486
Na+-Ca2+ Exchange in Cultured Vascular
Smooth Muscle Cells
Elizabeth G. Nabel, Bradford C. Berk, Tommy A. Brock, and Thomas W. Smith
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Vascular smooth muscle cells (VSMC) contract as intracellular free calcium ([Ca2+],) rises. While
Na + -Ca 2+ exchange has been proposed to contribute to transmembrane Ca2+ flux, its role in cultured
VSMC is unknown. Accordingly, we have investigated the role of Na + -Ca 2+ exchange in unidirectional
and net transmembrane Ca 2+ fluxes in cultured rat aortic VSMC under basal conditions and following
agonist-mediated stimulation. Transmembrane Ca 2+ uptake was significantly increased in response
to a low external Na + concentration ([Na + ]J compared with 140 mM [Na+],,. Na + -dependent Ca2+
uptake in response to low [Na + ] o was further increased by intracellular Na + loading by preincubation
of the VSMC with 1 mM ouabain. Under steady-state conditions, Ca 2+ content varied inversely with
[Na + ] o , increasing from 1.0 nmol Ca 2+ /mg protein at 140 mM [Na + ], to 4.0 nmol Ca 2+ /mg protein at
20 mM [Na + )0. Increasing [K + ], to 55 mM also enhanced Na+-dependent Ca2+ influx. Augmentation
of Ca2 + uptake with K + depolarization was not significantly inhibited by the calcium channel antagonist
verapamil. Transmembrane Ca2+ efflux was increased in response to 130 mM [Na + ) o compared with
zero [Na + ] o (iso-osmotic substitution with choline + ), and was further stimulated by the vasoconstrictor
angiotensin II, which is known to elevate [Ca 2+ ],. These changes in [Ca2*], were studied directly using
fura-2 fluorescence measurements. Elevated [Ca 2+ ], levels returned to baseline more rapidly in the
presence of normal (130 mM) [Na + ] 0 compared with zero [Na + ] o (iso-osmotic substitution with
choline + ). These findings suggest that a bidirectional Na*-Ca2+ exchange mechanism is present in
cultured rat aortic VSMC. Na + -Ca 2+ exchange appears to play a part in Ca2+ homeostasis, particularly
under conditions of altered intracellular Na + or increased [Ca2+], following agonist stimulation.
(Circulation Research 1988;62:486-493)
V
ascular smooth muscle cells (VSMC) contract
when intracellular free calcium concentration
([Ca2+],) is increased. The increase in [Ca 2+ ]|
may result from an influx of Ca2+ from the extracellular
compartment across the cell membrane and/or from a
release of Ca2+ from intracellular stores, presumably
the sarcoplasmic reticulum. The transmembrane influx
of Ca2+ has been proposed to occur through Ca2+
channels, the permeability of which is dependent on
membrane potential changes or hormonal stimulation
of specific receptors.1 It is also possible that Na + -Ca 2+
exchange could contribute to Ca2+ influx during depolarization, as occurs in cardiac muscle.2 Relaxation
of VSMC results from a decrease in [Ca2+]j, which may
occur by efflux across the cell membrane and/or by
sequestration of Ca2+ into intracellular organelles. In
a number of cell types, transmembrane Ca2+ efflux has
been proposed to occur via an ATP-dependent Ca2+
pump and a Na + -dependent Ca2+ exchange carrier.3-4
The existence of a Na + -Ca 2+ exchange mechanism
in VSMC is controversial. Using isolated sarcolemmal
vesicle preparations, Na + -Ca 2+ exchange has been
reported in rat myometrial and mesenteric arterial cells
From the Cardiovascular Division, Department of Medicine and
the Vascular Research Division, Department of Pathology,
(T.A.B.), Brigham and Women's Hospital and Harvard Medical
School, Boston, Masschusetts.
Supported by National Institutes of Health grants HL 36141 and
HL 18002. Dr. Berk is a Clinical Investigator of the National Heart,
Lung, and Blood Institute (HL 01831).
Address for reprints: Thomas W. Smith, MD, Cardiovascular
Division, Brigham and Wbmen's Hospital, 75 Francis Street,
Boston, MA 02115.
Received January 14, 1987; accepted October 1, 1987.
as a mechanism for Ca2+ influx and efflux.56 Blaustein
has suggested that a Na + -Ca 2+ exchange process exists
and may play a critical role in the regulation of [Ca 2+ ]|
and in the maintenance of resting vascular smooth
muscle tone.7-8 Others have argued that Na + -Ca 2+
exchange is a nonspecific process that does not play a
significant role in vascular smooth muscle contractility.9-10 In the present study, we have examined the
role of Na + -Ca 2+ exchange in the regulation of Ca2+
homeostasis in cultured rat aortic VSMC. In addition,
we have investigated Na + -dependent Ca2+ fluxes following intracellular Na + loading and angiotensin II
stimulation to investigate the involvement of Na + -Ca 2+
exchange during conditions of increased transmembrane Ca2+ flux. Finally, we have explored the dependence of steady-state VSMC intracellular Ca2+ content
the transsarcolemmal Na + gradient.
Materials and Methods
Cell Culture
Primary cultures of VSMC were obtained by enzymatic dissociation of aortic tissue from SpragueDawley male rats (200-300 g) as previously described."
Stock cultures (75-cm2 flasks) were passaged by
washing once with 2 ml Ca 2+ - and Mg2+-free Dulbecco's phosphate-buffered saline (Pj/NaCl) and incubating for 5 minutes at 37° C with 1 ml 0.05% trypsin
in P,/NaCl containing 0.02% Na2EDTA. The cultures
were passaged twice weekly and used for experiments
between the 4th and 15th passages. The stock cultures
were grown in Dulbecco's medium (GIBCO Laboratories) containing 10% calf serum (GIBCO), 100 U
Nabel el al
penicillin G/ml, and 100 (xg streptomycin/ml. Culture
dishes (100 mm, Falcon) containing 25-mm circular
glass coverslips were innoculated at a density of 1 x 105
cells/ml. The cells were grown at 37° C in a humidified
atmosphere of 5% CO2-95% air. Confluent monolayers
developed by 3 days of incubation.
45
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Ca2* Uptake and Content Measurements
For determination of Ca2+ uptake by cultured
VSMC, 25-mm circular glass coverslips with attached
monolayers of VSMC were obtained from each culture.
Twenty-four hours prior to the uptake experiment, cells
were exposed to L-[4,5-3H,N]leucine (0.2 ^Ci/ml).
[3H]Leucine was incorporated into cell protein, and
subsequent determination of 3 H + counts permitted
normalization of 45Ca2+ content relative to milligrams
of cell protein for each coverslip. Glass coverslips
(n = 1) were placed into small Lucite baskets and then
immersed in a 140-mM Na + solution (140 mM NaCl,
4 mM KC1, 0.9 mM CaCl2, 0.5 mM MgCl2, and 5 mM
HEPES, pH 7.4) for 5 minutes, followed by immersion
in a preincubation medium at 37° C for 10 minutes.
45
Ca2+ uptake was performed in Na+-free solution (140
mM choline Cl, 4 mM KC1, 0.9 mM CaCl2, 0.5 mM
MgCl2, and 5 mM HEPES, pH 7.4) or in a 140-mM Na+
solution in an incubation bath at 37° C for a designated
period of time (2 seconds to 1 hour). Additional studies
were performed by exposing cells to varying external
Na + concentrations (Na+-free solution, 10-mM Na+
solution, 20-mM Na+ solution, 50-mM Na+ solution,
100-mM Na+ solution, 140-mM Na + solution, isoosmotic substitution with choline + ) for 4 hours. After
the desired uptake period, the experiment was terminated by washing the coverslips twice for 7 seconds
each in two 50-m| volumes of Ca2+-free HEPESbuffered solution at 4° C. The VSMC monolayer was
removed from the coverslip, and the cells were placed
in 1.6 ml of a solution containing 1% sodium dodecyl
sulfate (SDS) and 10 mM sodium borate. An aliquot
was placed in 12 ml liquid scintillation fluid (New
England Nuclear, Boston, Massachusetts). Cells from
five coverslips were dissolved in 1.8 ml of SDS-sodium
borate solution, and 0.2 ml was used for determination
of protein content.12
Na+-CaJ+ Exchange in Vascular Smooth Muscle
487
coverslip. Twenty-four hours prior to study, cells were
incubated in 45Ca2+ (2 (iCi/ml). On the day of study,
coverslips with attached monolayers of VSMC (n = 7)
were placed into small Lucite baskets and washed three
times in a balanced salt solution (130 mM NaCl, 5 mM
KC1, 1.5 mM CaCl2, 1 mM MgCl2, and 20 mM
HEPES-Tris, pH 7.4) (TBSS) at 37° C. Additional cells
were washed in Na+-free TBSS in which choline
chloride isotonicalh/ replaced sodium chloride (130
mM choline Cl, 5 mM KC1, 1.5 mM CaCl2, 1 mM
MgCl2, and 20 mM HEPES-Tris, pH 7.4). Cells were
then exposed to angiotensin II (100 nM) for designated
time periods (1-5 minutes) in the presence or absence
of Na + . The experiment was terminated by washing the
cells four times with ice-cold, Ca2+-free TBSS containing 10 mM LaCl3, followed by a 5-minute incubation with the same buffer. The VSMC monolayer was
removed from the coverslip, and the cells were placed
in 1.6 ml of a 1% SDS and 10 mM sodium borate
solution. An aliquot was placed in 12 ml liquid
scintillation fluid. Measurement of 45Ca2+ and 3 H +
counts and determination of 45Ca2+ content were
performed as described above.
4S
[Ca2+], Measurements
[Ca 2+ ], was measured using the Ca 2+ -sensitive fluorescent dye fura-2. For these experiments, VSMC
were grown in 100-mm dishes. Four to eight dishes
(approximately 3.0-6.0 x 107 cells) were exposed for
5 minutes to modified Hanks' balanced salt solution
(GIBCO) containing cojlagenase (0.1 mg/ml), soybean
trypsin inhibitor (0.1 mg/ml), and bovine serum
albumin (0.3 mg/ml) (BSA) to detach the cells. Cells
were resuspended in Hanks' solution and an aliquot
was removed for determination of cellular autofluorescence. The eel 1 suspension was incubated with 2 ^.M
fura-2/AM for 20 minutes at 37° C. The cells were then
washed in TBSS containing 1 mg/ml BSA and stored
in 1-ml aliquots prior to use. Fluorescence measurements were carried out in a SPEX fluorolog-2 instrument equipped with magnetic stirrer and temperature
control. Fura-2 fluorescence was measured at 340 and
380 nm (excitation) and 505 nm (emission) with slit
band widths of 3.3 and 4.5 nm, respectively. The
fluorescence intensity ratio (340:380) was obtained
after subtracting the background fluorescence observed
in the absence of fura-2 in the cells. The fluorescence
intensity ratio was calibrated for each experiment using
30 M-M digitonin to permit equilibration of intracellular
and extracellular Ca2+ (maximum fluorescence), followed by addition of 12 mM EGTA, final pH >8.8
(minimum fluorescence) to give [Ca 2+ ] ; using equations
as previously described.13
Ca2+ efflux was determined using monolayers of
VSMC attached to 25-mm circular glass coverslips.
Thirty-six hours prior to the efflux study, cells were
exposed to L-[4,5-3H,N]leucine (0.2 \xCjjm\, 28 mg/1),
which was incorporated into cell protein. Determination of 3H+ counts allowed for normalization of 45Ca2+
content relative to milligrams of cell protein for each
[Na +], Content Measurements
For determination of [Na + ], content, VSMC were
grown to confluence in 35-mm dishes. Eight dishes
were exposed for 30 minutes to a 140-mM Na + solution
with 1 mM ouabain (four dishes) or without ouabain
(four dishes). In additional experiments, 10 dishes
were exposed for 4 hours to Na+-free solution, 10-mM
Simultaneous counting of 45Ca2+ and 3 H + counts was
performed using a Packard liquid scintillation spectrometer. Protein content for each coverslip was determined from the 3 H + cpm/mg protein ratio. Calcium
content was determined from the 45Ca2+ cpm from each
coverslip and from the known Ca2+ concentration of the
uptake medium. For normalization of each coverslip,
the data were calculated as nmol Ca 2+ /mg protein.
Ca2+ Efflux
488
Circulation Research
Vol 62, No 3, March 1988
Na + solution, 20-mM Na + solution, 50-mM Na 4
solution, 100-mM Na + solution, or 140-mM Na +
solution (iso-osmotic substitution with choline + ) (two
dishes per solution). Cells were quickly washed five
times with ice-cold 100 mM MgCl2 with 10 mM
HEPES (adjusted to pH 7.4 with Tris base) and air-dried
under sterile conditions. The cells were treated with 2
ml 0.02% Acationox (American Scientific Products,
McGraw Park, Illinois). Total [Na + ] in each 2-ml
aliquot was determined by atomic absorption spectrophotometry. Na + concentration was corrected for cell
volume and protein content to derive a final [Na + ] ;
value.
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Cell Volume
The equilibrium distribution of 3-0-[methyl- 14 C]D-glucose was used to measure cellular water space.1415
The cultures were incubated with 3-O-[methyl-14C]-Dglucose (1 (xCi/m)) and unlabeled 3-O-[methyl]-Dglucose (1 mM) in a 140-mM Na + solution with or
without 1 mM ouabain for 30 minutes at 37° C. The
cultures were then quickly washed five times with
ice-cold 0.1 M MgCl2 with 10 mM HEPES (adjusted
to pH 7.4 with Tris base). Cellular radioactivity was
counted in 10 ml scintillation fluid (New England
Nuclear) using a liquid scintillation spectrometer.
Volumes between 3 and 5 n,l of intracellular space
per milligram protein were obtained under these
conditions.
Statistical Analysis
Data are expressed as the mean±SEM. Tests of
significance were performed using Student's t test. A
p value of less than 0.05 was considered significant. A
kinetic analysis of 45Ca2+ efflux was performed using a
Na*-0
.E 3.0
e
a.
1
2.0
1.0
o
60
30
Time (sec)
FIGURE 1. Effect of extracellular Na* concentration on Ca2*
uptake. Monolayers of cultured rat aortic vascular smooth
muscle cells were equilibrated in HEPES-buffered solution and
then exposed to Na*-free solution or 140-mM Na*a solution
containing "Ca2*. "Ca2* content was assayed after the designated periods of uptake. Each point represents the mean ± SEM
of seven determinations. The two curves are significantly
different from each other (p < 0.05).
10
20
Na=0
I'
2
10
o
c
No ' 140 mM
c
o
o
o
o
I 5 10
30
60
Time (min)
FIGURE 2. Effect of extracellular Na* concentration on Ca2*
uptake. Cells were exposed to Na * -free solution or 140-mM Na*
solution containing "Ca2* for the intervals on the x axis. Ca2*
content was determined as described in "Materials and Methods. "
nonweighted, nonlinear least-squares fit program
(RSI).
Results
+
45
2+
Na -Dependent Ca Uptake
To study the influence of extracellular Na+ concentration ([Na+]0) on 45Ca2+ uptake, monolayers of
confluent cultured VSMC were abruptly exposed to
Na+-free solution or to a 140-mM Na + solution.
Figures 1 and 2 demonstrate 45Ca2+ uptake in the
presence of Na+-free solution and 140-mM Na +
solution. Intracellular 45Ca2+ uptake was significantly
augmented in the presence of zero [Na + ] 0 (/?<0.05)
between 2 and 60 seconds (Figure 1) and between 1 and
60 minutes (Figure 2).
Effect of Intracellular Na* Loading on 45Ca2+ Uptake
Ouabain is a highly specific Na,K-ATPase inhibitor
that causes an increase in [Na + ],. 16 This rise in [Na + ],
increases the outward transmembrane Na+ gradient
present during exposure to Na+-free solution. To
examine the effect of an increased [Na + ], on 45Ca2+
uptake, VSMC were preincubated in 1 mM ouabain for
30 minutes and then abruptly exposed to Na+-free
solution containing 45Ca2+. [Na + ]; increased by 63%
from 23.9 to 37.9 mM when VSMC were preincubated
with 1 mM ouabain. Figure 3 shows that VSMC
preincubated with ouabain demonstrated a significantly
greater 45Ca2+ uptake compared with control cells
(p<0.05). These results demonstrate that 45Ca2+ uptake is enhanced by intracellular Na+ loading. A
Na+-dependent Ca2+ uptake mechanism is suggested
by 1) increased transmembrane 45Ca2+ uptake in response to a low external Na + concentration and 2)
enhanced intracellular 45Ca2+ uptake following intracellular Na + loading, both of which induce a favorable
[Na + ] gradient to facilitate Ca2+ entry via Na + -Ca 2+
exchange.
Effect of Varying [Na+]o on 4SCa2+ Uptake
To examine the acute effect of different external Na +
Nabel et al
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3.0
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15
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Time (sec)
45
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FIGURE 3. Effect of Na* loading on Co1* uptake. Cells were
preincubated in control medium (HEPES-buffered solution) or
medium containing 1 mM ouabain for 10 minutes and then
exposed to Na*-free solution containing "Co1*. Each point is the
mean ± SEM of seven determinations. The two curves are
significantly different from each other (p<0.05) using Student's t test.
concentrations on 45Ca2+ uptake, monolayers of cultured VSMC were abruptly exposed to Na+-free solution, 140-mM Na + solution, or solutions with varying
intermediate Na + concentrations (10-100 mM) (isoosmotic substitution with choline + ). There was no
acute effect of varying [Na + ] 0 on [ C a 2 ^ measured by
45
Ca2+ uptake or fura-2 techniques up to 15 minutes at
intermediate [Na + ] o . Since little change in [Ca2+]; was
observed over short time periods under basal conditions, a detailed analysis of the dependence of 45Ca2+
fluxes on [Na], and [Na]0 could not be performed.
Therefore, we next studied 45Ca2+ content in response
to graded [Na + ] o values under steady-state conditions
in order to investigate a potential longer term modulatory role of Na + -Ca 2+ exchange in Ca2+ homeostasis.
Monolayers of confluent cultured VSMC were exposed
to graded [Na + ] 0 values for 4 hours. Pilot studies
demonstrated that a steady state had been reached by
4 hours. Figure 4A demonstrates that steady-state
45
Ca2+ content was dependent on [Na + ] 0 , with 45Ca2+
content being greatest following incubation in low
[Na + ] 0 . Additional studies were performed to determine 45Ca2+ uptake after a 2-hour exposure to Na+-free
Effect of K+ Depolarization and Verapamil
To investigate the role of K+ depolarization on Ca2+
uptake in cultured VSMC and Ca2+ entry via voltagedependent Ca2+ channels, 45Ca2+ uptake was determined after exposure to 55 mM K + o in the presence and
absence of verapamil. Figure 5 demonstrates the
time-dependent 45Ca2+ uptake upon exposure to 55 mM
K + o or 3 mM K + o . K+ depolarization induced a
significant increase in the rate of initial 45Ca2+ uptake
over 60 seconds (/?<0.05) and in the total accumulation of 45Ca2+ over 5 minutes (p <0.05). These findings
indicate that net Ca2+ influx in the cultured rat aortic
VSMC is augmented by depolarization.
Verapamil is a Ca2+ channel antagonist that produces
a concentration-dependent inhibition of slow channel
Ca2+ conductance. To study the effect of slow Ca2+
channel blockade on Ca2+ uptake, confluent monolayers of cells were preincubated with 10~6 M verapamil.
45
Ca2+ uptake was then measured in 55 mM K+o or 4
mM K+o. Figure 5 shows no significant difference in
45
Ca2+ uptake in the presence of verapamil compared
with control uptake in response to K + depolarization.
The small decrease in Ca2+ uptake from both 4-mM K +
and 55-mM K+ media observed with verapamil is
likely a nonspecific effect. These results indicate that
these cultured rat aortic VSMC lack functional Ca2+
channels, or that the channels in these cells are
insensitive to verapamil. Thus, Ca2+ uptake following
depolarization in this line of VSMC is not attributable
to L-type voltage-dependent Ca2+ channels.17 An al-
40.0
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50
100
[Ua*]0 (mM)
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489
solution or 140-mM Na + solution. Mean 4iCa2+ content
in Na+-free solution was 3.62 nmol/mg protein and in
140-mM Na+ solution was 2.64 nmol/mg protein.
These results are similar to 45Ca2+ content at 4 hoursj
confirming that steady-state conditions were present at
the 4-hour measurements.
To determine changes in [Na+]j at steady-state
conditions with varying [Na + ] 0 , [Na + ] i content was
measured at 4 hours for each of the six [Na + ] o values
used in the studies described above. Figure 4B demonstrates that at low [Na + ] 0 (0-20 mM), [Na + ], is also
low (12 mM); however, as [Na + ] 0 increases to 140 mM,
[Na + ], also rises in a sigmoidal manner to about 30 mM.
T
6
(nmol/nrvg prot(
ro
~
Na+-CaI+ Exchange in Vascular Smooth Muscle
100
[No*] o (mM)
FIGURE 4. Effect of varying [Na *]e on "Ca2*
uptake and content. A: Monolayers of cultured
rat aortic vascular smooth muscle cells were
exposed to varying [Na */„ (Na *-free solution,
10-mM Na* solution, 20-mM Na* solution,
50-mM Na* solution, 100-mM Na* solution,
140-mM Na * solution, iso-osmotic substitution
with choline*) for 4 hours, and "Ca2* content
was assayed. Each point represents the
mean ± SEM of seven determinations. vCa2*
content was greatest following incubation at
low [Na*Jr B: [No*], content measured in the
vascular smooth muscle cell monolayers at 4
hours for each of the six [Na*]o values described above. With increases in [Na*]o,
[Na*], also rises.
490
Circulation Research
Vol 62, No 3, March 1988
55mMK*
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Time (mini
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FIGURE 5. Effect of K* depolarization and verapamil on
"Ca2*uptake. Monolayers of vascular smooth muscle cells were
equilibrated in HEPES-buffered solution for 5 minutes and then
abruptly exposed to vCa2 * uptake medium containing 55 mM K*
or 4 mM K+. Additional cells were preincubated in 10'i M
verapamil for 10 minutes and also abruptly exposed to 55 mM
K* or 4 mM K*. "Ca2* content was assayed after the designated
periods of uptake. Each point is the mean ± SEM of seven
determinations. The 55-mM K* and 4-mMK* uptake curves are
significantly different (p<0.05), as are the 55-mM K* plus
verapamil and 4-mM K* plus verapamil curves.
II exposure as a percent of the peak [ C a 2 ^ was 57 ± 4%
in Na+-free TBSS and 28 ± 3 % in Na + -TBSS (n = 3),
indicating that an inwardly directed [Na + ] gradient
augments the rate of the return to basal [Ca 2+ ],,
presumably via Na + -Ca 2+ exchange.
A kinetic analysis of angiotensin II-stimulated Ca2+
efflux based on Figure 6 demonstrated that the data are
best fit by two exponential curves representing a rapid
and slow component of efflux ( F = 9 1 . 8 2 , p < 0 . 0 0 1 ) .
The rapid component of Ca2+ efflux has a rate constant
of 3.03/min (mean, n = 24) in the presence of 130 mM
Na + 0 and a rate constant of 1.33/min (mean, n = 24) in
the presence of zero Na + 0 (Table 1). The slower
component of Ca2+ efflux also demonstrated different
rate constants for efflux in the presence of 130 mM Na + 0
or zero Na+O. The efflux rate constants were 0.061/min
in 130 mM Na + 0 and 0.0014/min in zero Na + 0 . Under
basal conditions in the absence of angiotensin II, there
were no measurable differences between the rapid and
slow phases of Ca2+ efflux in the presence or absence
of Na+O. This suggests that the K,, of the Na + -Ca 2+
ternative pathway for depolarization-induced Ca2+
uptake may be Na + -Ca 2+ exchange, an exchange
mechanism in which Ca2+ entry would be expected to
be augmented by K + depolarization if the stoichiometry
of the process is such that three Na + ions exchange for
one Ca2+ ion.18
Na+-Dependent Ca2+ Efflux
To examine Na + -dependent Ca2+ efflux, we studied
angiotensin II-stimulated 45Ca2+ efflux in the presence
or absence of [Na + ] 0 . The cells were preincubated with
100 nM angiotensin II in Na + -free TBSS or Na + -TBSS.
Figure 6 demonstrates that 45Ca2+ efflux is significantly
greater (/?<0.05) following angiotensin II stimulation
compared with basal conditions. In addition, angiotensin II-stimulated Ca2+ efflux is significantly inCTeased (p<0.05) in the presence of Na+O compared
with its absence.
To analyze further the effect of extracellular Na + on
Ca2+ homeostasis, changes in [Ca 2+ ], were directly
studied using fura-2 fluorescence measurements. Since
bidirectional Na + -Ca 2+ exchange should be activated
by increasing [Ca 2+ ], (as well as by altering intracellular
Na + ), we used angiotensin II (100 nM), which is known
to elevate [Ca2+], to greater than 1,000 nM," to study
Na + -dependent changes in [Ca 2+ ],. When VSMC were
exposed to 100 nM angiotensin II in the absence of
extracellular Na + , the rate and magnitude of decline of
[Ca2+]i after the initial rise was markedly diminished
(Figure 7A) compared with cells stimulated in the
presence of physiological [Na + ] 0 (Figure 7B). The
amount of Ca2+ remaining 30 seconds after angiotensin
Time (min)
FIGURE 6. Effect of extracellular Na* concentration on basal
and angiotensin II-stimulated Ca1* efflux. Monolayers of
cultured vascular smooth muscle cells were loaded with "Cal*
for 24 hours. The cells were then washed in Na*-free TBSS or
130 mMNa*-TBSS. Cells were exposed to Na*-free TBSSorUO
mM Na*-TBSS for the designated efflux period. "Ca2* content
was then assayed. ''Ca1* content at control was 1.88±0.08
nmollmgprotein (mean ± SEM, n = 7). Results are expressed as
percent Ca2* remaining after the efflux period. Each point is the
mean ±SEM of seven determinations. Additional cells were
loaded with ''Ca2* for 24 hours and then exposed to 100 nM
angiotensin II (All) in Na*-free TBSS or 130 mM Na*-TBSS.
Angiotensin II significantly increased the KCa2* efflux, both in
Na * -free TBSS and 130mMNa*- TBSS (p < 0.05). In addition,
in the presence of angiotensin II, exposure to 130 mMNa*-TBSS
significantly increased Ca2* efflux compared with Na*-free TBSS
(p<0.05).
Nabel et al
630
o
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*\i
Na + =
14
0
300
Time (sec)
608 r
Na+ = !30mM
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300
B
Time (sec)
FIGURE 7. Effect of extracellular Na * concentration on angiotensin II'-stimulated changes in [Ca2*],. Cells were prepared
forfura-2 fluorescence measurements of Ca1* as described in
"Materials and Methods. "Approximately 2x10* cells/ml were
exposed to 100 nM angiotensin II (arrow) in Na * -free TBSS (A)
or in 130 mM Na *-TBSS (B). Measurement of [Ca2*!, remaining was determined at 30 seconds (vertical bar) following
angiotensin II stimulation. The tracings are representative of
three such experiments.
exchange carrier is considerably greater than resting [Ca2+]j, which is approximately 90 nM in these
cells.19 The major contribution of angiotensin II-stimulated Na + -dependent Ca2+ efflux occurs during
the initial minute of Ca2+ efflux, consistent with
the fura-2 data suggesting an increased rate of
return to basal [Ca 2+ ], with an inwardly directed [Na + ]
gradient.
Na+-CaI+ Exchange in Vascular Smooth Muscle
Discussion
There has been considerable controversy as to the
existence and the physiological role of a Na + -Ca 2+
exchange mechanism in VSMC. A Na + -dependent
Ca2+ uptake process has been described in isolated
membrane vesicles from rat mesenteric arteries6 and rat
aorta.20 Similarly, using rat myometrial plasma membrane vesicles, Grover et al5 described a Na + -Ca 2+
exchange mechanism in which high intravesicular Na +
promoted Ca2+ uptake by the vesicles, while a high
extravesicular Na + promoted Ca2+ release.5 Na + -Ca 2+
exchange-mediated Ca2+ influx has been well documented in other preparations, including cultured chick
cardiac cells,16 cardiac sarcolemmal vesicles,21 and
squid axon.22 Our results in cultured rat aortic VSMC
support the existence of a bidirectional Na + -Ca 2+
exchange mechanism. Furthermore, our evidence suggests potential roles for Na + -Ca 2+ exchange in both
acute and chronic physiological VSMC functions. We
also present data indicating hormone-mediated activation of Na + -Ca 2+ exchange by changes in both [Ca2*],
and [Na+]j at physiological levels.
Ca2+ efflux in cardiac muscle has been proposed to
be mediated by two principal mechanisms: a highaffinity, low-capacity ATP-driven sarcolemmal pump
(Ca 2+ -ATPase), and a low-affinity, high-capacity carrier, Na + -Ca 2+ exchange.123 The findings presented
here suggest that similar mechanisms exist in VSMC.
In VSMC, Na + -Ca 2+ exchange may be more important
in the regulation of [Ca2*]! under stimulated conditions
compared with the basal state. At high [Ca 2+ ], (1 JJLM)
(e.g., following angiotensin II stimulation), Na + -Ca 2+
exchange was activated and contributed to Ca2+ efflux.
Since mitochondria appear to sequester, at most, only
small amounts of Ca2+ under normal conditions,24"26
Na + -Ca 2+ exchange may well be a major pathway for
extrusion of Ca2+ at high [Ca2+]i5 as seen following a
vasoconstrictor stimulus. Since the sarcoplasmic reticulum (SR) is a major storage site of intracellular
Ca 2+ , SR storage likely contributes to regulation of
[Ca 2+ ]| homeostasis under both agonist-stimulated and
basal conditions.27-28 As mentioned above, basal Ca2+
efflux did not depend acutely on [Na + ] 0 , yet following
agonist-mediated increases in [Ca 2+ ] i; the slow component of Ca2+ efflux was markedly dependent on
[Na + ] 0 . Thus, Na + -Ca 2+ exchange appears to be
TABLE 1. Kinetics of Angiotensin H-Stlmulated **Ca*+ Efflux From Cultured Vascular Smooth Muscle Cells
nmol/mg protein
130 mM [Na + ] 0
zero [Na
b,
1.15±0.10
0.96±0.16
bz
0.87 ± 0 . 14
0.89±0. 16
491
min~'
3.03
1.33
K2
0.061
0.0014
F
91.82
44.33
P
/?<0.001
p<0.001
Data points from the experiment illustrated in Figure 5 were fitted to the following equation using a nonweighted,
nonlinear least-squares fit program (RSI):
r(t)=
where r(t) equals the total radioactivity present in the cells at a given time; x equals the number of exponential terms; b
is the amount of 4 3 Ca 2 + present in a given compartment; and k is the rate constant for 4 5 Ca 2 + efflux from each
compartment.
492
Circulation Research
Vol 62, No 3, March 1988
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involved in Ca2+ efflux at high levels of [Ca 2+ ],, while
other mechanisms such as SR uptake and the sarcolemmal Ca 2+ -ATPase are more important at normal
resting [ C a 2 ^ levels.
Na + -Ca 2+ exchange may also be activated by an
agonist-mediated rise in [Na + ] 1 . We have preliminary
evidence that numerous vasoconstrictor agonists, including angiotensin II and platelet-derived growth
factor, stimulate an amiloride-sensitive Na + -H + exchange in cultured rat aortic VSMC.29-30 This exchange
mechanism results in Na + influx of 30 nmol Na + /mg
protein/min measured at 2 minutes.31
Another major physiological role for Na + -Ca 2+ exchange may exist under chronic conditions of high [Na + ],.
That is, under conditions that promote elevation of [Na+],
(e.g., enhanced Na+ entry or inhibition of Na,K-ATPase
by cardiac grycosides or an endogenous natriuretic
hormone), Na + -Ca 2+ exchange may become activated
and will tend to decrease [Na + ],. Our results demonstrate
that varying [Na+]< alters Ca2+ content markedly under
steady-state conditions at 4 hours. Blaustein7 has hypothesized that in essential hypertension, failure of the
Na + -K + pump or inhibition by a natriuretic factor may
produce chronic elevation of [Na + ] ( , leading to activation
of Na + -Ca 2+ exchange in VSMC as a mechanism to
extrude Na + from the cell. The resulting rise in [Ca2+],
might augment tonic contraction of VSMC, perpetuating
the hypertensive state.
It is well known that inhibition of Na,K-ATPase by
ouabain increases [Na+]1.7-32 Pretreatment with ouabain
promoted Na + -dependent Ca2+ influx in our cultured
VSMC. The digitalis grycosides have been reported to
have a direct vasoconstrictor effect on vascular smooth
muscle.33-* Thus, Na + -Ca 2+ exchange may play a role
in the regulation of [Ca2+]j under conditions of intracellular Na + loading through a sequence of inhibition
of Na,K-ATPase activity, increased [Na + ],, and increased Ca2+ entry (or decreased Ca2+ extrusion) that
is consistent with the direct vasoconstrictor effect
observed with digitalis grycosides. It is unlikely that the
ouabain-promoted Na + -dependent Ca2+ influx in these
cultured VSMC was due to voltage-dependent Ca2+
entry since previous investigations in this laboratory
have failed to demonstrate a significant amount of Ca2+
entry through voltage-dependent Ca2+ channels in these
cells. The apparent lack of functional Ca2+ channels in
these cells is not unexpected since cultured VSMC
undergo phenotypic modulation in culture with loss of
contractile capability. Nonetheless, they maintain
many differential cellular functions including responsiveness to angiotensin II and expression of contractile
proteins (M. Taubman and B. Berk, unpublished
observations).
It has been argued that Na + -dependent Ca2+ flux may
be explained by Na + -Ca 2+ competition at extracellular
anionic sites. 910 However, under basal conditions in the
present study, the amount of Ca2+ efflux in Na + -free
TBSS and 130 mM Na + -TBSS was not significantly
different. Therefore, the magnitude of apparent Ca2+
efflux due to Na + binding to extracellular anionic sites
can be no greater than 10-15%. Thus, it is unlikely that
Na+-dependent Ca2+ flux could result simply from a
change in binding to external cell surface components.
The studies of cultured VSMC reported here indicate
that a Na + -Ca 2+ exchange mechanism does exist in
these cells and can be shown to mediate transmembrane
Ca2+ flux under the conditions of these experiments.
This exchange process may be particularly important
in the regulation of [Ca 2+ ], during agonist-mediated
increases in [Ca2+];. Hence, Na + -Ca 2+ exchange may
contribute to short-term regulation of [Ca2+],. In
addition, Na + -Ca 2+ exchange may play a role in
regulation of [Ca2+], during chronic changes in [Na+]i,
as has been suggested in chronic hypertension.8-35 This
demonstration of a bidirectional Na + -Ca 2+ exchange
mechanism in cultured rat aortic VSMC extends
findings from vesicle preparations to intact cells and
provides a basis for further studies of the role of this
pathway under physiological conditions in vivo.
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•
Na+-Ca2+ exchange
muscle • Ca2+ influx • Ca2+ efflux
KEYWORDS
vascular smooth
Na+-Ca2+ exchange in cultured vascular smooth muscle cells.
E G Nabel, B C Berk, T A Brock and T W Smith
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Circ Res. 1988;62:486-493
doi: 10.1161/01.RES.62.3.486
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