1494
Ouabain Increases the Calcium
Concentration in Intracellular Stores Involved
in Stimulus-Response Coupling in
Human Platelets
Peter Roevens and Didier de Chaffoy de Courcelles
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The effect of ouabain on Ca> homeostasis in human platelets was studied using both quin 2
and chlorotetracycline to monitor changes in cytosolic Ca>2 as well as changes in the amount
of Ca>2 accumulated in intracellular storage sites. In resting platelets, ouabain induces a
concentration- and time-dependent increase in cytosolic Ca21 concentration and a marked
elevation of Ca2 in the intracellular stores. The amount of Ca>2 mobilized from these stores
upon stimulation with thrombin, as well as thrombin-induced secretion of platelet 5-hydroxytryptamine, was increased after preincubation with the glycoside (3 x 10-6 M). These data show
that ouabain induces an elevation of intracellular Ca>2 levels, most likely mediated via
Na4-Ca' exchange, and that this incremental amount of Ca>2 is accumulated in an
intracellular store involved in stimulus-response coupling. This may explain the enhanced
functional responses of platelets to agonists in the presence of ouabain and suggests a role for
Na+-Ca'4 exchange in Ca>2 homeostasis of the human platelet. (Circulation Research
1990;67:1494-1502)
C ardiac glycosides are known to inhibit the
plasma membrane-associated Na+,K+ATPase, resulting in an increase in intracellular Na' and in the inhibition of K' uptake. The
increased intracellular Na+ has been suggested to reduce the gradient across the cell membrane and in turn
to reduce the magnitude of the Na+-Ca2` exchangecoupled calcium efflux.' The consequent net increase in
intracellular Ca`+ could then account for the increased
contractility induced by cardiac glycosides in cardiac
and smooth muscle.'-4 Ouabain, a typical cardiac glycoside, also enhances functional responses in the platelet,5-7 and both the rate and the amplitude of the
aggregatory response to agonists are increased.7 Recently Na4-Ca`+ exchange activity has been demonstrated in human platelet plasma membrane vesicles.8
In the unstimulated platelet, this Ca2+ transport mechanism may well contribute to the low cytosolic free Ca2`
concentration ([Ca'+]<,t) against the steep Ca`+ gradient. If this is the case, it should be expected that
lowering of the transmembrane Na+ gradient decreases
the driving force for outward Ca2+ flux by the exchanger
and that, as a consequence, [Ca]2+y increases. Indeed,
From the Department of Biochemistry II, Janssen Research
Foundation, Beerse, Belgium.
Address for correspondence: P. Roevens, Department of Biochemistry II, Janssen Research Foundation, 2340 Beerse, Belgium.
Received September 13, 1989; accepted August 2, 1990.
studies in which extracellular Na+ was replaced by
choline described a small increase in [Ca'+k, that was
measured using quin 2, but the results depended on
the kind and/or the purity of the substitute for Na+
that was used.910 The use of ouabain to increase
intracellular Na+ has the advantage that the Na+
gradient is lowered at extracellular Na+ in the physiological range. In smooth muscle cells, an ouabaininduced increase in [Ca 2],y, has been evidenced using
fluorescent probes.3"'1 However, in the platelet, quin 2
studies failed to demonstrate an effect of ouabain on
basal [Ca'+]@,,.9,12,13 These puzzling data and the lack
of effect of Na+ on total platelet Ca2+9 make the role
of Na+-Ca2+ exchange in platelet Ca'+ homeostasis
controversial,9"14-17 in spite of the reported effects of
Na+ on platelet intracellular Ca2+ pools, assessed by
45Ca2+ flux experiments.9 Nevertheless, it has been
generally accepted that Ca2+ plays a pivotal role as
an intracellular messenger for platelet excitatory
agonists. Its release from intracellular stores is
made possible by the breakdown of inositol-containing phospholipids.'7
Although there is much circumstantial evidence
indicating that in muscle cells ouabain increases Ca'
in intracellular stores that are involved in excitationresponse coupling, direct proof on the working mechanism is still absent. We have taken the human blood
platelet as a model to test this hypothesis. In platelets, the Ca'+-sensitive fluorescent probes quin 2 and,
Roevens and de Chaffoy de Courcelles Effect of Ouabain on Platelet Calcium Homeostasis
to a lesser extent, chlorotetracycline (CTC) have
been used to measure respectively changes in
[Ca2'], and changes in intracellular Ca'+ stores.18-20
We used both methods to study the effect of ouabain
on platelet Ca2' handling. We found that inhibition
of Na+,K+-ATPase activity leads to a concentrationand time-dependent increase in [Ca2], and in the
amount of Ca2' accumulated in an intracellular pool
that is involved in stimulus-response coupling. The
results can be explained by ascribing a role to Na+Ca2' exchange in platelet Ca2' homeostasis.
Materials and Methods
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CTC was obtained from Sigma Chemical Co., St.
Louis, ouabain from Janssen Chimica, Beerse, Belgium, and thrombin (Topostasine) from Roche Laboratories, Basel, Switzerland. They were all dissolved in
0.9% NaCl. Quin 2-AM came from Calbiochem, La
Jolla, Calif., and was dissolved in dimethyl sulfoxide.
Pure grade bovine serum albumin was obtained from
Serva, Heidelberg, FRG. Sepharose-CL2B came from
Pharmacia Laboratories, Uppsala, Sweden.
['2P]Orthophosphate and ['H]5-hydroxytryptamine
(5-HT) were purchased from Amersham, Buckinghamshire, England.
Platelet Isolation and Handling
Blood collection from healthy human volunteers
and preparation of platelet-rich plasma were carried
out as described previously.21 Except for quin 2
experiments, platelets were isolated by repeated centrifugation21 and suspended in buffer A containing
(mM) HEPES 25, NaCl 125, KCl 2.7, MgSO4 1,
glucose 10, EGTA 0.1, Na2HPO4 0.5, and 0.1%
bovine serum albumin at pH 7.4. In CTC experiments, MgSO4 and bovine serum albumin were omitted. In all experiments, except for those shown in
Figure 5, platelets were preincubated for 15 minutes
in buffer with 1 mM CaCl2 to allow them to recover
from the isolation procedure and to restore normal
Ca2' homeostasis before ouabain was added. All
incubations were at 370 C.
Fluorescence Measurements Using Quin 2 as a Probe
Platelet-rich plasma was incubated with 15 ,uM
quin 2 acetoxymethylester at 370 C for 12 minutes.
Platelets were separated from plasma and extracellular dye by gel filtration on a Sepharose-CL2B
column (2.5 x 18 cm) equilibrated with buffer A. The
platelet concentration was adjusted to 2.2 x 108/ml,
and 1.1 ,umol CaClJml was added. Fluorescence was
measured at 370 C using a thermostated cuvette in a
purpose-modified fluoronephelometer (excitation at
340 nm, emission at 490 nm; Technicon, Tarrytown,
N.Y.). Platelets (1 ml) were placed in the cuvette,
and fluorescence in the resting state was recorded.
Subsequently, 2 mM EGTA was added, and 1 minute
later, platelets were triggered with thrombin. Quantification of [Ca2'],, was performed as described by
Rink and Pozzan.22 The measurements were corrected for dye leakage in each sample using 100 uM
1495
Mn'+ and 1 mM diethylenetriaminepentaacetic acid
(DTPA). Unstimulated platelets were exposed to
free Mn' ions (100 ,M) for maximally 20 seconds
before they were chelated by an excess of DTPA (1
mM). Preliminary experiments had shown that neither this short exposure to free Mn2 ions nor the
presence of free DTPA and the Mn-DTPA complex
had any effect on platelet Ca2' response to stimulation by thrombin, in agreement with Rink and Pozzan.22
Calibration was also carried out on every platelet
sample studied. One measurement took 5 minutes.
The sensitivity of platelets to thrombin slightly decreased within the time of the experiment. Therefore, measurements on ouabain-treated platelets and
control platelets were performed alternately and
evaluated as paired samples.
Fluorescence Measurements Using CTC as a Probe
The mechanism by which platelet-associated CTC
responds to changes in Ca>2 in intracellular stores
has been described in detail by others.18 We used the
following procedure: 10 ,uM CTC was added to
washed platelet suspension (2 ml, 2 x 108 platelets/ml)
in the cuvette at 370 C in the presence of 1 mM
external Ca2. Fluorescence was measured at 390 nm
excitation and 530 nm emission in a fluorometer
(model 3C, Jobin-Yvon, Longjumeau, France)
equipped with a thermostatically controlled cell
holder (four cuvettes). The addition of CTC gives
rise to an instantaneous increase of fluorescence (fast
phase), followed by a slow increase of fluorescence,
which we monitored for 15 minutes (slow phase).
Other authors18 have shown that the rapid change is
due to fluorescence of the Ca-CTC complex in the
aqueous phase and binding of the Ca-CTC complex
on the outer surface of the plasma membrane. The
slow fluorescence increase has been shown to arise
primarily from the binding of the Ca-CTC complex to
the inner surfaces of Ca2+-sequestering organelles
and is proportional to the free Ca>2 concentration in
these organelles.18,23
We calculated the CTC ratio as the amplitude of
the slow phase (A,1Ow) divided by the amplitude of the
fast phase (Afast) taken 15 minutes after CTC addition. It represents a quantitative index of net Ca21
accumulated into intracellular Ca2+-sequestering
pools per unit surface area of platelets.24
Measurement of 5-HT Secretion
Platelet amine storage granules were loaded with
[3H]55-HT by incubating platelet-rich plasma for 20
minutes at 370 C with 0.3 ,Ci/ml [3H]5-HT. The
loaded platelets were washed as described above.
Secretion of [3H]5 -HT after stimulation was assessed
by removing 200 ,ul platelet suspension (2x 108/ml)
and adding it to 20 ,ul of 13.5% (wt/vol) formaldehyde in 0.9% NaCl on ice. After centrifugation at
10,OOOg for 2 minutes, aliquots (100 ,ul) of the supernatant were removed for liquid scintillation counting.
1496
Circulation Research Vol 67, No 6, December 1990
Results are expressed as a percentage of total
tritium in an equivalent volume of the platelet suspension lysed with 0.1% Triton X-100.
[32P]Orthophosphate Labeling Experiments
Isolated platelets (2x 108/ml) were incubated for
70 minutes with 50 litCi/ml [32P]orthophosphate.
Phospholipid analyses were performed exactly as
described previously.21
[3H]Inositol Labeling Experiments
Platelet isolation, labeling, and [3H]inositol phosphate analyses were performed exactly as described
previously.25
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Data Presentation
In each test, ouabain-treated platelets were compared with control platelets. Data are presented as
mean+ SEM. Unless indicated otherwise, the asterisks in the figures denote the statistical evaluation of
data performed by Student's t test for paired observations. Values of p<0.05 were considered significant. Dose-effect data were also evaluated by analysis of
variance.
For quin 2 experiments, each measurement on
ouabain-treated platelets was compared with either
the preceding control measurement or (only in Figure
6) the succeeding control measurement. In Figure 2,
control values corresponding with different ouabain
concentrations were not significantly different by analysis of variance and thus were pooled in the presentation of the results. Because the variance of [Ca2+],
data increased with the mean value, analysis was
performed on the logarithmic transformation.26
Where error bars do not appear on figures, errors
are within the symbol size.
Results
Ouabain-Induced Changes in Ca'2 Homeostasis
When quin 2 was used as an intracellular probe,
[Ca21 was 112+9 nM (mean+SEM; n=25). Incubation of platelets with ouabain during 75 minutes, in
the presence of extracellular Ca2+ (Ca2+'ex), increased
the resting [Ca21] in a concentration-dependent
manner (Figure 1).
Addition of excitatory platelet agonists induces an
elevation of [Ca2+]1,y, by mobilization from intracellular stores and by influx from the extracellular space.27
We investigated whether ouabain had any effect on
the mobilization of Ca21 from the intracellular stores.
Therefore, platelets were stimulated with thrombin
in the presence of an excess of extracellular EGTA
over Ca2+. Exposure to the chelator was brief in order
to avoid a time-dependent EGTA-induced modification of Ca2' homeostasis. Because of the virtual
absence of Ca2+ex at the moment of triggering, the
rise in fluorescence can be attributed to mobilization
from intracellular stores.20 In the absence of ouabain,
the increase in [Ca21]( after the addition of thrombin was 214± 16 nM (mean±SEM; n=25) (Figure 2).
1501400
0
,
120X
0
110-0(NS
100
,.
8
7
6
5
4
Ouabain concentration (-log M)
FIGURE 1. Graph showing the effect of ouabain on the
cytosolic free Ca2' concentration ([Ca2'],) in resting platelets. Quin 2-loaded platelets were preincubated with different
concentrations of ouabain or its solvent in the presence of 1
mM extracellular Ca2 , and basal [Ca2+/t was determined
after 75 minutes. The data shown (a) are mean +SEM of the
percent of control values from five experiments. [Ca2+/]t of
resting controlplatelets (100%) was 112+9 nM (mean ±SEM;
n=25; see also data presentation). NS, not significant;
*p <0.01 and **p <0.001 by analysis of variance for repeated
measures using a Bonferroni correction for the significance
level (i.e., overall significance level divided by number of
comparisons, in this case 0.05/5=0.01).
Preincubation with ouabain increased the amount of
Ca2' mobilized from intracellular stores in a concentration-dependent manner (Figure 2). Maximal elevation was obtained at 10-5 M of the glycoside. These
data indicate that more Ca21 was accumulated in
intracellular pools, due to ouabain treatment.
CTC is a probe that has been shown to monitor
changes in free Ca21 concentration in intracellular
storage sites, in which this concentration approaches
the millimolar range.18 We determined the platelet
CTC ratio after 75 minutes incubation, according to
quin 2 measurements. In the presence of ouabain
(3x10`6 M), A,10w was markedly higher than in its
absence (Figure 3), whereas pretreatment with ouabain did not significantly change the magnitude of
Afast. Af5St as well as A010w depend on platelet concentration, the concentration of Ca2'ex, and the amount
of CTC added. A.10W, however, has also been shown to
be proportional to the free Ca21 concentration in
Ca2+-sequestering organelles.18 The CTC ratio (A,10,/
AfaSt) can therefore be considered as an index of the
amount of Ca21 that is accumulated in these organelles. The CTC ratio increased from 0.68+±0.07 in the
control to 1.12±0.06 (mean±SEM; n=8) after 60
minutes of treatment with the glycoside. The relevance of the CTC ratio to measure the amount of
Ca2' accumulated in intracellular storage sites was
validated by the use of ionomycin. In the absence of
Ca2+ex ionomycin has been shown to discharge Ca2+
from intracellular stores.14,18 If the increased A,1,. in
ouabain-treated platelets indicates an increased
Roevens and de Chaffoy de Courcelles Effect of Ouabain on Platelet Calcium Homeostasis
A
900800-
1497
B
6-
Thrombin
0.5 UlmI
C
. 6005-
cuiNO
400(U
*
(NS)
en
OCD
Thrombin
0.5 UlmI
c
a)
0i
4-
200c)
c
c
0_
l
i
/
o)
4
5
7
6
8
Ouabain concentration (-log M)
0
1 min.
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FIGURE 2. Graph showing the effect of ouabain on the
increase in cytosolic free Ca21 concentration ([Ca21]13,) after
stimulation with thrombin in the absence of extracellular Ca2 .
Quin 2-loaded platelets were preincubated for 75 minutes
with different concentrations of ouabain (a) or its solvent (o)
in the presence of 1 mM extracellular Ca2+. Basal [Ca2+]1,Y
was determined and presented in Figure 1. Subsequently, 1
mM extracellular Ca21 was chelated by the addition of 2 mM
EGTA, and 1 minute later, platelets were stimulated with
thrombin (0.02 units/ml). Peak [Ca2+1]Y, was determined, and
the increase in [Ca2+],Y was calculated. The data shown are
mean +SEM from five experiments and from all the controls
(o; n=25). NS, not significant; *p<0.01 and **p<0.001 by
analysis of vaniance for repeated measures using a Bonferroni
correction for the significance level.
0
-G
3c
lonomycin
2mM EGTA
lonomycin
4
2mM EGTA
6
J
platelets, which we measured as an increase of the
CTC ratio, could be released by the ionophore.
The Ca2' accumulated in these stores could also be
mobilized on stimulation with thrombin. This is
shown in Figure 4. After 15 minutes of pretreatment
with CTC (10 ,M), platelets were triggered with
thrombin in the presence of 2 mM EGTA, as was the
case in quin 2 experiments. Upon the addition of
thrombin (0.5 units/ml), CTC fluorescence decreased
to a larger extent in ouabain-treated platelets than in
control platelets, indicating that thrombin indeed
could release more Ca2' out of the intracellular pool
in the presence of ouabain. The threshold level for
B
7
4
5.4.
aa
:p 3.2
1*
0
t
0
5
10
15
20
Time (min.)
25
30
CTC
0
1 min.
FIGURE 4. Tracings showing thrombin-induced changes in
platelet chlorotetracycline (CTC) fluorescence in the absence
of extracellular Ca2`. Washed platelets were preincubated for
60 minutes with solvent (panel A) or ouabain (3x10`6 M)
(panel B) in the presence of 1 mM Ca2`; 10 pM CTC was
added, and the incubation was continued for 15 minutes.
Then extracellular Ca2' was chelated by the addition of2 mM
EGTA, and 3 minutes later, fluorescence was recorded. Zero
fluorescence was set with platelet suspension without CTC.
The data represent 24 measurements.
amount of Ca2' in intracellular pools, complete discharge by ionomycin should result in a drop of CTC
fluorescence to a same level, independent of ouabain
treatment. Figure 3 shows that, upon the addition of
ionomycin (3 ,uM) together with EGTA to complex
Ca2+ ex the fluorescence of control platelets, as well as
ouabain-treated platelets, indeed falls to a common
steady-state level. Apparently, the increased amount
of Ca2+ in intracellular stores of ouabain-treated
A
3-
5
10
15
Time (min.)
20
25
FIGURE 3. Graphs showing
the effect of ouabain on the chlorotetracycline (CTC) ratio. A,10,
and Afast, amplitude of the slow
and fast phases of fluorescence
increase, respectively. Washed
platelets were preincubated for
60 minutes with solvent (panel
A) or ouabain (3 x 10 -6 M)
(panel B) in the presence of 1
mM Ca2`. CTC was added, and
fluorescence was recorded as described in "Materials and Methods." The CTC ratio is calculated as AsowlAf,fast. At indicated
3'0 time periods, ionomycin (3 MM)
together with 2 mM EGTA was
added. The data represent 30
measurements.
1498
Circulation Research Vol 67, No 6, December 1990
2
,
800-
900-
700 1
800-
600 1
700-
500-
600
400-
C
500-
CW
°
400 -
300-
(NS)
300-J
200-
'
200-
100-
(NS)
+
1000-
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Preincubation
Control Ouabain
Control Ouabain
[Ca +]ex
[Ca 2eX
<
10-M
=
1mM
FIGURE 5. Bar graphs showing the effect of ouabain on
resting cytosolic free Ca21 concentration ([Ca2'],,) and on the
thrombin-induced Ca2' mobilization from intracellular stores
after preincubation in either the presence or absence of
extracellular Ca21 ([Ca2+],). Quin 2-loaded platelets were
preincubated for 75 minutes with ouabain (3x10`6 M) or
solvent (control) in the presence of 1 mM Ca2+ or without
Ca2+. [Ca2+J1,1 was determined in the resting state (shaded
bars) and after stimulation with thrombin (0.02 units/ml) in
the presence of 2 mM EGTA, described in the legend of
Figure 2. Results represent mean ±SEM from nine measurements in three experiments. NS, not significant; **p<0.05 and
***p<O.OJ by Student's t test. The significance was venified on
the resting [Ca2'](, and on the increase in [Ca2+],, after the
addition of thrombin.
as
measuring thrombin-induced release of Ca2' from
this intracellular store, using CTC as a probe, was as
high as 0.25 units/ml (results not shown).
Dependence on Ca',
To determine the contribution of Ca21ex on [Ca2+]y
and on the thrombin-induced mobilization from intracellular stores, Ca2' was omitted during pretreatment
of quin 2-loaded platelets. Because buffer A contained 0.1 mM EGTA, Ca2+ex was virtually absent
under these conditions. The data presented in Figure
5 show that after 75 minutes of incubation in the
virtual absence of Ca2+ex platelet [Ca21] was markedly lower than after incubation with Ca2 ex in the
physiological range. Treatment with ouabain, in the
presence as well as in the absence of Ca2
slightly
augmented the resting [Ca2],y and markedly amplified the mobilization of intracellular Ca21 induced by
thrombin. Furthermore, Figure 5 illustrates that -the
amount of Ca21 mobilized from intracellular sources
on stimulation with thrombin increased with increasing resting [Ca2+]1M.
ex,
00
1015
25 30 40 45
60 65
Time (min)
80 85
FIGURE 6. Graph showing the time course of the effect of
ouabain on the cytosolic free Ca2+ concentration ([Ca2+j,,) in
resting platelets and after stimulation with thrombin in the
absence of extracellular Ca2 . Quin 2-loaded platelets preincubated in the presence of 1 mM Ca21 for 15 minutes were
divided into two batches. At zero time, ouabain (3 x10-6 M; ,
a) was added to one batch, while the control (o, r) received
solvent. At indicated time periods, samples were taken for
measurement. [Ca21q,t was determined in the resting state (n,
*) and after stimulation with thrombin (0.02 units/ml) (o, *)
in the presence of 2 mM EGTA, as described in the legend of
Figure 2. Results represent mean +SEM from three experiments. NS, not significant; *p<0.05, **p<0.03, and
***p<0.01 by Student's t test.
Time Dependence
In resting platelets, ouabain (3 x 10 6 M) increased
[Ca2'],Y, in a time-dependent manner, and changes
were significant within 10 minutes (Figure 6). After
35 minutes of contact with the glycoside, thrombininduced mobilization of intracellular Ca2' was significantly higher than in the controls. The most pronounced effect appeared only after 60 minutes
contact with ouabain. The sensitivity of control platelets to thrombin notably decreased as a function of
time, resulting in a 50% loss of the response after 85
minutes. The same experiments were performed
using CTC as a probe. Figure 7 illustrates that the
drop of thrombin-induced increase in [Ca2'],, in
control platelets, as shown in Figure 6, coincided with
a decrease in CTC ratio. Conversely, the CTC ratio
increased in the presence of ouabain and was obvious
after 30 minutes of contact with the glycoside. There
appeared to be no significant differences between
Afast of control platelets and ouabain-treated platelets within the course of the experiment (not shown).
Roevens and de Chaffoy de Courcelles Effect of Ouabain
Platelet Calcium Homeostasis
1499
1000-
1.5-
800-
1.2
c
*4
NS)
-5
+
0
on
cm
S)
0.9
600-
0
C:
()
400-
F-
0
0.6 200-
0.3-
0-
0.0025
0.0
0.01
0.02
0.03
g
9
0
15
a
30
45
Time (min)
Thrombin (U mr-1)
1
60
75
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FIGURE 7. Graph showing the time course of the effect of
ouabain on the chlorotetracycline (CTC) ratio. Washed platelets preincubated in the presence of 1 mM Ca2' for 15 minutes
were divided into two batches. At zero time, ouabain (3 X10 6
M, a) was added to one batch while the control (n) received
solvent, and incubation was continued. At indicated time
periods, samples were taken, and CTC ratio was determined.
Results represent mean +SEM offour experiments with duplicate detertninations. Four cuvettes were measured at the same
time; two were filled with control platelet suspension, and two
were filled with ouabain-treated suspension. The means of the
duplicate deterninations (n=4) were compared using Student's t test for paired data. NS, not significant; *p<0.05;
**p<0.03; and ***p<0.01.
Thrombin Concentration Dependence
Thrombin induced a concentration-dependent mobilization of intracellular Ca2' (Figure 8). Even an
agonist concentration as low as 0.0025 units/ml was
able to release more Ca2' from the intracellular
stores, in the presence of ouabain (3 x 106 M).
We verified whether ouabain also increased the
functional response of the platelet to stimulation in
our experimental conditions. Therefore, we measured the thrombin-induced secretion of dense granular 5 -HT. In the presence of the glycoside, the
concentration-response relation for thrombininduced 5 -HT release was shifted to the left (Figure
9), which substantiates that platelets are more sensitive to thrombin in the presence of ouabain.
Effect on the Signal Transducing System
As in other cell types, mobilization of intracellular
Ca' in platelets depends on the generation of second messengers. Thrombin activates an inositolphospholipid-specific phospholipase C.28 A stimulatory
effect of ouabain on the activation of this phospholipase C might thus be the underlying mechanism for
the thrombin-induced increase in [Ca2],,, and secretion. Therefore, we measured activation of the phospholipase C by the thrombin-induced increased formation of [32P]phosphatidic acid ([32P]PA) in
FIGURE 8. Graph showing the effect of ouabain on the
increase in cytosolic free Ca21 concentration ([Ca2 ]c,,) on
stimulation with different thrombin concentrations in the
absence of extracellular Ca2 Quin 2loaded platelets were
preincubated for 75 minutes with ouabain (3x10 'M) (-) or
its solvent (o) in the presence of 1 mM Ca2`. [Ca2+],,, was
deternined in the resting state (not shown) and after stimulation with indicated concentrations of thrombin in the presence
of 2 mM EGTA. Results shown are mean+±SEM of the
increase in [Ca2'],, from three to six measurements in three
experiments. *p<0.05 by Student's t test. The upper curve
differs significantly from the lower curve (p=0.0028 by oneway analysis of variance).
.
[32P]orthophosphate-prelabeled cells.* Table 1 shows
that the thrombin-induced [32P]PA formation was not
increased by ouabain (3 x 10 6 M). However, in resting platelets, ouabain significantly decreased the 32p
incorporation in this phospholipid during the prelabeling period (Table 1).
Discussion
Ouabain-Induced Changes in Ca2' Homeostasis
In the present communication, changes in platelet
Ca2' homeostasis were assessed with quin 2. Although fura 2 has several properties that are advantageous compared with those of quin 2,29 its accuracy
is strongly hampered after long incubation times, due
to dye leakage, especially at physiological temperature levels.16,30 In contrast, using quin 2, we were able
to quantify minor changes in cytosolic Ca2' even after
75 minutes of incubation at 370 C (Figure 1). In the
light of specific limitations that have been reported
the use of cytosolic Ca2+ indicators,30 we used
on
*Activation of an inositolphospholipid-specific phospholipase C
is usually quantified by measuring labeled inositolphosphate in
cells or tissue prelabeled with [3H]inositol. In accordance with data
presented earlier,28 the low concentration of thrombin used in our
study did not enable us to measure any changes in agonist-induced
[3H]inositolphosphate release. Therefore, as in most platelet studies,'7 cells were prelabeled with [32P]orthophosphate. Receptorinduced activation of phospholipase C results in the formation of
diacylglycerol, which is immediately phosphorylated by diacylglycerol kinase into [32P]PA using [32PIATP as a cosubstrate.25
Circulation Research Vol 67, No 6, December 1990
1500
10080-
CD)
a
60
40-
20(NS)
0*
I
0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05
Thrombin U mF'1
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FIGURE 9. Graph showing the effect of ouabain on the
thrombin-induced secretion of platelet 5-hydroxytryptamine (5
HT) in the absence of extracellular Ca2+. Washed platelets
loaded with [3H] 5 HT were preincubated for 75 minutes with
ouabain (3x10-6 M) (o) or its solvent (o) in the presence of 1
mM Ca2`. EGTA (2 mM) was added, and after 1 minute,
platelets were stimulated with different concentrations of thrombin. Samples were taken 5 minutes later. Preincubation with
ouabain did not affect theplatelet 5HTcontent. Results represent
mean +SEM of three experiments with duplicate samples. NS,
not significant; *p<0.05, **p<0a03, and ***p<O.0J by Student's t test. The upper curve differs significantly from the lower
curve (p=0.0043 by one-way analysis of variance).
CTC as an alternative probe. Although the parameters monitored by CTC are less clearly defined than
the more commonly used Ca2' indicators,2031 this
probe enabled us to estimate changes of Ca2, in
intracellular stores and could, as such, be regarded as
complementary to the cytosolic Ca2, measurements.
We found, using quin 2 as a probe, that ouabain
induced a concentration- and time-dependent increase
in resting [Ca2+], (Figures 1, 2, and 6). The effect of
ouabain was small; differences in platelet handling
and/or in the experimental procedures strongly affect
the precision of the quin 2 method, which might explain
the lack of effect of ouabain in earlier studies.912,13
TABLE 1. Effect of Ouabain on the Thrombin-Induced Activation
of Phospholipase C
[3"PiPhosphatidic acid formation
(cpm)
Saline
Ouabain (3 x 10`6 M)
Control
16,916+1,053*
11,756±930
Thrombin (0.03 units/ml) 41,613+9,044t
31,802+8,112
Values are mean±SEM from four experiments with duplicate
samples. Platelets were prelabeled for 75 minutes with ['2PJorthophosphate at 1 mM extracellular Ca'2, either in the presence or
absence of ouabain (3 x 10-6 M). One minute after the addition of
2 mM EGTA, control samples were taken, and platelets were
subsequently challenged with thrombin (0.03 units/ml). Samples of
the stimulated platelets were taken 35 seconds later.
*p<0.005 compared with ouabain-treated platelets by Student's
t test.
tp<0.05 compared with ouabain-treated platelets by Student's t
test.
Schaeffer and Blaustein16 though, using fura 2, demonstrated an ouabain-induced increase in [Ca2'], at low
extracellular Na+, and very recently, an ouabain-induced enhancement of basal and stimulus-induced
platelet [Ca'+]<, at physiological levels of extracellular
Na+ was reported.15 However, the authors were unable
to decide on which aspect of Ca'+ homeostasis the
ouabain-enhanced functional response is ultimately dependent. Since in their experiments platelets were only
triggered in the presence of Ca' ex, the release of Ca'
from intracellular stores upon stimulation with thrombin was, most likely, masked by influx of Ca`.
The use of CTC enabled us to demonstrate that
ouabain induced a marked elevation of Ca' in
intracellular stores (Figures 3 and 7). The importance of this store in stimulus-response coupling
became apparent on stimulation with thrombin in the
absence of Ca2`ex. Thrombin released Ca> from this
store (Figure 4) into the cytosol (Figure 5). This
phenomenon occurred in an ouabain concentrationand time-dependent manner (Figures 2 and 6) and
was quantitatively of much more importance than the
small ouabain-induced increases in resting [Ca'],yt
(Figure 1).
When prolonged incubation of control platelets
was performed in the absence of Ca>2ex, basal
[Ca21, was markedly lower (Figure 5). This phenomenon has been reported before.32,33 In these
conditions, both Na+ and Ca> can flow along their
chemical gradients. As a consequence, Ca>2 efflux via
Nat-Ca2 exchange is most likely facilitated and
reaches a new equilibrium at the lower level in
resting [Ca2+I<,. In the absence as well as in the
presence of Ca 'ex, the ouabain-induced decrease of
the transmembrane Na+ gradient resulted in an
increase in basal [Ca 21, compared with the control
level. Jy and Haynesl8 demonstrated, by the use of
CTC, that the resting [Ca2']<, determines the Ca>2
level in intracellular stores. This view was supported
by our finding that, as [Ca2+],, is elevated, more Ca>2
can be mobilized by thrombin from the intracellular
stores to the cytosol (Figure 5).
Using CTC as a probe, the thrombin-induced
release of Ca> from intracellular stores, which is
measured as a decrease in fluorescence, could only
be made evident at higher concentrations of the
agonist when compared with the quin 2 method; this
occurred in control as well as in ouabain-treated
platelets. Most likely, the thrombin releasable pool is
only a minor part of the total intracellular Ca>2 store.
Indeed, the release of a minor part of the intracellular pool that contains a millimolar concentration of
Ca> can produce relatively major elevations of Ca>2
in the cytosol, where it is present at submicromolar
concentrations. Furthermore, the Ca2'-buffering capacity of quin 2 may induce an exaggerated mobilization of Ca> out of intracellular stores. It has been
reported that, when CTC is used in combination with
the nonfluorescent intracellular Ca>2 chelator bis(oamino, n-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, which mimics this Ca2+-buffering capacity,
Roevens and de Chaffoy de Courcelles Effect of Ouabain on Platelet Calcium Homeostasis
the CTC method becomes more sensitive to lower
concentrations of thrombin.20
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Time-Dependent Aspects of the Ouabain Effect
The time course experiments (Figures 6 and 7)
indicate that prolonged incubations are needed to
demonstrate a marked effect of ouabain on platelet
Ca2' homeostasis. This is not surprising since the
influx of Na+ down its concentration gradient by
passive diffusion is the most likely rate-limiting step
in this phenomenon. Influx of Na+ via Na+-Ca2
exchange seems unlikely by the strong Ca2 gradient
that is directed inward, and influx via Na+-H+ exchange has been reported to be nearly quiescent in
the nonactivated platelet.34
The time-dependent fall of the CTC ratio (Figure
7) and of the thrombin-induced increase of [Ca2'],
in control platelets (Figure 6) indicate that there is a
gradual loss in the amount of Ca'+ that is accumulated in the intracellular stores. The release of Ca's
from intracellular stores into the cytosol is thought to
be one of the earliest steps in platelet activation.17,35
Decreased accumulation of Ca'+ in these stores may
thus be a part of the molecular mechanism behind
the previously reported time-dependent decline of
platelet reactivity during long-term experiments.7"16
The amplification of the platelets' functional responses to agonists after ouabain pretreatment, as
found by others5-7 as well as by ourselves (Figure 9),
is also in line with the idea that CTC-detectable Ca2
stores play an important role in the platelet response.
Taken together, these data point to the increased
intracellular accumulation of Ca2' as a likely explanation for the enhancement of platelet reactivity
after ouabain treatment.
Ouabain Does Not Affect the Signal-Transducing
System for Thrombin
The mobilization of Ca'4 from intracellular stores
in control platelets, as well as in ouabain-treated
platelets, was dependent on the agonist concentration (Figure 8), indicating the particular role of
receptor-induced second messenger formation in the
Ca`4-mobilizing process. The increased amount of
Ca'2 mobilized in ouabain-treated platelets could
therefore alternatively be explained by an ouabaininduced increase in the amount of second messenger
formed. In this context, our finding that the thrombin-induced activation of the phospholipase C (the
enzyme that generates the second messengers) was
not increased in the presence of ouabain (Table 1) is
of major importance. Although not conclusive, since
we were unable to measure inositol 1,4,5-trisphosphate (IP3) formation, these results indicate a lack of
effect of ouabain on the primary enzyme involved in
the signal transducing system in which Ca2 functions
as an intracellular messenger.
Platelet activation by thrombin and ADP has been
shown to induce a rapid increase in Na4 influx, which
could imply that Na4 plays a role in stimulusresponse coupling.5,36 Although the effect of ouabain
1501
on platelet Ca`4 homeostasis is evident, a separate
role for intracellular Na4 in the ouabain-induced
effects on stimulus-response coupling cannot be excluded. The increase of the cytosolic Na4 concentration in the resting platelet most likely causes acidification of the cytosol via Na+-H+ exchange." By
increasing the sensitivity of the dense tubular system
to 1P3, these changes in cytosolic pH and/or cytosolic
Na4 concentration may also explain the ouabaininduced enhancement of the platelet response to
agonists. This possibility has been addressed by Brass
and Joseph,35 who studied the effect of pH and Na4
on 1P3-induced 45Ca`4 release in permeabilized platelets. They demonstrated that Na+ concentration
(10-90 mM) was without effect and that a decrease
of pH from 7.1 to 6.7 resulted in a marked decrease
of 1P3-induced 45Ca`+ release. Neither of these observations is likely to explain the ouabain-induced enhancement of the physiological response nor the
increased mobilization of Ca`4 from intracellular
stores in ouabain-treated platelets.
Our finding that the 32p incorporation in PA (Table
1) was significantly lower when platelets were prelabeled in the presence of ouabain when compared
with the control is noteworthy. After 70 minutes
incubation with [32P]orthophosphate, PA is labeled
near the isotopic equilibrium37; therefore, changes in
32p incorporation reflect changes in mass. Philipson
and Nishimoto38 demonstrated a positive correlation
between Na+-Ca2+ exchange activity and the amount
of PA present in sarcolemmal vesicles. Thus, PA
metabolism could be involved in Na4-Ca2+ exchange
activity in the intact cell. Although we have no direct
evidence, the observation that the change in Na+Ca2+ exchange activity, which presumably occurs
during ouabain treatment, coincides with a change in
the amount of PA in the resting platelet tends to
favor this view.
Concluding Remarks
As far as we know, our data demonstrate for the
first time the ouabain-induced accumulation of Ca2+
into intracellular stores and its importance in stimulus-response coupling. In cardiac and smooth muscle
cells, as well as in platelets, the same biochemical
entities regulate Ca2+ homeostasis (ATPase-driven
ion pumps and Na+-Ca2+ exchanger and mitochondrial ion transport mechanisms). In all these cell
types, ouabain amplifies the Ca2+-mediated functional responses. Therefore, the increased contractility provoked by cardiac glycosides in cardiac and
smooth muscle cells is probably based on the same
working mechanism as the one we identified in
platelets. We have to admit that the weakest link in
explaining the effect of ouabain is the role of Na+Ca`+ exchange in elevating cytosolic Ca'4. Conclusive
evidence on the importance of this transport mechanism in intact cells will have to await development
of selective inhibitors.
Elevation of cell Na+, increased Ca2+ storage, and
increased aggregation of platelets were found in
1502
Circulation Research Vol 67, No 6, December 1990
untreated mildly hypertensive patients.39 Furthersimilar abnormal Ca2+ handling and hyperaggregability were reported for platelets from patients
with a thrombotic disorder.24,40 In view of striking
analogies between observations that were made in
these pathological conditions and in our in vitro data
from platelets upon treatment with ouabain, the use
of cardiac glycosides in this type of patient needs
particular care since it might aggravate the already
existing acute risk of thrombosis.
more,
Acknowledgments
The authors thank H. Van Belle for his continuous
interest and advice during the course of this work.
We are indebted to Y. Dillen, C. Geentjens, and A.
Nuyts for the careful preparation of this manuscript
and to L. Vanherle for her linguistic advice.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
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KEY WORDS * ouabain * quin 2 * chlorotetracycline
stimulus-response coupling * human platelets
Ouabain increases the calcium concentration in intracellular stores involved in
stimulus-response coupling in human platelets.
P Roevens and D de Chaffoy de Courcelles
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Circ Res. 1990;67:1494-1502
doi: 10.1161/01.RES.67.6.1494
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