Physiological effects of
Na+/Ca2+
exchanger knockdown by antisense oligodeoxynucleotides in arterial
myocytes
Martin K.
Slodzinski1,3 and
Mordecai P.
Blaustein1,2,3
Departments of 1 Physiology and
2 Medicine and the
3 Center for Vascular Biology
and Hypertension, University of Maryland School of Medicine,
Baltimore, Maryland 21201
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ABSTRACT |
Antisense oligodeoxynucleotides (AS-oligos) targeted to the
Na+/Ca2+
exchanger (NCX) inhibit NCX-mediated
Ca2+ influx in mesenteric artery
(MA) myocytes [Am. J. Physiol.
269 (Cell Physiol. 38):
C1340-C1345, 1995]. Here, we show AS-oligo knockdown of
NCX-mediated Ca2+ efflux. In
initial experiments, the cytosolic free
Ca2+ concentration
([Ca2+]cyt)
was raised, and sarcoplasmic reticulum (SR)
Ca2+ sequestration was blocked
with caffeine and cyclopiazonic acid; the extracellular
Na+-dependent (NCX) component of
Ca2+ efflux was then selectively
inhibited in AS-oligo-treated cells but not in controls (no oligos or
nonsense oligos). In contrast, the
La3+-sensitive (plasmalemma
Ca2+ pump) component of
Ca2+ efflux was unaffected in
AS-oligo-treated cells. Knockdown of NCX activity was reversed by
incubating AS-oligo-treated cells in normal media for 5 days. Transient
[Ca2+]cyt
elevations evoked by serotonin (5-HT) at 15-min intervals in
AS-oligo-treated cells were indistinguishable from those in controls.
When cells were stimulated every 3 min, however, the peak amplitudes of
the second and third responses were larger, and
[Ca2+]cyt
returned to baseline more slowly, in AS-oligo-treated cells than in
controls. Peak 5-HT-evoked responses in the controls, but not
AS-oligo-treated cells, were augmented more than twofold in
Na+-free media. This implies that
NCX is involved in Na+ gradient
modulation of SR Ca2+ stores and
cell responsiveness. The repetitive stimulation data suggest that the
NCX may be important during tonic activation of arterial myocytes.
serotonin; lanthanum; caffeine; cyclopiazonic acid
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INTRODUCTION |
CYTOSOLIC CALCIUM is a ubiquitous signal
transduction element. In vascular smooth muscle (VSM), agonist- and
depolarization-triggered vasoconstrictions are initiated by an increase
in the cytosolic free Ca2+
concentration
([Ca2+]cyt)
(20, 28). This "activator
Ca2+" is mobilized from
Ca2+ stores in the sarcoplasmic
reticulum (SR) and/or enters the cells from the extracellular
fluid (ECF) (20, 28). Conversely, reduction of
[Ca2+]cyt
promotes vasodilation (20, 28).
Ca2+ is removed from the
contractile apparatus by resequestration into the SR by the SR
Ca2+ (SERCA) pump and/or
extrusion to the ECF (17, 20, 28). Ca2+ extrusion is mediated by the
plasma membrane (PM) Ca2+ (PMCA)
pump (17) and the
Na+/Ca2+
exchanger (NCX) (3, 19, 28). The relative roles of the PMCA pump
and NCX in VSM are controver- sial. Some investigators have suggested
that the NCX in VSM is "latent" (26), or that it plays only a
minor role in VSM Ca2+ homeostasis
(18, 27). In contrast, others have proposed that the NCX plays a key
role in modulating SR Ca2+ and,
thus, vascular reactivity (1, 3, 19).
One method to measure the influence of a particular ion transporter is
to study the physiology of the cell or organ of interest under normal
conditions and after selective block of the transporter. Unfortunately,
no sufficiently selective antagonists of the NCX have yet been
identified. Removal of extracellular
Na+ can inhibit NCX-mediated
Ca2+ efflux, but this manipulation
also affects the cytosolic Na+
concentration
([Na+]cyt)
and alters other Na+-dependent
processes such as
Na+/H+
exchange (see Ref. 25). Moreover, this reduction in the
Na+ electrochemical gradient may
promote Ca2+ entry via the NCX.
Amiloride analogs (e.g., 3,4-dichlorobenzamil) and a recently described
isothiourea derivative (compound 7943) are not sufficiently selective
inhibitors of NCX (8, 25, 30). The exchanger inhibitory peptide (XIP),
which must be injected into cells, targets the NCX putative calmodulin
binding domain on the large cytoplasmic loop of the exchanger (13), but
XIP also affects other calmodulin-dependent systems including, for example, the PMCA pump (5). A specific inhibitor of NCX would be
especially helpful for resolving questions about NCX function in VSM
under physiological conditions.
Recently, we described antisense oligodeoxynucleotide (AS-oligo)
knockdown of NCX in primary cultured arterial myocytes (24). In those
experiments, AS-oligo-treated VSM cells proliferated normally,
maintained normal morphology, and responded appropriately to
physiological agonists when compared with "control" cells grown either without oligos, or with scrambled (NS) oligos. [NS-oligos had the same base composition as AS-oligos, but the bases were mismatched.]
[Ca2+]cyt
was only slightly elevated in quiescent VSM cells in which NCX was
knocked down with AS-oligos. In striking contrast to control cells or
those treated with NS-oligos, however, AS-oligo-treated cells did not
exhibit an increase in
[Ca2+]cyt
when external Na+ was removed to
promote Ca2+ influx via NCX, even
after exposure to ouabain (to raise
[Na+]cyt).
Yet, serotonin (5-HT) was able to evoke a rise in
[Ca2+]cyt
in AS-oligo-treated cells as well as in control groups. In controls,
however, but not in AS-oligo-treated cells, the transient rise in
[Ca2+]cyt
in response to 5-HT (applied in
Ca2+-free media) was augmented
after treatment of the cells with ouabain. This implies that the NCX
plays a role in ouabain-induced augmentation of SR
Ca2+ stores. Taken together, these
earlier results demonstrate that AS-oligos targeted to the NCX mRNA can
be used to knock down NCX function selectively in arterial myocytes.
To distinguish between the roles of the PMCA pump and the NCX in
Ca2+ extrusion, independent
inhibition of the PMCA pump and the NCX is required. Eosin can inhibit
the PMCA pump, but eosin acts on the cytoplasmic face (9) and also
affects other P-type ATPases such as the
Na+ pump and the SERCA pump (9).
Vanadate inhibits the PMCA pump and other ATPases and may also affect
NCX (4). In contrast, low concentrations of
La3+ (0.03-0.25 mM) appear to
inhibit the PMCA pump selectively in VSM (7, 22). These low
La3+ concentrations do not affect
the extracellular Na+-dependent
(NCX-mediated) component of Ca2+
removal from the cytosol of primary cultured rat VSM cells.
In this report, we address the aforementioned controversy about the
physiological role of NCX in VSM. These two tools, AS-oligos and
low-dose La3+, were used to
identify the mechanisms involved in
[Ca2+]cyt
recovery after evoked elevation of
[Ca2+]cyt.
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MATERIALS AND METHODS |
Primary cell culture.
Primary cultured arterial myocytes were prepared from rat mesenteric
arteries (MA). The arteries were aseptically excised and placed in
Hanks' solution containing a 1% dilution of penicillin/streptomycin solution (Sigma, St. Louis, MO). After removal of connective tissue, the blood vessels were incubated for 35 min at 37°C in Hanks' solution with collagenase (2 mg/ml). The adventitia was then separated from the arterial media; the latter was incubated overnight in DMEM
containing 10% FCS (95% air-5%
CO2, 37°C). The next day the vessels were placed in Hanks' balanced salt solution containing collagenase (2 mg/ml) and elastase (0.5 mg/ml) and incubated for 40 min
(95% air-5% CO2, 37°C). The
dissociated cells were triturated with a Pasteur pipette and
resuspended in DMEM containing heat-inactivated 10% FCS and the
appropriate oligos (the FCS was heated 55°C for 4 h to minimize
exonuclease activity; Ref. 21). The suspended cells were plated onto
glass coverslips on 35-mm culture plates. The medium was changed three
times weekly.
VSM cell culture purity was determined immediately after physiological
experiments. Cells were fixed in 95% ice-cold ethanol. A nuclear
stain, 4,6-diamidino-2-phenylindole (DAPI), was used to label all
cells. Myocytes were then identified by immunochemical methods with
mouse anti-rat smooth muscle
-actin antibodies (Boehringer Mannheim); FITC-conjugated goat anti-mouse antibodies were used as the
secondary stain (Jackson ImmunoResearch Laboratories, West Grove, PA).
AS-oligos.
A tandem pair of chimeric phosphorothioated AS-oligos was designed to
target the region upstream (5') to, and encompassing the ATG
start codon (15) of the NCX1 mRNA (from base
26 to base
10, and from base
9 to base +6) (24). These chimeric phosphorothioated oligos have natural deoxynucleotides flanked by four
phosphorothioate modified deoxynucleic acids at the 3'- and
5'-ends
(5'-TGAGACTTCCAATTGTT-3'
and
5'-AAGCATG
CAA-3', where bold letters refer to phosphorothioated bases and underlined region corresponds to the location of the start codon). Chimeric phosphorothioated oligos are stable (exonuclease resistant) and are
transported into cells better than unmodified oligos (6, 14, 31).
A second set of oligos, with the same base composition, but with
scrambled (nonsense, NS-oligos) sequence
(5'-AGTACCTTCTATGAGT-3' and
5'-CAGATATATCAGATG-3'),
was used to control for nonspecific or toxic effects of the oligos. To
ensure that the antisense knockdown of extracellular
Na+-dependent
Ca2+ efflux effect was dependent
on sequence and not on secondary structure, a sense (S-oligos) sequence
(5'-AACAATTGGAAGTCTCA-3' and
5'-TTGTACAACATGCTT-3')
of complementary base pair composition to our antisense probe served as
an additional control in some experiments. All probes were compared
with known sequences in GenBank and European Molecular Biology
Laboratories using the "Wisconsin Package" sequence analysis
program; no significant homologies to other sequences were found.
Control cells were grown without oligos. In parallel, cells were grown
in media containing the AS-oligo pair, the S-oligo pair, or the
NS-oligo pair. The final concentration of the oligos was 0.5 µM from
the time of initial plating until imaging 7-10 days later.
[Ca2+]cyt
determination using fura 2.
Details of the imaging methods are published (3). Cells were incubated
(30 min, 20-22°C, 5%
CO2-95%
O2) in culture media containing
4.0 µM fura 2-AM, the membrane-permeable acetoxymethyl ester of fura
2. The coverslips were transferred to a tissue chamber mounted on a
Nikon Diaphot microscope stage. The cells were superfused with standard
physiological salt solution (PSS) for 30 min at 32-34°C to
wash away extracellular dye and to allow time for cellular esterases to
hydrolyze the fura 2-AM.
The imaging system was designed around a Nikon Diaphot microscope
optimized for ultraviolet transmission. Fura 2 fluorescence (510-nm
light emission; 380- and 360-nm excitation) and background fluorescence
were imaged using a Nikon UV-Fluor objective (×40, numerical
aperture 1.3). Fluorescent images were obtained using a micro-channel
plate image intensifier (Amperex XX1381, Opelco, Washington, DC)
coupled, by fiber optics, to a Pulnix charge-coupled device video
camera (Stanford Photonics, Stanford, CA).
Images were acquired, background subtracted, and transformed to
Ca2+ images using the MetaFluor
imaging system (Universal Imaging, West Chester, PA). Video frames were
digitized at a resolution of 512 horizontal × 480 vertical pixels
and 8 bits using a Matrox LC imaging board. To improve the
signal-to-noise ratio, 16 consecutive video frames were averaged.
[Ca2+]cyt
was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (10); 360 nm is the fura 2 isosbestic point.
Ca2+ images were acquired at a
rate of one averaged image every 3 s to one every 60 s
depending on the experimental protocol. The [Ca2+]cyt
measurements described in RESULTS were
limited to small (~2 µm2)
peripheral cytosolic "areas of interest" so as to avoid the nucleus and dense perinuclear organelles. Each image contained 6-12 VSM cells per field; one area of interest was studied in each
cell.
Solutions.
PSS contained (in mM) 140 NaCl, 5.9 KCl, 1.2 NaH2PO4,
5 NaHCO3, 1.4 MgCl2, 1.8 CaCl2, 11.5 glucose, and 10 HEPES,
titrated to pH 7.4 with NaOH. For
Ca2+-free PSS,
CaCl2 was replaced by 1.8 mM
MgCl2 (total = 3.2 mM), and 0.05 mM EGTA was added. In Na+-free
PSS, the NaCl and NaHCO3 were
isosmotically replaced by N-methyl-D-glucamine
(NMDG), and the pH was adjusted with HCl. In the
La3+ PSS, phosphate and
bicarbonate salts were replaced by chloride salts, and EGTA was
omitted. LaCl3 was added to
solutions immediately before use to minimize the formation of
La2(CO3)3.
Stock solution containing fura 2-AM (1 mM) was prepared in DMSO.
Cyclopiazonic acid (CPA, 5 µM), an inhibitor of the SERCA pump, and
caffeine (10 mM) were added to the bathing solution to increase
[Ca2+]cyt
in certain experiments.
Materials.
Chimeric phosphorothioated oligos were synthesized by Oligos Etc.
(Wilsonville, OR). FCS was obtained from Hyclone (Logan, UT). Fura 2-AM
was obtained from Molecular Probes (Eugene, OR). NMDG, DAPI, 5-HT,
elastase, Hanks' solution, LaCl3,
CPA, caffeine, verapamil, and ouabain were purchased from Sigma. DMEM
was obtained from GIBCO BRL (Grand Island, NY). Collagenase was
obtained from Worthington Biochemical (Freehold, NJ). All other
reagents were analytical grade or the highest purity available.
Statistical analysis and data presentation.
Numerical data are presented as means ± SE of a total of
n single cells from at least three
independent experiments. Where appropriate, ANOVA was used to calculate
the significance of the differences of the means. As noted in
RESULTS and the legends to Figs.
1-6, fluorescence data (reported as
[Ca2+]cyt)
correspond to values from 1) a
representative single cell, 2) the
averaged values for 6-12 cells in a single field on one coverslip,
or 3) the means of values from a
number of cells on each of several coverslips from different
experiments. In graphs of
[Ca2+]cyt
vs. time, the data points were connected, and the curves were smoothed
using a Fourier transform with discontinuities at the times of solution
changes. Microcal Origin software (Northhampton, MA) was used for
calculations, graphics, and curve smoothing.
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RESULTS |
Do AS-oligos affect the
La3+-sensitive
reduction of
[Ca2+]cyt?
Previously, we described AS-oligo knockdown of the cytosolic
Na+-dependent increase of
[Ca2+]cyt
in Na+-free media (24); this rise
in
[Ca2+]cyt
is a manifestation of NCX-mediated
Ca2+ influx. To determine the role
of NCX in removal of Ca2+ from the
cytosol, however, we must distinguish between
Ca2+ extrusion via the PMCA and
the NCX. Previous studies indicate that the PMCA pump (7, 22) and NCX
(24) can be independently inhibited, by
La3+ and AS-oligos, respectively,
in MA myocytes.
Figure
1A
illustrates the reversible inhibitory effect of
La3+ on the removal of
Ca2+ from the cytosol in
representative single cells. Initially, cells were superfused with
Na+- and
Ca2+-free medium to block
Ca2+ entry and inhibit
extracellular extracellular
Na+-dependent
Ca2+ efflux via NCX. This medium
also contained 0.25 mM LaCl3 to
inhibit the PMCA pump and 10 µM verapamil to block
La3+ entry (22). Application of
CPA and caffeine increased
[Ca2+]cyt
in these cells by mobilizing SR
Ca2+ and blocking resequestration.
The elevated
[Ca2+]cyt
was sustained because the major mechanisms involved in removing Ca2+ from the cytosol were then
inhibited. When the La3+ was
subsequently removed from the perfusion medium,
[Ca2+]cyt
rapidly declined as a result of reactivation of the PMCA pump (22).
This effect, about an 8- to 10-fold increase in the rate of
[Ca2+]cyt
decline, was observed in AS-oligo-treated cells as well as in controls
and NS-oligo-treated cells (Fig.
1B). Apparently, neither of these
oligos adversely affected the activity of the PMCA pump; indeed, the
slightly faster recovery rate in AS-oligo-treated cells (Fig.
1B) could have been the result of
upregulation of the PMCA pump.

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Fig. 1.
Effects of antisense oligonucleotides (AS-oligos) and
La3+ on rate of decline of
cytosolic Ca2+ concentration
([Ca2+]cyt)
after mobilization of Ca2+ from
sarcoplasmic reticulum (SR). A:
representative original
[Ca2+]cyt
data from single cells cultured for 9 days in control medium (no
oligos) or in medium containing AS-oligos or nonsense oligonucleotides
(NS-oligos). Each curve corresponds to a single representative cell
that was superfused in Na+- and
Ca2+-free medium to inhibit
Ca2+ entry and extracellular
Na+-dependent
Ca2+ efflux [via
Na+/Ca2+
exchange (NCX)]. La3+ (0.25 mM) was included to inhibit plasma membrane
Ca2+ (PMCA) pump and, as
indicated, intracellular Ca2+
stores were mobilized with 10 µM cyclopiazonic acid (CPA) and 5 mM
caffeine (CAF) to raise
[Ca2+]cyt;
10 µM verapamil was also present, to block
La3+ entry (22, and see text). At
time indicated, La3+ was washed
out to reactivate PMCA pump. B: pooled
data from 3 experiments similar to and including the one in
A. Ordinate corresponds to rate of
[Ca2+]cyt
decline during 30 s preceding La3+
washout (crosshatched bars) and 30 s immediately after initiating
La3+ washout (solid bars). Rate
constant for washout of chamber was 0.3 s 1 (22), ~10 times faster
than rates of
[Ca2+]cyt
decline measured here. In all 3 groups of cells, increase in rate of
[Ca2+]cyt
decline after La3+ washout was
significantly greater (ANOVA, P < 0.001) than rate before washout.
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Do AS-oligos affect the extracellular
Na+-dependent
reduction of
[Ca2+]cyt?
The effects of AS-oligos on the extracellular
Na+-dependent (NCX-mediated)
Ca2+ efflux in MA myocytes are
shown in Fig. 2. In these experiments [Ca2+]cyt
was again elevated by CPA plus caffeine in
Na+- and
Ca2+-free medium containing 0.25 mM La3+ and 10 µM verapamil. In
this case, however, external Na+
was reintroduced to reactivate NCX-mediated
Ca2+ extrusion while the PMCA pump
was still inhibited by the continued presence of
La3+. Under these circumstances,
the rate of
[Ca2+]cyt
decline was accelerated by ~10- to 16-fold in control cells and in
those treated with S- or NS-oligos, but not in myocytes treated with
AS-oligos, in which the rate increased only 1.7-fold (Fig. 2,
A and
B). Thus
Ca2+ extrusion by NCX was markedly
reduced in the AS-oligo-treated cells. The AS-oligos did not, however,
significantly affect the ability of the SR to store
Ca2+, or the ability of CPA and
caffeine to mobilize the stored
Ca2+. Furthermore, the
extracellular Na+-dependent
decline in
[Ca2+]cyt,
presumably a manifestation of NCX activity (22), in the control,
NS-oligo, and S-oligo groups was apparently unaffected by 0.25 mM
La3+.

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Fig. 2.
Effects of AS-oligos and Na+ on
rate of decline of
[Ca2+]cyt
after mobilization of Ca2+ from
SR. A: representative original
[Ca2+]cyt
data from single cell cultured for 7 days in control medium (no oligos)
or in medium with AS-, NS-, or sense (S)-oligos. Cells were superfused
in Na+- and
Ca2+-free medium to inhibit
Ca2+ entry and extracellular
Na+-dependent
Ca2+ efflux (via NCX).
La3+ (0.25 mM) was included to
inhibit PMCA pump and, as indicated, intracellular
Ca2+ stores were mobilized with
CPA + caffeine to raise
[Ca2+]cyt;
10 µM verapamil was also present. At time indicated,
Na+ was added back to medium to
reactivate NCX. B: pooled data from 3 similar experiments (including one in
A). Ordinate corresponds to rate of
[Ca2+]cyt
decline during 30 s before adding back
Na+ (crosshatched bars) and 30 s
immediately after adding back Na+
(solid bars). Rate of
[Ca2+]cyt
decline accelerated markedly (ANOVA, P < 0.001) in control cells and in those treated with NS- or S-oligos
when external Na+ was added back.
Rate accelerated only minimally (and insignificantly,
P > 0.05) when
Na+ was added back to
AS-oligo-treated cells.
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As illustrated in Figs. 1B and
2B, the rates of recovery from the
elevated
[Ca2+]cyt
in control and NS-oligo-treated cells were faster after
Na+ replacement (Fig.
2B) than after
La3+ washout (Fig.
1B). With the assumption that
La3+ dissociation from the PMCA
pump molecules was not rate limiting, these data suggest that the NCX
in these MA myocytes may have been able to extrude
Ca2+ a little faster than the PMCA
pump under the conditions of these experiments.
Can the effects of AS-oligos be reversed?
An important control for the specific antagonism by AS-oligos is
reversibility. Figure 3 shows data from
cells that were treated with either AS-oligos or no oligos (controls)
for 7 days. Then, on day 7, some of
the cells that had been treated with DMEM plus 10% FCS containing
AS-oligos were returned to normal (oligo-free) DMEM plus 10% FCS
medium (the "recovery" group). The three groups were continued in
culture for an additional 5 days. Extracellular Na+-dependent reduction of
[Ca2+]cyt
was then measured, using the protocol illustrated by the experiment in
Fig. 2A. External
Na+ greatly accelerated the
decline in
[Ca2+]cyt
in recovery cells (by 8-fold) as well as in the controls (by 15-fold),
but not in the cells incubated with AS-oligos for all 12 days (Fig. 3).
The slower recovery rates of the control cells in these experiments,
compared with those of Fig. 2B, may be
attributed to the older age of the cultures in the experiments of Fig.
3.

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Fig. 3.
Recovery of NCX activity after treatment with AS-oligos. Cells were
divided into 3 groups: 1 group (control) was continuously incubated in
culture medium without oligos for 12 days; the other 2 groups were
incubated with AS-oligos for 7 days, then 1 of the groups (recovery)
was returned to oligo-free medium for next 5 days of culture, whereas
the other group (antisense) was incubated with AS-oligos for all 12 days. Protocol for testing NCX activity (extracellular
Na+-dependent decline in
[Ca2+]cyt)
was identical to that shown in experiment of Fig.
2A. Results shown here are pooled data
from 3 independent experiments. "Antisense" group exhibited no
detectable NCX activity, whereas both "control" and
"recovery" groups showed large (ANOVA,
P < 0.001) extracellular
Na+-dependent declines in
[Ca2+]cyt.
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Little recovery was observed in AS-oligo-treated cells that were
subsequently incubated without AS-oligos for only 1 or 2 days (data not
shown). Thus the knockdown of NCX function by AS-oligos appears to be
reversible in MA myocytes, but only after more than 2 days of recovery
without the oligos.
Does NCX knockdown affect 5-HT-evoked
Ca2+ transients?
To determine the role that NCX plays in the response to physiological
agonists, control cells and AS- or NS-oligo-treated cells were
stimulated with brief (30 s) applications of 10 µM 5-HT (a
maximal effective dose). The Ca2+
transients in AS-oligo-treated cells evoked by this single, brief exposure to 5-HT were indistinguishable from those in control cells
(Fig.
4A and
see Table 1). Furthermore, the
Ca2+ transients in controls
(including NS-oligo-treated cells) and AS-oligo-treated cells were
negligibly affected if external
Ca2+ was removed 30 s
before the 5-HT was introduced (Fig.
5; and see Table 2, no
La3+). Thus these transients can
be attributed primarily to Ca2+
mobilization from the SR, and not to
Ca2+ entry from the extracellular
fluid (2).

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Fig. 4.
Ca2+ transients evoked by
repetitive applications of serotonin (5-HT) in primary cultured
mesenteric artery myocytes. Representative original
[Ca2+]cyt
data from single cells cultured for 9 days without oligos (control) or
with AS- or NS-oligos. 5-HT (10 µM) was superfused for 30 s at 10-min
intervals (A) and at 3-min intervals
(B). Pooled results from several
such experiments are presented in Tables 1 and 3, respectively.
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Table 1.
Effects of AS-oligo knockdown of
Na+/Ca2+ exchange on 5-HT-evoked
[Ca2+]cyt transients with 30-s
5-HT application at 15-min intervals (see Fig. 4A)
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Fig. 5.
Effects of AS-oligos on 5-HT-evoked
Ca2+ transients in
Ca2+-free media in absence and
presence of 0.125 mM La3+.
A: representative original
[Ca2+]cyt
data from single cells cultured for 9 days in medium without oligos
(control) or with AS- or NS-oligos.
Ca2+ transients were evoked with
10 µM 5-HT in Ca2+-free media to
prevent Ca2+ entry. During second
exposure to 5-HT, 0.125 mM La3+
was added to inhibit PMCA pump; at this
La3+ concentration, no verapamil
was needed to inhibit La3+ entry
(22). B: expanded time scale
(abscissa) to show first-order exponential curve fits (heavy lines) to
decline of
[Ca2+]cyt
during first 30 s after peaks of
Ca2+ transients in absence
(left) and presence
(right) of 0.125 mM
La3+.
C: time constants ( ) for decline of
[Ca2+]cyt
during first 30 s after peak Ca2+
transients; time constants were measured from curve fits such as those
illustrated in B. Bar graph shows
pooled data from 3 independent experiments similar to (and including)
one illustrated in A and
B. In absence of
La3+, time constants for three
groups of cells were not significantly different from one another (see
Table 2 and text). In presence of 0.125 mM
La3+, mean time constant for
AS-oligo-treated cells was significantly larger (ANOVA,
P < 0.01) than time constants for
other 2 groups of cells.
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Table 2.
Effects of AS-oligo knockdown of
Na+/Ca2+ exchange on 5-HT-evoked
[Ca2+]cyt transient in absence and
presence of La3+ (see Fig. 5A)
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Does NCX knockdown affect responses to repetitive applications of
5-HT?
Even when 5-HT was repetitively applied at 15-min intervals, the
amplitudes of the three consecutive
[Ca2+]cyt
peaks were relatively constant and did not differ significantly in
cells from each of the three groups (controls, AS-oligos, and NS-oligos; Fig. 4A and Table 1). These
results demonstrate that the responses to 5-HT were reproducible and
that there were no substantial differences among the three groups when
the cells were given sufficient time to recover.
When 5-HT was repetitively applied at much shorter intervals, however
(e.g., every 3 min, as in Fig. 4B and
Table 3), the peak amplitudes of the
Ca2+ transients declined
progressively (peak 1 > peak 2 > peak
3) in control cells and in those treated with
NS-oligos. Explanations for this decline include the possibility that
the 5-HT receptor/intracellular signaling (inositol trisphosphate)
pathway was temporarily downregulated or that the SR did not have
sufficient time to refill between exposures to 5-HT at this higher rate
of stimulation.
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Table 3.
Effect of AS-oligo knock down of
Na+/Ca2+ exchange on 5-HT-evoked
[Ca2+]cyt transients with 30-s
5-HT application at 3-min intervals (see Fig. 4B)
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|
The responses of the AS-oligo-treated cells differed from those of the
controls and NS-oligo-treated cells activated at 3-min intervals. One
difference was a slower recovery to baseline
[Ca2+]cyt
of the AS-oligo-treated cells; for example, 2.9 min after each exposure
to 5-HT (i.e., just before the next stimulus),
[Ca2+]cyt
remained higher in the AS-oligo-treated cells than in the controls
(Fig. 4B and Table 3, prestimulation).
Although these data were obtained in standard PSS, comparable results
were obtained when external Ca2+
was temporarily removed during each application of 5-HT (data not
shown). These data indicate that the NCX plays a role in the rapid
removal of Ca2+ from the cytosol
and recovery of
[Ca2+]cyt
after Ca2+ transients in MA
myocytes.
A second difference between the AS-oligo-treated cells and controls in
these experiments concerns the amplitudes of the
Ca2+ transients. There was a
relatively smaller decline in the peak amplitudes of the second and
third 5-HT-evoked Ca2+ transients
in AS-oligo-treated cells (~10 and 25% decline, compared with the
initial response) than in the controls and NS-oligo-treated cells
(~35 and 50%, respectively; Figs.
4B and Table 3). This, too, seems
consistent with inhibition of NCX-mediated
Ca2+ extrusion during recovery
after Ca2+ transients; under these
circumstances, the SR Ca2+ stores
may have been able to refill more rapidly than normal because of the
slower extrusion of Ca2+ from the
cytosol.
Effects of application of 5-HT on MA myocytes incubated in
Na+-free media.
If external Na+ was removed at the
time that 5-HT was applied, the evoked
Ca2+ transients in control and
NS-oligo-treated cells were significantly greater than those evoked in
these cells in standard PSS (Fig. 6B). In
contrast, the removal of external
Na+ had little effect on the
5-HT-evoked Ca2+ transients in the
AS-oligo-treated cells (Fig. 6, A and
B). This can be explained if the
reversal of the Na+
electrochemical gradient across the plasma membrane promoted Ca2+ entry via NCX only in the
control and NS-oligo-treated cells and thereby augmented the
Ca2+ mobilized from the SR stores
and directly contributed to the larger
Ca2+ transients in these cells (2,
24).

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Fig. 6.
Effects of AS-oligos on 5-HT-evoked
Ca2+ transients in
Na+-free medium.
A: representative original
[Ca2+]cyt
data from single cells cultured for 9 days in medium without oligos
(control) or medium containing AS- or NS-oligos.
Na+-free medium was introduced at
same time that 10 µM 5-HT was added to medium. 5-HT was removed after
30 s, but superfusion with
Na+-free medium was continued for
remainder of recording period. B:
pooled data from 3 similar,
Na+-free experiments (Na-free PSS,
right). Peak
[Ca2+]cyt
both in control cells and in those treated with NS-oligos were
significantly greater than peak
[Ca2+]cyt
in AS-oligo-treated cells (ANOVA, P < 0.001 in both cases). Peak
[Ca2+]cyt
in control and NS-oligo-treated myocytes during inital 5-HT-evoked
responses was also significantly greater
(P < 0.001) when cells were bathed
in Na+-free medium
(right) than in standard PSS
(left). The latter data are pooled
results from initial 5-HT-evoked transients described in Tables 1 and
3.
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|
Effects of
La3+ on
5-HT-evoked Ca2+
transients.
To evaluate, further, the effect of AS-oligos on the rate of
[Ca2+]cyt
recovery from activation by 5-HT,
Ca2+ responses evoked in the
absence and presence of La3+ were
compared. Myocytes were first exposed to 5-HT in
Ca2+-free medium to ensure that
the evoked increase in
[Ca2+]cyt
was the result of mobilization of intracellular
Ca2+. After recovery from the
initial exposure to 5-HT, 0.125 mM
La3+ was added to the medium to
block the PMCA pump, and 5-HT was reapplied (Fig.
5A); at this
La3+ concentration, verapamil is
not needed to block La3+ entry
(22). The initial 30-s decline of
[Ca2+]cyt
from the peak of each 5-HT-evoked transient was fitted to a first-order
exponential (Fig. 5B). The time
constants (
) for the decline of
[Ca2+]cyt
in the absence and presence of
La3+ were then compared (Fig. 5,
B and
C). In the absence of
La3+ (i.e., with the PMCA pump
functioning), there were no significant differences in the
values
among the three groups of cells (controls and AS-oligo- and
NS-oligo-treated cells), although
was slightly higher in these
AS-oligo-treated cells (Fig. 5C; and
see Table 2). In other experiments, such as those illustrated in Fig.
4, A and
B, however, no differences in
values were detected (0.18-0.21 min in controls, 0.18-0.25
min in NS-oligo-treated cells, and 0.19-0.26 min in
AS-oligo-treated cells). In contrast, when the PMCA pump was inhibited
with La3+, the
for recovery of
AS-oligo-treated cells increased markedly and was ~10-fold larger
than that of the control cells and ~3-fold larger than the
of the
NS-oligo-treated cells. The implication is that during a single
exposure to 5-HT, the PMCA pump is able to remove
Ca2+ from the cytosol at a rapid
rate so that the NCX appears to be superfluous under these conditions.
Nevertheless, as noted above, the NCX clearly plays a role in
Ca2+ removal from the cytosol
after rapid, repetitive 5-HT stimuli.
 |
DISCUSSION |
AS-oligos inhibit NCX-mediated
Ca2+ extrusion
from VSM cells.
Previously, we reported that AS-oligos targeted to the NCX inhibit,
selectively, the extracellular
Na+-dependent component of
Ca2+ influx, which is mediated by
the NCX, in primary cultured rat VSM cells (24). The AS-oligos did not
interfere with normal growth or other physiological functions of these
cells. Moreover, other studies have demonstrated that low
concentrations of La3+
(0.03-0.25 mM) block, selectively, the extracellular
Na+-independent component of
Ca2+ efflux from arterial myocytes
that is mediated by the PMCA pump (9, 22). Thus, by employing
La3+ in conjunction with
AS-oligos, we were able to test the role of the NCX in
Ca2+ extrusion from VSM cells.
As shown in Figs. 1 and 2, there are two main components of
Ca2+ efflux. When
Ca2+ was mobilized from the SR and
resequestration was prevented, with CPA plus caffeine,
[Ca2+]cyt
remained elevated if both efflux mechanisms were inhibited (with
Na+-free media and
La3+). In initial experiments
(Fig. 1), we observed that treatment with either AS- or NS-oligos did
not significantly affect the La3+-inhibitable (PMCA pump)
component of Ca2+ efflux.
Selective La3+ inhibition of the
PMCA pump enabled us to evaluate the extracellular
Na+-dependent
Ca2+ efflux directly (Fig. 2).
After an evoked rise in
[Ca2+]cyt,
replacement of extracellular Na+
did not accelerate the removal of
Ca2+ from the cytosol of
AS-oligo-treated cells. In marked contrast, untreated control,
NS-oligo-treated, and S-oligo-treated cells exhibited a 10- to 16-fold
increase in the rate of decline of [Ca2+]cyt
after the replacement of extracellular
Na+. These results indicate that
the AS-oligos knocked down the extracellular Na+-dependent
Ca2+ efflux. The fact that the NCX
functioned as well in S-oligo- as in NS-oligo-treated cells implies
that AS-oligos are sequence specific and that NCX knockdown by
AS-oligos was not a result of differences in oligo secondary structure
(29).
The knockdown of NCX activity by AS-oligos was reversible (Fig. 3).
This is a further indication that the effect of the AS-oligos was
specific. Whether this recovery, which was observed at 5 days, but not
at 1 or 2 days, was the result of a relatively long AS-oligo half-life,
a long NCX protein half-life, and/or other factors is unknown.
This result is, however, consistent with the evidence, from neonatal
rat cardiac myocytes, that the NCX protein half-life is ~33-36 h
(23, and W. J. Lederer and T. B. Rogers, personal communication).
The preceding observations confirm that the MA myocytes have two major
mechanisms for extruding Ca2+.
This apparent redundancy may, at first, seem surprising. Other studies,
however, demonstrate that the PMCA pump and the NCX are distributed
differently in the plasma membrane of MA myocytes: the NCX is confined
to "junctional" domains of the plasma membrane that overlie
junctional SR (11), whereas the PMCA pump appears to be more
ubiquitously distributed over the cell surface (12). This differential
distribution of these two Ca2+
transport systems is circumstantial evidence that they may play different roles in the physiology (and, perhaps, pathophysiology) of
the arterial myocytes.
Physiological role of the NCX in arterial myocytes.
To elucidate the physiological role of the NCX, the influence of
AS-oligos on the responses to a physiological agonist, 5-HT, was
studied. The AS-oligos had no detectable effects on the
Ca2+ transients evoked by single
brief exposures to 10 µM 5-HT or on transients evoked by repetitive
applications of 5-HT when the stimuli were presented at a low frequency
(every 15 min).
The experiments with La3+ (Fig. 5
and Table 2) also demonstrate that recovery after 5-HT-evoked
[Ca2+]cyt
elevation was only minimally slowed when either the PMCA pump was
inhibited (Fig. 5C, control + La3+) or the NCX was knocked
down as a result of AS-oligo treatment (Fig.
5C, AS-oligos, no
La3+). When, however, the PMCA
pump was blocked in AS-oligo-treated myocytes, the recovery of
cytosolic Ca2+ was greatly slowed.
These results all appear to indicate that the NCX may play only a
limited role in Ca2+ homeostasis
when the cells are not burdened with a large
Ca2+ load. On the contrary,
however, the augmentation of 5-HT-evoked Ca2+ tranients by removal of
external Na+ in controls, but not
AS-oligo-treated cells (Fig. 6), implies that NCX normally mediates the
Na+ electrochemical
gradient-dependent modulation of SR
Ca2+ stores and the resultant cell
responsiveness to agonists.
In another series of experiments, 5-HT was applied repetitively at
short (3 min) intervals. The amplitudes of the
Ca2+ transients declined
progressively (i.e., peak 1 > peak 2 > peak 3) under these circumstances, but the decline in peak
amplitude was attenuated in the AS-oligo-treated cells. Moreover,
[Ca2+]cyt
returned to baseline more slowly in the AS-oligo-treated cells than in
controls. These observations are consistent with the view that
Ca2+ was extruded from the cells
more slowly when the NCX was knocked down. The smaller decline in peak
height in the AS-oligo-treated cells (in standard PSS) can be
attributed to more complete refilling of the SR
Ca2+ stores. The larger (relative
to control and NS-oligo) second and third responses in the
AS-oligo-treated cells (Fig. 4B)
indicate that downregulation of the 5-HT receptor/inositol
trisphosphate signaling pathway is probably not the explanation for the
marked decline in amplitude of the
Ca2+ transients in the control and
NS-oligo-treated cells.
Taken together, our results with AS-oligo knockdown of the NCX (this
report and 24) and La3+ inhibition
of the PMCA pump (22, and this report) indicate that quiescent arterial
myocytes, and cells that are stimulated infrequently, can maintain
reasonable Ca2+ balance with only
one of the two Ca2+ transport
systems functioning. When the myocytes are activated at short
intervals, however, the effects of NCX knockdown become much more
obvious. Under normal physiological conditions, the smooth muscle in
most arterial beds is tonically activated by local, neurogenic, and
hormonal factors (16). Thus the present results suggest that the NCX
plays an important role in Ca2+
homeostasis in arterial smooth muscle in vivo.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Magdalena Juhaszova for critical comments on an early
version of this manuscript.
 |
FOOTNOTES |
This work was supported by National Institutes of Health (NIH) Grant
HL-45215, by NIH Training Grant GM-025-22580, and by intramural
research funds from the University of Maryland School of Medicine and
the University of Maryland-Baltimore Graduate School.
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. §1734 solely to indicate this fact.
Address for reprint requests: M. P. Blaustein, Dept. of Physiology,
Univ. of Maryland School of Medicine, 655 West Baltimore St.,
Baltimore, MD 21201.
Received 6 January 1998; accepted in final form 6 April 1998.
 |
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