-h inhibition of the Na+,K+ pump in
Jurkat cells are consistent with recent data from Olej et
al. (32) on the induction of apoptotic DNA degradation in human peripheral lymphocytes treated with ouabain for 48 h. The potentiation of base-line apoptosis after long term treatment of immune
system cells with ouabain may be caused by the rise of intracellular
Ca2+ concentration due to activation of the
Na+i/Ca2+o mode of operation of
the Na+/Ca2+ exchanger, which is highly active
in human T-lymphocytes (33) and in Jurkat cells (34). This hypothesis
is supported by prevention of the induction of base-line apoptosis in
Jurkat cells treated with ouabain in Na+-depleted medium
(Table IV), i.e. under inhibition of the
Na+i/Ca2+o mode of operation of
the Na+/Ca2+ exchanger, and is consistent with
numerous data on the implication of sustained elevation of
[Ca2+]i in triggering apoptosis in immune system
cells (35, 36). In contrast to immune system cells, we did not observe any effect of moderate elevation of [Ca2+]i by
thapsigargin and ionomycin on VSMC apoptosis (37).
Our results show that inhibition of apoptosis by ouabain in VSMC is
caused by inversion of the
[Na+]i/[K+]i ratio rather
than by Na+i/K+i-independent
modulation of ion current and membrane potential mediated by
electrogenic Na+,K+ pump. Indeed, suppression
of VSMC apoptosis by ouabain was abolished in K+-enriched
Na+-depleted medium, i.e. when inhibition of the
Na+,K+ pump did not affect intracellular
Na+ and K+ content (Table III). As with
ouabain, VSMC apoptosis was blocked by inhibition of the
Na+,K+ pump in K+-depleted medium
(Table I). We did not observe any significant effect of an 8-h
incubation of VSMC with ouabain on lactate dehydrogenase release (Table
II), ATP content, and protein synthesis (data not shown). DNA synthesis
in VSMC, measured as serum-induced [3H]thymidine
incorporation, was also insensitive to a 5-h preincubation with ouabain
and was decreased by ~60% only after 48 h of the Na+,K+ pump inhibition (data not presented).
Viewed collectively, these results rule out the possible side effects
of ouabain on VSMC as well as the toxic effect of inversion of the
[Na+]i/[K+]i ratio.
It may be assumed that inversion of the
[Na+]i/[K+]i ratio blocks
VSMC apoptosis via inhibition of the net K+ efflux involved
in cell shrinkage revealed in most of the cells undergoing programmed
cell death, including the VSMC (10). This hypothesis is based on the
induction of apoptotic DNA degradation in hyperosmotically shrunken
mouse lymphoma cells, rat thymocytes (27), and Jurkat cells (14).
Similar results were obtained in the present study by analysis of the
effect of mannitol-induced shrinkage on chromatin cleavage in Jurkat
cells (Fig. 4b). Moreover, we report here that like other
inducers of apoptosis, hyperosmotic shrinkage led to activation of
Caspase-3 in Jurkat cells (Fig. 5B). However, neither
chromatin cleavage (Fig. 3B) nor Caspase-3 activity (Fig.
5A) was affected by mannitol-induced shrinkage of VSMC-E1A.
These results underlie a cell-specific impact of shrinkage in apoptosis
and demonstrate a cell volume-independent mechanism of inhibition of
VSMC apoptosis by inversion of the [Na+]i/[K+]i ratio. Under
analysis of these data it should be mentioned that hyperosmotic
shrinkage leads to rapid clustering and ligand-independent activation
of receptor tyrosine kinases, including tumor necrosis factor receptors
(38). Based on these results, the induction of apoptosis in immune
system cells in hyperosmotic medium may be viewed as a consequence of
the clustering and activation of Fas receptors (16). Considering this
fact, it may be suggested that the lack of effect of hyperosmotic
medium on VSMC apoptosis is caused by the absence of functionally
active death domain receptors. Indeed, neither Fas nor tumor necrosis
factor-
were able to trigger apoptosis in VSMC from normal vessels
(39, 40) or in E1A-transfected VSMC.2
Dissipation of the transmembrane gradient of monovalent cations caused
by Na+,K+ pump inhibition can affect apoptosis
via membrane depolarization and conformational transition of
membrane-bound proteins involved in the triggering of apoptotic
machinery. In this case, it can be predicted that similar to
ouabain-treated VSMC, apoptosis can also be blocked by a rise of
extracellular K+ concentration. Indeed, suppression of
apoptosis by equimolar substitution of Na+o with
K+o was observed in Jurkat cells treated with 10 ng/ml anti-human Fas IgM (14). In our study, equimolar substitution of
Na+o with K+o led to a 30-40%
inhibition of the increment of chromatin cleavage triggered by
treatment of Jurkat cells with 100 ng/ml anti-human Fas IgM (Table IV).
However, in contrast to Jurkat cells, equimolar substitution of
Na+o by K+o did not protect
ouabain-untreated VSMC-E1A from apoptosis triggered by serum withdrawal
(Table III). These results strongly suggest that inversion of the
[Na+]i/[K+]i ratio blocks
VSMC apoptosis independently of modulation of the Na+ and
K+ electrochemical gradient across the plasma membrane.
We report here that suppression of apoptosis in ouabain-treated
VSMC-E1A is accompanied by inhibition of Caspase-3 activity (Fig.
5A). Thus, it may be assumed that the increased
[Na+]i/[K+]i ratio blocks
VSMC apoptosis via direct inhibition of the activity of this enzyme. To
examine this hypothesis, we measured the activity of Caspases in
lysates of VSMC-E1A subjected to 6 h of serum withdrawal. Both
Caspase-3 and Caspase-1 activity was decreased by 10-20% by the
addition of 100 mM KCl. However, the same level of
inhibition was also observed with the addition of 100 mM
NaCl or choline chloride (data not shown). Thus, it may be concluded
that the rise in ionic strength slightly inhibits Caspases, whereas the
Na+/K+ ratio does not affect enzyme activity.
It is well documented that activation of Caspase-3 by Fas ligand is
caused by cleavage of pro-Caspase-3 triggered by Fas-activated death
domain-mediated activation of Caspase-8 (41) and is independent of
cytochrome c release from mitochondria (42). However, as mentioned above, the Fas ligand signaling pathway is quenched in VSMC
and VSMC-E1A. In several cell types, Fas-independent cleavage of
pro-Caspase-3 is triggered by Caspase-9, which in turn is activated by
the Apaf-1/cytochrome c complex (41). At least in HeLa
cells, cytochrome c release is caused by Caspase-8-mediated
proteolysis of Bid, a BH3 domain-containing protein that interacts with
both Bax and Bcl-2 (43) and is independent of dissipation of the mitochondrial transmembrane potential (44). Recently, it was proposed
that pro-Caspase-3 activity is controlled by the
Na+/K+ ratio (31). This hypothesis was based on
the selective inhibition by KCl of Caspase-3 activity in rat thymocyte
lysates treated with 10 µg/ml cytochrome c and 1 mM dATP. However, we failed to detect any effect of these
compounds at the same concentration on Caspase-3 activity in VSMC-E1A
lysates in the absence of monovalent cations or in the presence of 100 mM KCl or NaCl (data not shown). Viewed collectively, these
results demonstrate that the
[Na+]i/[K+]i ratio blocks
VSMC apoptosis at a site upstream of Caspase-3.
In conclusion, our results show that inversion of the
[Na+]i/[K+]i ratio blocks
apoptosis in VSMC via the inhibition of trigger-independent steps of
the programmed death machinery at a site upstream of the
Caspase-3-triggering cascade. Comparison of the requirements for
Caspase-9 and Caspase-3 in fibroblasts and immune system cells treated
with UV and
-irradiation, staurosporine, or anti-CD95 indicates the
existence of at least four different types of apoptosis (45). Data
obtained in the present study show that Caspase-3-dependent
apoptosis may be further subclassified based on its sensitivity to the
[Na+]i/[K+]i ratio and cell
volume. In VSMC, Caspase-3-dependent apoptosis can be
blocked by increment of the
[Na+]i/[K+]i ratio but is
insensitive to cell shrinkage. In contrast, in Jurkat cells, cell
shrinkage activates Caspase-3-dependent apoptosis, whereas
the [Na+]i/[K+]i ratio does
not affect apoptosis triggered by Fas ligand, staurosporine, or cell
shrinkage. Is this mechanism limited to VSMC, or is it also expressed
in other electrical excitable tissue subjected to modulation of the
[Na+]i/[K+]i ratio under
sustained depolarization? Which molecules are involved in the sensoring
of intracellular Na+(K+) concentration(s) and
transduction of the antiapoptotic signal? These questions will be
addressed in future studies.
 |
ACKNOWLEDGEMENTS |
We are very much obliged to Dr. Martin R. Bennett (University of Cambridge, UK) for providing us with
VSMC-E1A and VSMC-MYC. We thank Monique Poirier for excellent technical
assistance and Ovid Da Silva for editing this manuscript.
 |
FOOTNOTES |
*
This study was supported by grants from the Medical Research
Council, Canada (to P. H. and S. N. O.), Bayer Canada (to P. H.),
Pfizer Canada (to P. H. and S. N. O.), and the Russian Foundation for Fundamental Research (to S. V. K. and S. N. O.).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: Centre de
Recherche-CHUM, Campus Hôtel-Dieu, Laboratory of Molecular
Medicine, 3850 rue St. Urbain, Montréal, Québec H2W 1T8,
Canada. Tel.: 514-843-2737; Fax: 514-843-2753; E-mail:
hamet{at}ere.umontreal.ca.
2
S. N. Orlov, N. Thorin-Trescases, J. Tremblay, and P. Hamet, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
VSMC, vascular
smooth muscle cell(s);
DMEM, Dulbecco's modified Eagle's medium;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
CHO, Chinese hamster ovary;
AMC, 7-amino-4-methylcoumarin;
YVAD, N-acetyl-Tyr-Val-Ala-Asp;
DEVD, N-acetyl-Asp-Glu-Val-Asp.
 |
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