Clinical Science (1996) #), 9-12 (Printed in Great Britain)
9
Low concentrations of ouabain increase cytosolic free
calcium concentration in rat vascular smooth muscle cells
Zhiming ZHU, Martin TEPEL, Marcus NEUSSER and Walter ZIDEK
Medizinische Universitats-Poliklinik, University of Munster. Munster. Germany
(Received 3 October 1994/7 September 1995; accepted 28 September 1995)
1. Low ouabain concentrations in the nanomolar
range significantly increased cytosolic free calcium
concentration.
2. The ouabain-induced cytosolic free calcium concentration increase was due to transplasmamembrane
calcium influx, which could be prevented in the
absence of extracellular calcium or by addition of the
calcium channel blocker nifedipine.
3. The amount of stored cellular Ca2+, as determined by the thapsigargin-induced cytosolic free calcium concentration increase, was also enhanced by
1 nmol/l ouabain.
4. It is concluded that low ouabain concentrations
affect intracellular cytosolic free calcium concentration homoeostasis.
INTRODUCTION
An endogenous inhibitor of Na+-K+-ATPase has
been proposed to contribute to the development of
hypertension in volume- and salt-dependent types of
hypertension [I]. The inhibition of Na+-K+ATPase by ouabain has been shown to increase
cardiac contractility [2], vasoconstriction [3] and
responsiveness of blood vessels to vasoactive agents
131. Long-term administration of ouabain produces
hypertension in rats [4]. The vasoconstriction may
be mediated by several mechanisms including depolarization, changes of membrane fluidity, changes
of Na +-Ca2 +-exchange activity, or by modulatory
effects on sarcoplasmic calcium stores (for review,
see [S]).
Recently, the concentrations of endogenous
ouabain in human plasma were reported to be
0.138+0.043 x 10-9mol/l or 0.44+_0.20x10-9mol/l
[6, 71, although other authors did not detect
measurable levels of endogenous ouabain in human
plasma [8]. The therapeutic concentration of
digoxin is generally accepted to be in the range of
1.28 to 2.56 x
mol/l. Furthermore, the endogenous ouabain plasma concentration in rats was
0.927 x 10-9mol/l [4]. O n the other hand, only a
few studies have examined the effects of nanomolar
ouabain concentrations on vascular smooth muscle
[9]. Since the cytosolic free calcium Concentration
([Ca' +Ii) plays a pivotal role in signal transduction
and force development on vascular smooth muscle,
in the present study the effects of low ouabain
concentrations on [Ca2 +Ii in rat vascular smooth
muscle cells (VSMCs) were investigated. The study
indicates that nanomolar ouabain concentrations
increase both [Ca2+li and the amount of C a 2 +
stored in the sarcoplasmic reticulum.
METHODS
Aortic smooth muscle cells from 6-month-old
male normotensive Wistar-Kyoto rats were cultured
and loaded with fura-2 for measurements of [Ca2+],
and with merocyanine 540 for measurements of
membrane potential as described previously [lo,
1 I]. Cultured cells up to the eighth passage were
used. In accordance with another study [12], within
the first eight passages no significant differences in
cellular Ca2+ handling were observed. For [Ca"],
measurements a spectrofluorophotometer RF-5001
PC (Shimadzu, Tokyo, Japan) equipped with a
thermostatically controlled cuvette holder, and with
intracellular
calcium
measurement
software
(Shimadzu) was used. Calibration of the fluorescence signal in terms of [Ca2+li was performed
with digitonin and EGTA at each cover slip as
described earlier [1 I].
Substances were purchased from Sigma Chemical
Co. (Deisenhofen, Germany) unless indicated otherwise. A physiological salt solution was used, which
contained the following: 135 mmol/l NaCI, 5 mmol/l
KCl, 1 mmol/l CaCI,, 1 mmol/l MgCI, and
10mmol/l Hepes, pH 7.4. Ouabain and digoxin were
dissolved in physiological salt solution with dimethylsulphoxide in a final concentration of 0.01%.
Calcium-free medium contained 5 mmol/l EGTA at
pH 7.4. EGTA was added to the VSMCs only 30s
before administration of the ouabain. In accordance
with earlier reports [I 31, [Ca2+Ii was not significantly changed by this procedure (56 f 5 nmol/l
compared with 62 4 nmol/l under control conditions). To block calcium channels in some experiments, nifedipine was used at a concentration of
0.1 ,umol/l. VSMCs were incubated with nifedipine
IOmin before the [Ca2 +Ii measurements. To avoid
Key words: calcium. ouabain. vascular smooth muscle.
Abbreviations: [Gal-I,. cytosolic free calcium concentration; PMA, phorbol I Z-myristate 13acetate; VSMG. vascular smooth muscle cells.
Cormpondcnte: Professor W. Zidek. Med. Univ.-Poliklinik. Albert-Schweiner-Str. 33, M I 2 9 Munster. Germany.
10
Z. Zhu et al
interference of nifedipine fluorescence with the Ca2
signals, nifedipine was washed out immediately
before administration of the agonists. Thapsigargin,
a selective inhibitor of sarcoplasmic Ca2+-ATPase,
was used at a concentration of 500nmol/l, which
in previous experiments was shown to exert maximal effects on cellular C a 2 + release [14]. The
thapsigargin-induced [Ca’ ‘Ii increase in Ca2+-free
medium was used to estimate the amount of cellular
stored Ca2+ in the presence and absence of
ouabain. In these experiments, VSMCs were preincubated with ouabain 30 min before addition of
thapsigargin. On the other hand, the ouabaininduced Ca2 influx after administration of thapsigargin was tested in the presence of extracellular
Ca2+ ( 1 mmol/l), and ouabain was added 200s after
addition of thapsigargin. Furthermore, VSMCs were
preincubated for 30s with l00nmol/l phorbol 12myristate 13-acetate (PMA), or for 30min in a
medium containing 135 mmol/l KCl, 5 mmol/l NaCl
and the other constituents as indicated above. The
influence of Na+-free medium was tested by
exchanging N a + for choline. Finally, 6 mmol/l
NiCl, was added to block transmembrane C a 2 +
fluxes including the Na+-Ca2 exchanger [15, 161.
In the presence of NiCI,, maximal and minimal
fluorescence intensities were changed (F340/F380
ratio: maximal 1.55 f0.08 and minimal 0.88 f0.02
compared with 2.77 k 0.1 1 and 0.99 f0.01, respectively). These changes due to the affinity of Ni2+ to
fura-2 were considered when calculating the [Ca”Ii
values in the presence of Ni2+.
+
1
f I nmol/l ouabain
m
loo
0
Time (I)
+
Fig. 1. Effect of ourbain on [Cr*+]i in VSMCr. The tracing is
representative of 22 experiments
+
Statistical analysis
Data are presented as means fSEM. Where error
bars do not appear on figures, errors were within
the symbol size. Results were tested for statistical
significance using analysis of variance with the
Bonferroni post-hoc test (computer software: Instat
2.02; Graph Pad, San Diego, CA, U.S.A.). Two-tailed
P values less than 0.05 were considered to be
significant.
“LI,
0
Ctrl
-13
I
I
I
-11
-9
-7
,
-5
Log [ouabain] (mol/l)
Fig. 2. Ourbaireinduccd increase in [Cr*’]i with increasing ourbain
concentrations. Ctrl denotes [Ca”], in control medium. Data are
mean f SEM from 18 to 25 measurements for each ouabain concentration.
Statistical significance: *P (0.05 compared with control conditions.
u)o
I
Ouobain
RESULTS
Ouabain-induced [Ca*+Ii increase in VSMCs
As shown in Fig. 1, the addition of 1 nmol/l
ouabain significantly increased [Ca’ +Ii in VSMCs.
( 1 nmol/l)
increased
[Ca2+Ii by
Ouabain
73 & 14 nmol/l (n = 22). The concentration-response
curve of ouabain-induced [Ca’ +Ii increase in
VSMCs is depicted in Fig. 2.
Comparison of ouabaie and digoxinjnduced [Ca*+Ii
increase
It was then evaluated whether the inhibition of
Na+-K -ATPase by digoxin produces a similar
[ C a Z + l i increase in VSMCs compared with
+
10-9
10-5
10-9
10-5
Concn. (mot/l)
Fig. 3. Increase in [Ca*+]i induced by ourbrin and digoxin in
VSMCr. Data are mean kSEM ( n = 17). Statistical significance: *P<O.OS,
**P<O.OI compared with ouabain.
ouabain. The [Ca2+li increase in VSMCs induced
by addition of ouabain was significantly higher
when compared with the addition of an equimolar
digoxin concentration (Fig. 3).
Ouabain and cytosolic free calcium in vascular smooth muscle cells
II
DISCUSSION
Fig. 4. [Ca*']i in the presence.( ) and absence (0) of I nmol/l
ouabain in control medium (buffer), in the presence of 5Wnmol/l
thapsigargin, and of 135mmol/l K' (high K'), in Na*-free
medium, and in the presence of 6mmol/l NiCII, of Immol/l EGTA
in Ca*'hee medium, of O.Ipnol/l nifedipine, and of 100nmol/l
PMA. Data are means f SEM from 6 t o 22 experiments. Statistical rignifk
cance: *P <O.OS. **P < 0.01 compared with the ouabain-induced changes in
buffer alone.
Mechanism of ouabain-induced [Ca*+],increase
In the absence of external calcium the [Ca2+Ii
increase in VSMCs induced by 1 nmol/l ouabain
was significantly lower compared with control conditions [24 f5 nmol/l ( n = 16) compared with
73 f.I4 nmol/l ( n = 22); P < 0.01, Fig. 41. This result
indicates that the ouabain-induced [Ca2 +Iiincrease
is mainly due to transplasmamembrane calcium
influx.
After preincubation with 0.1 pmol/l nifedipine to
block calcium channels, the [Ca2+Ii increase in
VSMCs induced by 1 nmol/l ouabain was significantly reduced to 27+4nmol/l (n=7; P <0.05,Fig.
4). These findings indicate that the ouabain-induced
[Ca2+Ii increase may be due to transplasmamembrane calcium influx through calcium channels.
The ouabain-induced [Ca2+Ii response could be
abrogated by increasing extracellular K concentrations, by preincubation with thapsigargin, and by
removing extracellular Na+ (Fig. 4). Also, by adding
extracellular N i 2 + the [Ca2+Iiresponse to ouabain
was significantly decreased [ - 1 5 nmol/l ( n = 6);
P <0.05, Fig. 41. Furthermore, PMA significantly
increased the [Ca"], response to ouabain (Fig. 4).
Using the potential-sensitive dye merocyanine 540,
no significant changes in membrane potential were
observed after administration of ouabain in low
concentrations (3.1 f.0.2 compared with 3.2f.0.3
arbitrary fluorescence units, n = 5).
To evaluate whether ouabain also increases the
calcium content in the intracellular sarcoplasmic
calcium stores, the rise in [Ca2+Ii after specific
inhibition of sarcoplasmic Ca2'-ATPase by
500 nmol/l thapsigargin was measured. After administration of 1 nmol/l ouabain the thapsigargininduced [Ca2+Iiincrease in the absence of external
Ca2 + was significantly enhanced from 53 f.5 nmol/l
to 91 f7 nmol/l ( n = 7, P <0.05).
+
+
Several investigators reported that low ouabain
concentrations affect VSMCs. Weiss et al. [9]
showed that a reversible increase of caffeine-evoked
contractions of rat small mesenteric arteries could
be induced with ouabain concentrations as low as
10-'omol/l [9]. Woolfson et al. [3] showed that
1 x 10-6mol/l ouabain significantly increased the
tension of human resistance arteries. Further, the
addition of 1 1 x l o v 9mol/l ouabain significantly
potentiated the tone of human resistance arteries
submaximally precontracted with noradrenaline [3].
The addition of ouabain in concentrations ranging
from 5 x lo-* to 5 x 10-6mol/l caused a doserelated contraction of cerebral arteries isolated from
dogs [17]. On the other hand, Moromizato et al.
[I81 did not find any effect of ouabain on the
contractile response to noradrenaline of isolated rat
aorta. Therefore, up to now no direct contractile
effect of those low ouabain concentrations has been
demonstrated, and it remains open whether the
[Ca2+Ii increase of about l00nmol/l induced by
1 nmol/l ouabain is relevant for vascular contraction
in vivo.
In contrast to earlier views assuming the relative
insensitivity of rat tissue to cardiac glycosides [19],
Blanco et al. [20] and Blaustein [21] established
that some rat tissues are very sensitive to ouabain,
probably due to the different distribution of isoforms of the Na+-K +-ATPase in several tissues.
Apparently, nanomolar concentrations of ouabain
increase [Ca2 +Ii by stimulating Ca2+ influx, since
extracellular Ca2
deprivation abrogates the
[Ca2+li response to ouabain. Since EGTA at a
concentration of 5 mmol/l may also bind other
divalent ions such as Mg2+ and may also affect
membrane structure, experiments with the Ca2+
channel blocker nifedipine were additionally performed, and yielded essentially the same findings.
Similar results have been reported earlier for millimolar ouabain concentrations [l 1, 223. The
enhanced ouabain effect after short-term incubation
with PMA is in accordance with similar PMA
interactions with other agonists such as transforming growth factor 1 and is most probably due to
activation of Ca2+ channels [23].
However, in contrast to the observations with
higher ouabain concentrations [l I], no significant
changes in membrane potential were found using a
potential-sensitive fluorescent dye. This finding suggests that a depolarization due to inhibition of the
Na+-K+-ATPase and hence a decrease in intracellular K + concentration or a decrease in electrogenic Na+ outward transport may not be the
crucial mechanism initiating the transmembrane
Ca2+ influx. Furthermore, compared with the
ouabain effect observed with higher concentrations,
other mechanisms may be prevalent with nanomolar
concentrations. Apparently, the Ca2 influx induced
by low concentrations of ouabain can be inhibited
+
+
Z.Zhu et al.
12
by several manoeuvres increasing [Ca2 + I i , such as
depolarization by a high extracellular K concentration, release of cellular Ca" stores by thapsigargin or extracellular N a + deprivation inhibiting
Na+-Ca'+ exchange. Although the [Ca2+], response to low concentrations of ouabain is inhibited
by nifedipine. no significant changes of membrane
potential were found. Since nifedipine may not be a
specific inhibitor of potential-dependent Ca2 channels, these findings are not necessarily contradictory.
Interestingly, the effects of ouabain and digoxin
on C a Z + influx arc different. Therefore, a different
effect on Ca" channels may be discussed. On the
other hand, a different affinity of Na+-K +-ATPase
to both glycosides cannot be excluded. The truncated X I isoform of Na+-K+-ATPase, which is
predominantly expressed in V S M C s [24], has not
been extensively studied with respect to different
glycoside affinities. However, myocardial tissue,
which expresses both the XIand x 2 subunit isoform,
shows similar affinities to ouabain and digoxin [ 2 5 ] .
Ouabain is known to be more hydrophilic than
digoxin, and the different hydrophobicity of the
molecules could account for a different binding to
Ca' + channels.
In summary. the present study shows that nanomolar concentrations of ouabain not only modulate
the effects of vasoactive agonists, but can also
directly increase Ca'
influx in VSMCs. This
property is not equally shared by other cardiac
glycosides. but may be more pronounced with the
hydrophilic glycoside, ouabain. The mechanisms
underlying the Ca'+ influx are different from those
operating with higher concentrations of ouabain.
+
+
+
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