Peroxide resistance of ER
Ca2+ pump in endothelium:
implications to coronary artery function
Ashok K.
Grover and
Sue E.
Samson
Department of Biomedical Sciences, McMaster University, Hamilton,
Ontario, Canada L8N 3Z5
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ABSTRACT |
We examined the effects of peroxide on the sarco(endo)plasmic
reticulum Ca2+ (SERCA) pump in pig
coronary artery endothelium and smooth muscle at three organizational
levels: Ca2+ transport in
permeabilized cells, cytosolic
Ca2+ concentration in intact
cells, and contractile function of artery rings. We monitored the
ATP-dependent, azide-insensitive, oxalate-stimulated 45Ca2+
uptake by saponin-permeabilized cultured cells. Low concentrations of
peroxide inhibited the uptake less effectively in endothelium than in
smooth muscle whether we added the peroxide directly to the
Ca2+ uptake solution or treated
intact cells with peroxide and washed them before the permeabilization.
An acylphosphate formation assay confirmed the greater resistance of
the SERCA pump in endothelial cells than in smooth muscle cells.
Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolic
Ca2+ concentration in a
Ca2+-free solution, but it did not
affect the endothelial cells. Peroxide pretreatment inhibited the
CPA-induced contraction in deendothelialized arteries with a 50%
inhibitory concentration of 97 ± 13 µM, but up to 500 µM
peroxide did not affect the endothelium-dependent, CPA-induced
relaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,
endothelium-dependent relaxation by only 40 ± 13%. The greater
resistance of the endothelium to reactive oxygen may be important
during ischemia-reperfusion or in the postinfection immune response.
free radicals; adenosinetriphosphatase; bradykinin; angiotensin; cyclopiazonic acid; thapsigargin; fluorescence; ischemia ; endoplasmic
reticulum
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INTRODUCTION |
THE SARCO(ENDO)PLASMIC reticulum
Ca2+ (SERCA) pool plays a key role
in signal transduction in vascular smooth muscle and endothelial cells
(21, 25). Several agents such as angiotensin II, endothelins, and
prostanoids act on vascular smooth muscle, at least in part, by
producing second messengers [such as inositol 1,4,5-trisphosphate (IP3)] that release
Ca2+ stored in the endoplasmic
reticulum, whereas other agents may act mainly via entry of
extracellular Ca2+ (24, 29). The
net result is an increase in intracellular Ca2+ concentration
([Ca2+]i)
and contraction of the smooth muscle. In contrast, agents such as
acetylcholine and bradykinin act on endothelium by releasing Ca2+ from the endoplasmic
reticulum and via extracellular entry (21). The consequence in this
instance is an increase in
[Ca2+]i
in the endothelial cells. This results in the activation of pathways
that may lead to the release of endothelial relaxation factors,
especially nitric oxide (NO) because endothelial cells contain a
Ca2+/calmodulin-stimulated NO
synthase (6). The deactivation of smooth muscle and endothelial cells
involves a decrease in
[Ca2+]i,
and the SERCA pumps play an important role in it. The SERCA pumps also
play a role in refilling the endoplasmic reticulum along with the
extracellular Ca2+ entry (24, 29).
The SERCA pump in the arterial smooth muscle can be readily damaged by
reactive oxygen species (ROS, free radicals) that are generated during
ischemia and reperfusion (17-19, 33). Similar damage also occurs
in the sarcoplasmic reticulum Ca2+
pump in the heart (6). The SERCA pump in smooth muscle is more
sensitive to ROS than are the
IP3-sensitive
Ca2+ channels, the L-type
voltage-sensitive Ca2+ channels,
or the Na+ pump (9, 10). Damage to
the SERCA pump in smooth muscle leads to an impairment of the ability
of the arteries to contract with agents such as angiotensin II (16).
The effects of ROS on some of the functions of endothelium have also
been reported (7, 8, 23, 30). Many agents working on endothelium also act by mobilizing an intracellular
Ca2+ pool (7, 8, 13). Although the
smooth muscle contains SERCA2b, endothelium has been suggested to
express the SERCA pump isoform SERCA3 (1-3, 19, 31, 32), which is
approximately three times more resistant to ROS than SERCA2b (18).
However, to our knowledge this is the first study comparing the effects of ROS on the SERCA pumps in endothelium and smooth muscle.
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EXPERIMENTAL METHODS |
Coronary artery smooth muscle and endothelial cell cultures.
Pig coronary artery smooth muscle cells were isolated and plated in
Dulbecco's modified Eagle's medium (DMEM) (GIBCO 12800-017) supplemented with 0.5 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) (pH 7.4), 2 mM glutamine, 50 mg/l gentamicin, 0.125 mg/l
amphotericin B, and 10% fetal bovine serum and then allowed to grow to
confluence (16). The cells were removed from the plates by
trypsinization (0.25% trypsin, 1 mM EDTA in
Ca2+- and
Mg2+-free Hanks' balanced salt
solution, GIBCO) for 4 min at 37°C and then replated. At the third
passage, a large stock of cells was frozen into aliquots each
containing 2 × 106 cells.
An aliquot of cells was thawed and grown. After the cells had grown to
confluence, they were trypsinized and replated at a density of 2 × 104
cells/cm2. The medium was
changed after 2 days. The cells were used 5-8 days after
plating. Thus all of the experiments on smooth muscle cells were
performed on cells in passage 4.
Endothelial cells were dislodged from the inner surface of an artery by
using a sterile cotton swab. All the cells were cultured in DMEM.
Aliquots of the frozen cells were thawed and used in
passage 5.
Western blots.
Western blot studies were carried out either using whole cell material
or microsomes. For whole cell studies, typically
107 cells were suspended in 100 µl of the Laemmli sample buffer and boiled for 2 min. From this
suspension, 1 µl (105 cells) was
applied on a gel. Electrophoresis was carried out in 7.5% Laemmli
gels, which were then electroblotted onto nitrocellulose, and the blots
were treated with a 500-2,000× dilution of the primary antibodies. Three primary antibodies were used: a mouse monoclonal anti-smooth muscle
-actin, the mouse anti-endothelial NO synthase, and a rabbit anti-von Willebrand factor. The antibody binding was
visualized by enhanced chemiluminescence using an Amersham kit,
following the instructions of the manufacturer.
Peroxide treatment and permeabilization of cells.
The cultured smooth muscle cells were harvested by trypsinizing for 4 min as described above and suspended in physiological saline solution
(PSS) containing (in mM) 140 NaCl, 5 KCl, 10 NaHEPES at pH 7.4, 1 MgCl2, and 1.5 CaCl2. The cells were diluted at a concentration of ~106 cells/ml
in PSS containing specified concentrations of hydrogen peroxide. The
cell suspensions were incubated for 30 min at 37°C with different
concentrations of peroxide and then centrifuged to remove the peroxide.
The cells were resuspended in the skinning solution without saponin and
then washed in it. The skinning solution contained (in mM) 165 KCl, 0.4 MgCl2, 5 sodium azide, 1 dithiothreitol (DTT), and 20 sodium
3-(N-morpholino)propanesulfonic acid (MOPS) at pH 6.8 (11).
The cells were permeabilized at a concentration of
107 cells/ml in the skinning
solution containing 250 µg saponin/ml for 15 min at 25°C. The
same method was also used for permeabilizing the endothelial cells,
with the only difference being that a higher concentration of
endothelial cells (2 × 107
cells/ml) was found to be optimum, since these cells are much smaller
than smooth muscle cells. The permeabilized cells (>95%) were
permeable to trypan blue. The permeabilized cells were placed on ice
and used within several minutes.
Ca2+
transport.
Typically, 50 µl of the permeabilized cells (25-75 µg protein)
were added to 150 µl of a
45Ca2+
uptake medium so that the final composition of the solutions was 30 mM
imidazole-HCl (pH 6.8 at 37°C), 100 mM KCl, 1 mM
MgCl2, 5 mM sodium azide, 1 mM
ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.85 mM CaCl2 (plus
trace amounts of
45CaCl2), 5 mM
ATP, 10 mM creatine phosphate, 50-80 U/ml creatine kinase, 5 mM
oxalate, and 15-30 µg of cell protein (11). This resulted in a
Ca2+ concentration of 5 µM. In
an initial experiment, it was determined that the uptake was linear for
up to 30 min. Routinely, the samples were incubated for 30 min at
37°C and then filtered through 0.45-µm nitrocellulose filters
under suction. The filters were washed three times with a chilled
solution containing 8% sucrose, 0.5 mM EGTA, and 40 mM imidazole-HCl
(pH 7.0). The filters were then placed in vials containing Beckman
Ready Safe cocktail, and the amount of radioactivity in the filters was
determined with a scintillation counter.
Acylphosphates.
The acylphosphate experiments were carried out on microsomes prepared
from the cultured endothelial or smooth muscle cells (16). The cells
were suspended in chilled homogenization solution (8% sucrose, 1 mM
phenylmethysulfonyl fluoride, and 2 mM DTT). They were homogenized for
4 × 5 s with a Polytron PT20. The homogenates were centrifuged at
10,000 g for 2 min to remove
mitochondria and nuclei. KCl was added to the supernatant to get a
final concentration of 0.7 M, and the suspension was placed on ice for
10 min and then centrifuged at 550,000 g for 15 min. The microsomal pellets were rinsed with the suspension solution (8% sucrose and 5 mM sodium
azide) to remove traces of DTT and then suspended in it. The microsomes
were incubated in different concentrations of hydrogen peroxide for 30 min at 37°C, placed back on ice, and then used immediately. The
Ca2+-dependent acylphosphate
formation by the microsomes was examined as follows (16). Routinely, 24 µl of the microsomal suspension were added to 12 µl of a buffer
solution and incubated in ice water for 10 min, and then an equal
volume of radioactive ATP was added. Typically, the final reaction
mixtures contained 100 mM KCl, 30 mM imidazole-HCl (pH 6.8), 50 µM
CaCl2, 5 µM unlabeled ATP, 20 µCi of [
-32P]ATP,
and 10-15 µg of the microsomal protein. The reactions were started by adding ATP and stopped after 60 s by adding 250 µl of
ice-cold TCAP (10% trichloroacetic acid, 50 mM phosphoric acid, and 1 mM ATP). The samples were vortexed, placed on ice for 5 min, and then
centrifuged at 4°C. The pellets were washed once with 1,500 µl
TCAP and then suspended in 70 µl of freshly prepared sample buffer
containing 10 mM NaMOPS (pH 5.5), 1 mM EDTA, 3% sodium dodecyl sulfate
(SDS), 10% sucrose, 20 mM DTT, and 0.1% of the tracking dye methylene
green. The samples were vortexed vigorously for 15-20 min and then
electrophoresed immediately in 7.5% SDS-polyacrylamide gels at pH 4 as
described previously (16). The gels were autoradiographed at room
temperature, and the optical densities of the acylphosphate bands were
monitored by image analysis.
[Ca2+]i
measurements.
Cells were cultured on coverslips for the
[Ca2+]i
measurement experiments (16). The coverslips coated with the cultured
cells were removed from the culture medium and rinsed with a solution containing (in mM) 115 NaCl, 5.8 KCl, 2 CaCl2, 0.6 MgCl2, 12 glucose, and 25 NaHEPES
(pH 7.4 at 37°C). These coverslips were then preincubated in this
buffer at 37°C for 30 min in the presence of specified concentrations of peroxide, washed three times to remove peroxide, incubated for 40 min at 22-23°C in 3 µM fluo 3-acetoxymethyl
ester (AM) and 2 mM probenecid dissolved in the above buffer, washed three times to remove the excess dye, and then incubated in a solution
without the dye and with probenecid for 30 min at 22-23°C to
allow for hydrolysis of the dye. The coverslips were then rinsed with
the same buffer containing probenecid but with 1 mM
Ca2+ and placed in 2 ml of the
same in a stirred thermostated cuvette at 37°C for fluorescence
measurement using a SPEX Fluorolog 112. Thus the added peroxide was
removed by washing before the cells were loaded with fluo 3-AM and the
fluorescence was monitored. This protocol was important because
peroxide itself may interact with the added agents. For measurements in
Ca2+-free solutions, 1 mM
Ca2+ was replaced by 0.5 mM EGTA
in the last step. The fluorescence was monitored at excitation and
emission wavelengths of 490 and 530 nm, respectively. After the basal
fluorescence and the effect of cyclopiazonic acid (CPA) on the
fluorescence were monitored, calibration was carried out and the
fluorescence values converted to
[Ca2+]i
as described previously (16).
Contractility experiments.
The pig left coronary artery along with the surrounding cardiac cells
was excised from the heart and placed in a Krebs solution bubbled with
95% CO2-5% O2 (16). The Krebs
solution contained the following (in mM): 115 NaCl, 5 KCl, 22 NaHCO3, 1.7 CaCl2, 1.1 MgCl2, 1.1 KH2PO4,
0.03 EDTA, and 7.7 glucose. The cardiac tissue and fat were removed,
and the arteries were dissected as described previously (16). To obtain
the deendothelialized arteries, the endothelium was removed by drawing
a piece of cotton through each artery. The endothelium removal was
monitored as a loss of bradykinin relaxation. Each artery was cut into
3-mm-long rings and suspended between two hooks in an organ bath. The
tissues were placed under an initial tension of 3 g, which was
corrected 30 min later. A typical contraction experiment using
deendothelialized arteries consisted of the following steps:
1) monitor for 30 min the force
generated by adding 60 mM KCl to the bathing Krebs solution, 2) over 20-30 min wash the
arteries two times in normal Krebs solution,
3) treat the arteries for 30 min
with specified concentrations of peroxide,
4) wash three times in normal Krebs
solution for over a total of 60 min, and
5) test the effects of CPA or other agents. This experimental protocol was chosen to ensure that the results depended on the damage of peroxide on the tissues and not on
the effects of these agents on the assay solutions. The contraction to
60 mM KCl in each tissue was taken as 100%, and the subsequent CPA or
KCl was compared with it. For monitoring the endothelium-dependent
relaxation, the arteries were not deendothelialized. A typical
relaxation experiment consisted of the following steps: 1) monitor for 30 min the force
generated by adding 60 mM KCl to the bathing Krebs solution,
2) over 20-30 min wash the
arteries two times in normal Krebs solution,
3) treat the arteries for 30 min
with specified concentrations of peroxide,
4) wash three times in normal Krebs
solution for over a total of 60 min,
5) produce a contraction with 30 mM
KCl, and 6) at the peak of this contraction add CPA. The relaxation was expressed as a percentage of
the contraction. At the end of the experiment, the tissues were blotted
and weighed. In the control experiments with peroxide, the tissues were
treated identically except that peroxide was not added during the
preincubation period.
Data analysis.
The curve fitting was carried out with FigP (Biosoft). Null hypotheses
were tested with a Student's t-test,
and P values of <0.05 were
considered to be statistically significant.
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RESULTS |
The effect of peroxide was examined at three different organizational
levels: Ca2+ pump activities in
permeabilized cells or microsomes,
Ca2+ mobilization in intact cells,
and contraction and relaxation of artery rings. Except where specified,
the cells or tissues were preincubated with peroxide, washed to remove
the peroxide, and then assayed. This protocol eliminated any direct
interactions between peroxide and the components of the assay systems,
thus assuring that only the damage that remained when peroxide was washed off was monitored.
Characterization of cultured cells.
The antibody reactivity of the whole cell lysates was examined in
Western blots, and the results are shown in Fig.
1. The endothelial cells
reacted positively to anti-von Willebrand factor and anti-endothelial
NO synthase but negatively to anti-smooth muscle
-actin. Conversely,
the smooth muscle cells reacted positively to anti-smooth muscle
-actin but not to anti-von Willebrand factor or anti-endothelial NO
synthase.

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Fig. 1.
Western blots for characterizing cultured cells.
A, C,
and E: endothelial cells.
B, D,
and F: smooth muscle cells. Antibodies used were anti-von Willebrand factor
(A and
B), anti-endothelial nitric oxide
synthase (C and
D), and anti-smooth muscle -actin (E and
F). See
EXPERIMENTAL METHODS for further
details. The following were also used as positive controls but are not
shown here: human platelet lysate for von Willebrand factor, human
umbilical vein endothelial cultured cells for endothelial nitric oxide
synthase, and freshly isolated smooth muscle from pig coronary artery
for smooth muscle -actin.
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Characterization of SERCA pump.
Figure 2 shows the time course of the
ATP-dependent azide-insensitive oxalate-stimulated
Ca2+ uptake by cells that had been
cultured from endothelium and smooth muscle of pig coronary artery and
then permeabilized. The oxalate-stimulated uptake was linear for up to
30 min. In 30 min, the uptake in the absence of ATP or oxalate was
<10% of the total uptake. The value of the uptake observed was
consistently about threefold higher in the smooth muscle cells than in
the endothelial cells. In all subsequent experiments, the uptake was
examined over 30 min. Figure 3 shows the
effects of the sarcoplasmic reticulum
Ca2+ pump inhibitors thapsigargin
and CPA on the ATP-dependent, azide-insensitive, oxalate-stimulated
Ca2+ uptake by the permeabilized
cells (5, 11, 12, 22). Both agents inhibited the uptake nearly
completely, consistent with the uptake being due to the SERCA pumps.
Furthermore, consistent with the literature (11, 12), thapsigargin was
a more potent inhibitor than CPA (Fig. 3).

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Fig. 2.
Time course of Ca2+ uptake by
permeabilized cells. Cultured cells from smooth muscle and endothelium
were permeabilized with saponin and used for monitoring uptake of
45Ca2+
in the presence of ATP-regenerating system and sodium azide (to inhibit
uptake by mitochondria) with (+) and without ( ) 5 mM oxalate.
Samples were filtered after the specified intervals of time. Values are
means ± SE of 4 replicates. See EXPERIMENTAL METHODS for further details.
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Fig. 3.
Effects of cyclopiazonic acid (CPA) and thapsigargin.
A: smooth muscle cells.
B: endothelial cells. Permeabilized
cells were incubated with the ATP-regenerating system, 5 mM oxalate,
and 5 mM sodium azide for 30 min and then filtered. Data are from 2 experiments and represent means ± SE of 8-16 replicates. In each experiment, mean value of the uptake in the absence of CPA or
thapsigargin was taken as 100% and all values were expressed relative
to it. See EXPERIMENTAL METHODS for
further details.
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Effect of peroxide on SERCA pump.
Two assays of the Ca2+ pump were
used: transport of
45Ca2+
and Ca2+-dependent formation of
acylphosphates. For the first assay, the cells were permeabilized in
the absence of DTT because peroxide reacts instantaneously with this
reagent. The permeabilized cells were used for
Ca2+ uptake in the presence of
different concentrations of peroxide (Fig.
4). Although azide was included in these
assays to inhibit the mitochondrial
Ca2+ transport, it would also
inhibit any intrinsic catalase activities of these cells and hence
prevent a rapid degradation of peroxide. Peroxide decreased the
Ca2+ uptake in a
concentration-dependent manner. At low concentrations of peroxide,
however, the inhibition due to peroxide was significantly (P < 0.05) less in the endothelial
cells than in the smooth muscle cells (Fig. 4).

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Fig. 4.
Effect of adding peroxide to Ca2+
uptake reaction mixture on Ca2+
transport. For this experiment, cells were permeabilized in the absence
of dithiothreitol. Permeabilized cells were used for
Ca2+ uptake in the presence of
specified concentrations of peroxide, the ATP-regenerating system, 5 mM
oxalate, and 5 mM sodium azide. After 30 min, uptake reaction was ended
by filtration. Data are from 2 experiments and represent means ± SE
of 12 replicates. In each experiment, mean value of the uptake in the
absence of peroxide was taken as 100% and all values were expressed
relative to it. See EXPERIMENTAL
METHODS for further details.
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SERCA pumps bind Ca2+ and
[
-32P]ATP, forming
32P-labeled acylphosphates of
110-115 kDa that are acid stable. Permeabilized cells gave a very
faint band compared with the background, and, hence, purified
microsomes were used in this experiment. Microsomes prepared from the
cultured cells were treated with different concentrations of peroxide
and then used for the acylphosphate experiments. Only one major band
was seen in autoradiograms of the acylphosphate gels. In the presence
of added EGTA instead of CaCl2, no
bands were observed, indicating that the acylphosphate formation was Ca2+ dependent. Figure
5 shows that the intensities of these bands decreased when microsomes from the cells treated with peroxide were
used. Again, in this assay, the
Ca2+ pump was significantly
(P < 0.05) more sensitive to
peroxide in smooth muscle cells [50% inhibitory concentration
(IC50) = 97 ± 36 µM] than in endothelial cells
(IC50 = 704 ± 112 µM).

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Fig. 5.
Effect of treating microsomes with peroxide on acylphosphate formation.
Microsomes were incubated in peroxide and then used to examine the
acylphosphate formation as described in EXPERIMENTAL METHODS. A: each lane
showed only one major band at 110-115 kDa. In some gels, molecular
mass of this band was established with the cardiac sarcoplasmic
reticulum acylphosphates. Under these experimental conditions,
mobilities were similar for the acylphosphates formed from the cardiac
sarcoplasmic reticulum and the microsomes of smooth muscle and
endothelial cells. Optical densities of the acylphosphate bands from
gels as the ones shown were monitored. Within each gel, control
(without peroxide) was carried out in duplicate. Mean intensity of
control lanes was taken as 100%. For
B, data are means ± SE of 4 gels
for each cell type. See EXPERIMENTAL METHODS for detailed protocols.
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In the subsequent experiments, we examined the effects of preincubating
intact cells with peroxide and washing them before permeabilizing them
to examine the Ca2+ uptake. In
these experiments, azide was excluded from the initial incubation steps
but included in the Ca2+ uptake
solution to prevent the mitochondrial
Ca2+ uptake. In one experiment, we
incubated the cells at 37°C with 50 µM peroxide for different
lengths of time; then we washed and permeabilized these cells and
examined the ATP-dependent, azide-insensitive, oxalate-stimulated
Ca2+ uptake. The experimental
protocol was such that peroxide was present in the incubation mixtures
only for the specified length of time, but all the cells were incubated
at 37°C for 60 min, and also that peroxide was not present in the
Ca2+ uptake solution. There was a
decline in the Ca2+ uptake with
the preincubation time with peroxide, but this decline was much slower
in the endothelial cells than in the smooth muscle cells (Fig.
6).

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Fig. 6.
Effect of pretreating intact cells with 50 µM peroxide for different
times before permeabilization and
Ca2+ uptake. Cells were
trypsinized and then incubated in the presence of 50 µM peroxide for
the specified times at 37°C. Cells were then centrifuged, washed,
permeabilized, and used for the uptake reaction. Uptake was monitored
in the presence of the ATP-regenerating system, 5 mM oxalate, and 5 mM
sodium azide for 30 min. Data are from 1 experiment and represent means ± SE of 6 replicates. Mean value of the uptake in the absence of
peroxide was taken as 100% for each cell type, and all values were
expressed relative to it.
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In yet another set of experiments, we preincubated the cells with
different concentrations of peroxide at 37°C for 30 min, washed the
cells, permeabilized them, and examined the
Ca2+ uptake. As shown in Fig.
7, the oxalate-stimulated
Ca2+ uptake by the permeabilized
smooth muscle cells decreased with increasing concentrations of
peroxide (IC50 = 43 ± 10 µM). However, the endothelial cells were significantly
(P < 0.05) more resistant to the
peroxide treatment (IC50 = 2,215 ± 537 µM).

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Fig. 7.
Effect of pretreating intact cells with different concentrations of
peroxide before permeabilization and
Ca2+ uptake. Cells were
trypsinized and then incubated in the presence of different
concentrations of peroxide for 30 min at 37°C. Cells were then
centrifuged, washed, permeabilized, and used for the uptake reaction.
Uptake was monitored in the presence of the ATP-regenerating system, 5 mM oxalate, and 5 mM sodium azide for 30 min. Data are from 3 experiments and represent means ± SE of 12-24 replicates. In
each experiment, mean value of the uptake in the absence of peroxide
was taken as 100% and all values were expressed relative to it.
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Effect of peroxide on CPA-induced
Ca2+ increase.
Figure 8 contains representative tracings
showing basal
[Ca2+]i
levels, measured using the fluorescence dye fluo 3, and the effects of
inhibiting the SERCA pumps with CPA (11, 23). In the
Ca2+-free solution, smooth muscle
cells had a basal
[Ca2+]i
of 110 ± 16 nM and adding 10 µM CPA increased it by 152 ± 17 nM. As reported previously, pretreating the cells with peroxide produced a decrease in basal
[Ca2+]i,
and it inhibited the CPA-induced increase in
[Ca2+]i;
300 µM peroxide produced 77 ± 2% inhibition (Fig. 8).
Endothelial cells showed basal
[Ca2+]i
of 67 ± 9 nM, and CPA produced an increase of 76 ± 15 nM. In endothelial cells pretreated with peroxide, the basal
[Ca2+]i
(60 ± 10 nM) and the CPA-induced increase (75 ± 12 nM) in
[Ca2+]i
did not differ significantly from the control
(P > 0.05). Thus the peroxide
pretreatment inhibited the effect of CPA on the smooth muscle cells but
not on the endothelial cells. This result is consistent with the
Ca2+ pump being more resistant to
peroxide in the endothelial cells than in the smooth muscle cells.

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Fig. 8.
Effect of peroxide on CPA-induced increase in intracellular
Ca2+ concentration
([Ca2+]i).
Cultured cells were treated with 300 µM peroxide, washed, loaded with
fluo 3-acetoxymethyl ester and, for monitoring
[Ca2+]i,
placed in Ca2+-free solution.
A: representative tracings. Arrow
indicates the time of addition of 10 µM CPA. For
B, values are means ± SE of 18 replicates from smooth muscle cells and 9 from endothelial cells, each
carried out in at least 2 different experiments. See EXPERIMENTAL METHODS for detailed
protocols.
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Effect of peroxide on CPA-induced contraction and relaxation.
Inhibiting the SERCA pump with CPA produced an increase in
[Ca2+]i
in smooth muscle and endothelial cells. However, the increase in
[Ca2+]i
in the two cell types had different effects on contractility. In
deendothelialized arteries, CPA produced a contraction that was 36 ± 6% of that produced by 60 mM KCl (Fig.
9). CPA relaxed the arteries by 41 ± 4% when the endothelium was intact, and the arteries were
precontracted with 30 mM KCl (Fig. 9). The CPA-induced relaxation was
blocked by the NO synthase inhibitor, nitro-L-arginine methyl ester (L-NAME) (Table 1),
indicating that it involved the NO synthesis pathway.

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Fig. 9.
Effect of peroxide on CPA-induced contraction and relaxation of the
coronary artery rings. A: contraction
of deendothelialized arteries. Artery rings were contracted with 60 mM
KCl, washed, and then treated with 0 or 500 µM peroxide for 30 min
and then washed again. At this time, CPA was added to deendothelialized arteries to monitor the resulting contraction.
B: relaxation of arteries with intact
endothelium. Arteries with intact endothelium were contracted with 30 mM KCl. CPA was then added at the peak of the contraction, and
relaxation was monitored. Representative tracings are shown on
left. A summary (values are means ± SE) from several experiments is shown on
right.
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Peroxide, when added to deendothelialized arteries, produced a small
(5-10% of the contraction produced by KCl) transient contraction
lasting 5-10 min. Pretreating the deendothelialized arteries with
peroxide for 30 min, followed by washing them to remove the peroxide,
decreased the subsequent contractions by CPA with an
IC50 value of 97 ± 13 µM
(Fig. 9). Pretreating the deendothelialized rings with 500 µM
peroxide diminished this contraction to 4 ± 1% of the 60 mM
contraction (90% inhibition). The contraction produced by 30 mM KCl
was 93 ± 4% of the 60 mM KCl contraction. Treatment with 500 µM
peroxide decreased the contraction significantly (P < 0.05) but only marginally to 73 ± 4% (22% inhibition). Thus the peroxide treatment preferentially
inhibited the CPA contractions over the KCl contractions. Figure 9
shows a comparison of the effects of 500 µM peroxide on the
CPA-induced contractions in deendothelialized arteries and the
CPA-induced endothelium-dependent relaxation. Whereas the peroxide
pretreatment inhibited the CPA-induced contraction by 90%, it did not
significantly (P > 0.05) alter the
endothelium-dependent relaxation (Table 1).
We also examined the effects of peroxide on the action of peptide
hormones on the artery. Angiotensin II contracted the deendothelialized arteries, producing 22 ± 3% of the 60 mM KCl contraction.
Pretreatment with 500 µM peroxide diminished this contraction to 1.5 ± 0.7% of the 60 mM KCl contraction (93 ± 4% inhibition).
Bradykinin relaxed the precontracted arteries with intact endothelium
by 52 ± 5%. In arteries pretreated with 500 µM peroxide, the
corresponding relaxation was 28 ± 6% (40 ± 13% inhibition).
Thus peroxide inhibited the angiotensin II contraction in the
deendothelialized arteries significantly
(P < 0.05) more than it inhibited
the bradykinin relaxation in the arteries with intact endothelium
(Table 1).
 |
DISCUSSION |
In the RESULTS, we demonstrated that
the SERCA pump is more resistant to peroxide in the endothelial cells
than in smooth muscle. We also showed that the increase in
[Ca2+]i
produced by inhibiting the SERCA pump with CPA is more resistant to
peroxide in endothelium than in smooth muscle and that the CPA-induced
endothelium-dependent relaxation is more resistant to peroxide than the
CPA-induced contraction in deendothelialized artery rings. The
DISCUSSION will focus on an
interpretation of these observations, the limitations of the methods in
obtaining such interpretations, a comparison of these results with
those reported previously, and a working model based on these
observations and the literature.
ATP-dependent, oxalate-stimulated, azide-insensitive
45Ca2+
uptake by the saponin-permeabilized cells was used to demonstrate that
pretreating the cells with peroxide preferentially inhibited the SERCA
pump in smooth muscle cells over that in endothelial cells. The higher
potency of thapsigargin than that of CPA in inhibiting the
45Ca2+
uptake indicates that it is the SERCA pump that is inhibited (11),
since these agents do not affect other pumps. However, a SERCA pump has
also been reported to be present in the nuclear membrane, which is
present in the permeabilized cell preparation (20). In theory, the
SERCA pump activity can also be monitored as
Ca2+-Mg2+-ATPase
activity, but these measurements are not possible, since the smooth
muscle cells contain a very high level of
Mg2+-ATPase activity that
interferes (17). Therefore, we used the partial
Ca2+-Mg2+-ATPase
reaction of forming acylphosphates instead of
Ca2+-Mg2+-ATPase.
Peroxide preferentially inhibited the
Ca2+-dependent acylphosphate
formation in microsomes isolated from smooth muscle cells over those
from the endothelial cells. Because nuclei are discarded during the
preparation of the microsomal fraction, the preferentially inhibited
SERCA pump is unlikely to be nuclear. Furthermore, because both the
acylphosphate formation and the uptake were inhibited, the
possibilities that the observed results reflect only an effect of
peroxide on membrane permeability or an uncoupling of the pump are
ruled out. Because saponin was not used in the experiments with the
microsomes, the possibility is also ruled out that the differences
observed between the two cell types in the permeabilization experiments
are mainly due to differences in their interactions with saponin. The
observed effects of peroxide on the CPA-induced
[Ca2+]i
transients in Ca2+-free solution
are also consistent with the greater resistance of the SERCA pump in
endothelial cells. The possibility remained that all the transport and
the
[Ca2+]i
measurement studies were simply artifacts due to phenotypic expression
changes on culturing the cells. Therefore, it was empiric to carry out
the functional studies in the artery rings.
Peroxide inhibited the contractions by agents, such as angiotensin II
or CPA, whose actions involved intracellular
Ca2+ mobilization (16). Relaxation
experiments required precontracted tissues, and hence they could not be
carried out in a Ca2+-free
solution. The contraction due to extracellular
Ca2+ entry on membrane
depolarization with KCl can better withstand peroxide (16). Therefore,
although the results of the contractility experiments are consistent
with greater peroxide resistance of the SERCA pump in the endothelial
cells than in smooth muscle cells, this interpretation is not unique,
since a possible contribution of
Ca2+ entry from extracellular
space cannot be ruled out.
In this study, we examined the damage caused by pretreating the tissues
with peroxide and not reversed when they are washed. ROS may also
produce direct actions on the contractility assays. For instance, ROS
may react directly with NO (26). Our results on the damage remaining
after the removal of peroxide are consistent with the previous studies
in the literature (7, 8, 15, 16, 27). We and others have
shown that the SERCA pump in smooth muscle is inactivated by
ROS (7, 8, 15, 16, 27). However, this is the first study comparing
smooth muscle and endothelium or examining the effect of ROS directly
on the SERCA activity. The effect of
t-butyl-hydroxy-peroxide on
[Ca2+]i
has been reported for bovine aortic endothelial cells (8). Whereas
t-butyl-hydroxy-peroxide inhibited
bradykinin-stimulated Ca2+ influx
from extracellular space, it had little effect on the agonist-induced
release of Ca2+ from intracellular
stores. Our finding that peroxide only partially inhibited the
bradykinin relaxation is also consistent with this observation. The
SERCA pump inhibitors thapsigargin and
2,5-di-t-butylhydroquinone were shown
to produce an increase in
[Ca2+]i
in endothelial cells in Ca2+-free
solution, but they also caused a
Ca2+ entry when
Ca2+ was reintroduced to the
solution (7). ROS generated by
t-butyl-hydroxy-peroxide affected the
SERCA inhibitor-induced Ca2+ entry
only on prolonged exposure to ROS, but the
Ca2+ entry was affected more
readily. These results are also consistent with the resistance of the
endothelial SERCA pump to ROS. It has been reported that, in isolated
ischemic- and hypoxic-perfused hearts, the endothelium-dependent
relaxation was impaired before the endothelium-independent relaxation
(14, 28). These studies are consistent with ours, since the contractile
apparatus (as shown by contractions to KCl) is relatively resistant to
peroxide. It is the ability to contract by mobilizing intracellular
Ca2+ that is impaired in the
smooth muscle, and this was not examined in these studies. Furthermore,
it should be taken into consideration that the present study has been
carried out with only peroxide; the resistance to other ROS may be
different.
What is the cause of the greater resistance of SERCA pumps in
endothelium than in smooth muscle? Whereas different smooth muscles
express the splice variant SERCA2b coded by the gene
SERCA2, rat aortic endothelium has
been reported to express the gene
SERCA3 (1-3, 19, 31, 32). The
SERCA3 gene is also expressed in other
surface cells such as tracheal epithelium, mast cells, lymphoid cells,
and platelets (32). In microsomes from HEK293 cells overexpressing SERCA3, the IC50 value for
peroxide to inhibit the Ca2+ pump
is approximately three times higher than that for the cells overexpressing SERCA2b (18). Therefore, one of the possible reasons for
the differences in the sensitivity to peroxide between endothelial and
smooth muscle cells may be the expression of different SERCA isoforms.
However, there are some caveats with this interpretation. First, SERCA3
expression has not yet been demonstrated in the pig coronary artery
endothelium and it has been suggested that not all the vascular
endothelium may express SERCA3 (2). Second, the only evidence for the
existence of SERCA3 in the endothelium is in the in situ hybridization
studies (1, 2) and no other evidence has been presented, although it
has been stated in another study that vascular endothelium expresses
SERCA3 (31). The third and final caveat is that we observed a larger
difference in the resistance to peroxide between endothelium and smooth
muscle in the studies with intact cells or artery rings than in those
with permeabilized cells or microsomes. These caveats detract from the
simple interpretation that endothelium expresses SERCA3. Smooth muscle
expresses SERCA2b, and SERCA3 is more resistant to peroxide than
SERCA2b. Factors other than the differences in the SERCA isoforms may
also be involved. We propose a working model in which the resistance of
SERCA isoforms to ROS and the ability to scavenge ROS more efficiently
allow the endothelium to be better protected than the smooth muscle.
Because the ROS may be generated more readily in the arterial lumen,
these scavenging mechanisms may also allow the endothelium to act as a
protective barrier. It has been suggested that endothelium contains
mechanisms for scavenging ROS, such as catalase, superoxide dismutase,
glutathione, vitamin C, and vitamin E (4, 6, 21). Whereas this
observation is consistent with this working model, it must be pointed
out that just about every cell contains mechanisms to scavenge ROS, and
we do not know if endothelium is any better at it than smooth muscle.
 |
ACKNOWLEDGEMENTS |
We thank Dr. E. S. Werstiuk for reviewing the manuscript and C. M. Misquitta for assistance with some of the experiments.
 |
FOOTNOTES |
This work was supported by a Grant-in-Aid from the Heart and Stroke
Foundation of Ontario.
Address for reprint requests: A. K. Grover, Dept. of Biomedical
Sciences, HSC 4N41, McMaster Univ., 1200 Main St. West, Hamilton,
Ontario, Canada L8N 3Z5.
Received 6 January 1997; accepted in final form 19 June 1997.
 |
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