Mechanism of depression in cardiac sarcolemmal
Na+-K+-ATPase
by hypochlorous acid
Kiminori
Kato,
Qiming
Shao,
Vijayan
Elimban,
Anton
Lukas, and
Naranjan S.
Dhalla
Institute of Cardiovascular Sciences, St. Boniface General Hospital
Research Centre, and Department of Physiology, Faculty of Medicine,
University of Manitoba, Winnipeg, Manitoba, Canada R2H 2A6
 |
ABSTRACT |
Oxidative stress during pathological conditions
such as ischemia-reperfusion is known to promote the formation
of hypochlorous acid (HOCl) in the heart and to result in depression of
cardiac sarcolemmal (SL)
Na+-K+-ATPase
activity. In this study, we examined the direct effects of HOCl on SL
Na+-K+-ATPase
from porcine heart. HOCl decreased SL
Na+-K+-ATPase
activity in a concentration- and time-dependent manner. Characterization of
Na+-K+-ATPase
activity in the presence of different concentrations of MgATP revealed
a decrease in the maximal velocity
(Vmax) value, without a change in affinity for MgATP on treatment of SL membranes with 0.1 mM HOCl. The
Vmax value of
Na+-K+-ATPase,
when determined in the presence of different concentrations of
Na+, was also decreased, but
affinity for Na+ was increased
when treated with HOCl. Formation of acylphosphate by SL
Na+-K+-ATPase
was not affected by HOCl. Scatchard plot analysis of
[3H]ouabain binding
data indicated no significant change in the affinity or maximum binding
capacity value for ouabain binding following treatment of SL membranes
with HOCl. Western blot analysis of
Na+-K+-ATPase
subunits in HOCl-treated SL membranes showed a decrease (34 ± 9%
of control) in the
1-subunit
without any change in the
1- or
2-subunits. These data suggest
that the HOCl-induced decrease in SL
Na+-K+-ATPase
activity may be due to a depression in the
1-subunit of the enzyme.
oxidative stress; sarcolemmal
sodium-potassium-adenosinetriphosphatase; sarcolemmal ouabain binding; pig heart; sodium-potassium-adenosinetriphosphatase subunits
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INTRODUCTION |
ISCHEMIA-REPERFUSION IS KNOWN to produce free radicals
and oxidants (9, 13, 27, 28). Previously, we examined the effect of an
oxyradical-generating system (xanthine-xanthine oxidase) on the cardiac
sarcolemmal (SL)
Na+-K+-ATPase
(26) and found that oxyradical-induced depression of Na+-K+-ATPase
activity was due to a decrease in the maximal velocity of reaction
(Vmax) of the
enzyme, when measured in the presence of varying concentrations of
Na+ and MgATP. Furthermore, the
affinity of
Na+-K+-ATPase
for MgATP was decreased but the affinity for
Na+ was increased following the
treatment of SL membranes with xanthine plus xanthine oxidase (26).
Some reports indicate that hypochlorous acid (HOCl) decreases
Na+-K+-ATPase
activity (13, 17), but the mechanism by which HOCl alters this enzyme
is not clear. HOCl is one of the major oxidants formed by
polymorphonuclear leukocytes in the ischemic-reperfused areas of the
myocardium (13). The present study was therefore undertaken to examine
the direct effect of HOCl on cardiac SL Na+-K+-ATPase
to further clarify the mechanism of depression of this enzyme. Two agents, dithiothreitol (DTT) and
L-methionine, which possess antioxidant properties (4, 17, 29), were employed to
confirm that the changes due to HOCl were mediated through oxidative
stress.
The SL
Na+-K+-ATPase
is an important target of ischemia-reperfusion injury, since it
regulates the intracellular concentration of
Na+ and contributes to the resting
membrane potential in the myocardium (13, 17, 26, 27). Furthermore, a
depression of
Na+-K+-ATPase
may contribute to Ca2+ overload in
cardiomyocytes via
Na+/Ca2+
exchange during ischemia-reperfusion (14, 27). The
Na+-K+-ATPase
consists of
- and
-subunits (20, 30); the
-subunits determine
the catalytic activity of the enzyme, whereas the
-subunit is
necessary for functional integrity of the
Na+-K+-ATPase
(18, 24, 30). Recently, Huang et al. (8) reported that
2- and
3-subunits of
Na+-K+-ATPase
were more sensitive than
1-subunit to the effects of ischemia-reperfusion on ouabain-binding. This study tests that effect of HOCl on the
1- and
2-subunits of
Na+-K+-ATPase
in addition to investigating changes in the
-subunit of the enzyme.
 |
METHODS |
Preparation of cardiac SL membrane.
Porcine hearts were obtained from a slaughterhouse or were freshly
obtained in our animal holding facility. Hearts were cut into small
pieces and frozen (
70°C). SL membrane was isolated according
to the method of Pitts (23) as modified by Kaneko et al. (9). Marker
enzyme activities (2, 9, 21) revealed a 16- to 18-fold purification of
membranes with respect to
Na+-K+-ATPase
activity and minimal (2-4%) cross contamination with other subcellular organelles.
Either L-methionine (10 mM) or
DTT (1 mM) was used as an antioxidant for HOCl (29). The SL membrane
(0.4 mg/ml) was incubated separately with 0.1 mM HOCl in the absence
and presence of antioxidant for 10 min. HOCl was prepared by vacuum
distillation of sodium hypochlorite after adjusting the pH to 6.2 with
dilute sulfuric acid.
Measurement of
Na+-K+-ATPase
and K+-dependent
p-nitrophenylphosphatase activities.
Estimation of
Na+-K+-ATPase
activity was carried out as previously described (2). Briefly, SL
membranes (10 µg) were preincubated at 37°C with 1.0 mM EGTA
(Tris), pH 7.4, 5 mM NaN3, 6 mM
MgCl2, 100 mM NaCl, 10 mM KCl, 2.5 mM phosphoenolpyruvate (PEP), and 10 IU/ml pyruvate kinase. PEP and pyruvate were used as an
ATP-regenerating system to maintain the ATP concentration in the
incubation medium. The reaction was started by the addition of 0.025 ml
of 80 mM Na2ATP (pH 7.4) and
stopped after 10 min with 0.5 ml ice-cold 12% TCA. Liberated phosphate
was measured by the method of Taussky and Shorr (31). In experiments
using different concentrations of MgATP, the amounts of
Mg2+ and ATP required to achieve
the final concentration of MgATP were calculated using the SPECS
Fortran program developed by Fabiato (7). In parallel experiments,
either Na+ plus
K+ or
Mg2+ was omitted from the reaction
medium.
Na+-K+-ATPase
activity was calculated as the difference between activities with and
without Na+ plus
K+.
Mg2+-ATPase activity was estimated
as the difference between activities obtained with and without
Mg2+, in the absence of
Na+ plus
K+, in the medium.
The K+-dependent
p-nitrophenylphosphatase
(K+-pNPPase) activity was
determined at 37°C in 1.0 ml of reaction volume using a modified
method of Pierce et al. (21, 22). The assay medium contained 30 mM
imidazole-HCl, pH 7.8, 5 mM MgCl2,
1 mM EGTA, 20 mM KCl, and 10 µg SL membrane. The reaction was started
by adding 5 mM p-nitrophosphate and
stopped after 10 min by addition of 2 ml 1 N NaOH. The amount of
p-nitrophenol formed was measured at
410 nm.
Measurement of lipid peroxidation and
sulfhydryl group content. Lipid
peroxidation in the SL membrane was estimated from the malondialdehyde
(MDA) concentration by using the thiobarbituric acid method (28). Total
sulfhydryl (SH) groups in the SL were determined with DTNB (1).
Measurement of acylphosphate. A
modified method of Elmoselhi et al. (6) was used for the formation of
acylphosphate. Total reaction volume was 100 µl containing 1 mg/ml SL
membrane, 0.25 M sucrose, and 10 mM histidine, pH 7.0, with or without
0.1 mM HOCl, 10 mM L-methionine,
and/or 1 mM DTT according to each group. Solutions were
incubated at 37°C for 10 min and then immediately cooled to 4°C
to stop the reaction. Fifteen microliters of this solution were added
to 25 µl of a mixture containing (in mM) 10 NaCl, 30 imidazole-HCl
(pH 7.0), and 15 Na-EGTA (pH 7.0). The reaction was started with the
addition of
[
-32P]ATP (8 µCi/test tube; 3,000 Ci/mmol) and incubated at 0°C for 1 min. The
reaction was stopped by adding 250 µl of ice-cold TCA and 15 mM
phosphoric acid (TCAP), and the tubes were placed on ice for 5 min and
then centrifuged at 4°C for 10 min at 10,000 rpm. The pellets were
washed once with 1,250 µl TCAP and were suspended in 70 µl of
freshly prepared digestion buffer containing 10 mM MOPS (pH 5.5), 1 mM
EDTA, 3% SDS, 10% sucrose, 40 mM DTT, and 0.1% methylene green.
Samples were shaken vigorously on the vortex for 5-10 min and then
electrophoresed immediately in 7.5% polyacrylamide gels at pH 4. Electrophoresis was carried out at 10-12°C using a current of
20 mA/gel for 4 h. The gels were dried, autoradiographed, and exposed
overnight at
70°C to Kodak XAR-5 film backed by DuPont
lighting screen.
Determination of [3H]ouabain
binding.
Experiments were carried out according to a method previously described
(2). The SL membrane (1.0 mg/ml) with or without 0.1 mM HOCl treatment
was resuspended in 10 mM Tris · HCl (pH 7.5), and 50 µl of the suspension were transferred to the reaction mixture (0.5 ml
final vol). The reaction mixture contained 1.5 mM
MgCl2, 1.0 mM phosphate, 10 mM
Tris · HCl (pH 7.5, at 37°C), and 0.5-500 nM
[3H]ouabain (18.0 Ci/mmol, New England Nuclear) in the absence or presence of 2.0 mM
ouabain, a concentration sufficient to inhibit >95% of specific
[3H]ouabain binding.
SDS (9 µg/ml) was added to the incubation medium to permeabilize the
SL vesicles to ouabain. After 1 h at 37°C, the reaction was
terminated by filtration (pore size, 0.45 µm; Millipore, Bedford,
MA). Filters were washed three times with 2.5 ml ice-cold buffered
washing solution containing 50 mM Tris · HCl, pH 5.0, 0.1 mM ouabain, and 15.0 mM KCl, and the radioactivity of the filters
was counted. The nonspecific
[3H]ouabain binding
(in the presence of excess unlabeled ouabain) was subtracted from the
total binding (in the absence of unlabeled ouabain) to obtain the
specific binding of
[3H]ouabain.
Western blot analysis. Relative
Na+-K+-ATPase
content was measured by SDS-PAGE (15). The SL proteins were then
electroblotted to Immobilon-P transfer membrane (Millipore). Monoclonal
anti-
1-subunit of
Na+-K+-ATPase
mouse IgG (1:10,000), polyclonal
anti-
2-subunit rabbit IgG
(1:2,000), or polyclonal
anti-
1-subunit rabbit IgG
(1:2,000) from Upstate Biotechnology (Lake Placid, NY) were used to
detect subunits. The membranes were subsequently incubated for 1 h with biotinylated anti-mouse IgG (1:1,000) for
1-subunit and biotinylated anti-rabbit IgG antibody (1:3,000) for
2- and
1-subunits (Amersham, Arlington
Heights, IL). Finally, the membranes were incubated with
strepdavidin-conjugated horseradish peroxidase (1:5,000; Amersham) and
processed for chemiluminescence (ECL kit, Amersham) using Hyperfilm-ECL
(Amersham).
Na+-K+-ATPase
bands were analyzed using a model GS-670 imaging densitometer (Bio-Rad,
Mississauga, ON, Canada) with Image Analysis software version 1.0 and
expressed in relation to control values.
Statistical analysis. All values are
presented as means ± SE. Statistical analyses were conducted using
Student's t-test, one-way ANOVA, and
Scheffe's F test where appropriate. A
value of P < 0.05 was considered
significant. The Michaelis-Menten constant (Km), apparent
rate constant
(Ka),
and Vmax were
calculated using Lineweaver-Burk plots for the data of
Na+-K+-ATPase
activity in the presence of different concentrations of MgATP or
Na+. Estimates of kinetic
parameters [dissociation constant
(Kd) and
maximal density (Bmax)]
were obtained from Scatchard plot analysis in a
[3H]ouabain binding
study. The data on
[3H]ouabain binding
were analyzed according to the LIGAND computer program of McPherson
(19), which uses the F test for the
best fit.
 |
RESULTS |
Catalytic activity of
Na+-K+-ATPase.
Incubation of cardiac SL membranes with HOCl diminished the
Na+-K+-ATPase
activity with respect to concentration and time (Fig. 1). The data in Fig.
2 show the kinetic characteristics of
Na+-K+-ATPase
for MgATP, whereas Fig. 3 shows the
characteristics for Na+. The
results summarized in Table 1 indicate that
Vmax, when determined in the presence of varying concentrations of MgATP, was
decreased by HOCl, but
Km was not
changed significantly between control and HOCl-treated preparations. In
the kinetic study employing different concentrations of
Na+, both
Vmax and
Ka were
significantly decreased by HOCl. The data in Table
2 show that the HOCl-induced depression in
Na+-K+-ATPase
activity was partially prevented by 10 mM
L-methionine or 1 mM DTT. On the
other hand, the decrease in
K+-pNPPase activity by HOCl was
completely prevented by either 10 mM
L-methionine or 1 mM DTT.
L-Methionine or DTT alone did
not exert any effect on the
Na+-K+-ATPase
or K+-pNPPase activities (data not
shown). HOCl increased the SL MDA formation; both
L-methionine and DTT prevented
this effect (Table 2). HOCl decreased the SL SH group content and
L-methionine prevented this
effect (Table 2); DTT was not used in this experiment because it
interferes with the SH group content estimation.

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Fig. 1.
Effect of hypochlorous acid (HOCl) on porcine heart sarcolemmal
Na+-K+-ATPase
( ) and Mg2+-ATPase ( )
activities at different times of treatment with 0.1 mM HOCl
(A) and different concentrations of
HOCl (B). Each value is mean ± SE of 5 experiments. A:
* P < 0.05 compared with value
in 0 min incubation time. B:
* P < 0.05 compared with value
in 0 mM HOCl.
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Fig. 2.
Effect of 0.1 mM HOCl on porcine heart sarcolemmal
Na+-K+-ATPase
activities at different concentrations of MgATP. Each value is mean ± SE of 5 experiments. Inset:
Lineweaver-Burk plot of a representative experiment. , Control; ,
0.1 mM HOCl. * P < 0.05 compared with control.
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Fig. 3.
Effect of 0.1 mM HOCl on porcine heart sarcolemmal
Na+-K+-ATPase
activities at different concentrations of
Na+. Each value is mean ± SE
of 5 experiments. Inset:
Lineweaver-Burk plot of a representative experiment. , Control; ,
0.1 mM HOCl. * P < 0.05 compared with control.
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Table 1.
Kinetic characteristics of porcine cardiac sarcolemmal
Na+-K+-ATPase treated with or
without 0.1 mM HOCl
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Table 2.
Effect of 0.1 mM HOCl on porcine heart sarcolemmal
Na+-K+-ATPase,
K+-pNPPase, MDA content, and SH group content
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Acylphosphates and ouabain binding.
Figure 4 illustrates the results of
treatment with 0.1 mM HOCl on acylphosphate formation. These bands were
located at ~110 kDa. Treatment of SL membranes with 0.1 mM HOCl in
the absence and presence of 10 mM
L-methionine or 1 mM DTT did not
produce any significant effect on the formation of acylphosphate.
Figure 5 shows the effect of HOCl on
ouabain binding. As indicated, there are no changes in either
Bmax or Kd values between
control and 0.1 mM HOCl-treated preparation.

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Fig. 4.
Effect of HOCl (0.1 mM),
L-methionine (LM; 10 mM), and
dithiothreitol (DTT; 1 mM) on acylphosphate intermediate of porcine
heart sarcolemmal
Na+-K+-ATPase.
A representative autoradiograph is shown at
top.
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Fig. 5.
Scatchard plot analysis of specific
[3H]ouabain binding to
porcine heart sarcolemmal preparation. Values are taken from a typical
experiment in which 3 control and 3 HOCl treatment heart preparations
were employed. Inset: results of
dissociation constant
(Kd) and
maximum binding capacity (Bmax).
Each value is a mean of 3 experiments. , Control; , 0.1 mM
HOCl.
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Western blot analysis of
Na+-K+-ATPase
subunits.
The bands of both the
1- and
2-subunits of the SL
Na+-K+-ATPase
in our study are located at ~110 kDa (Fig.
6), which agrees with previous reports (see
review in Ref. 30). On the other hand, the
-subunit band was located
at ~55 kDa rather than at 35 kDa as reported by others (30). However,
Pedemonte and Kaplan (20) reported the band of the
Na+-K+-ATPase
-subunit to be at 55 kDa, which agrees with our results. Such a
difference was explained by the fact that the electrophoretic mobility
of the
-subunit was influenced largely by
N-linked carbohydrate groups (30).
Figure 7 shows that HOCl decreased the
density of
1-subunit of
Na+-K+-ATPase
and that L-methionine or DTT
significantly prevented this effect. On the other hand, HOCl had no
effect on both
1- and
2-subunits of the SL
Na+-K+-ATPase.

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Fig. 6.
Representative Western blots depicting the effect of treatment with
HOCl on expression of the 1-,
2-, and
1-subunits of porcine heart
sarcolemmal
Na+-K+-ATPase.
Lane 1, molecular mass markers (kDa);
lane 2, control; lane
3, 0.1 mM HOCl; lane
4, HOCl + 10 mM LM; lane
5, HOCl + 1 mM DTT. A band of nonspecific binding was
seen at ~75 kDa in all blots.
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Fig. 7.
Summary of Western blot analysis of subunit expression of porcine heart
sarcolemmal
Na+-K+-ATPase.
Bars, data obtained from 4 Western blots for each subunit. I, control;
II, 0.1 mM HOCl; III, HOCl + 10 mM LM; IV, HOCl + 1 mM DTT.
* P < 0.05 compared with
control.
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 |
DISCUSSION |
This study demonstrates that treatment of SL membranes with HOCl causes
a decrease in
Na+-K+-ATPase
activity and SH group content but an increase in MDA content. Other
studies (13, 17) also report a depression in cardiac Na+-K+-ATPase
by HOCl, which is a highly reactive oxidant (5, 25). Oxyradical
generation under in vitro conditions also decreases Na+-K+-ATPase
activity and SH group content of SL membranes but increases formation
of MDA (12, 26). It is likely that the observed depression in the SL
Na+-K+-ATPase
activity by HOCl may be due to a decrease in the SH group content of
the enzyme, since SH groups are important for the activity of
Na+-K+-ATPase
(12, 25). On the other hand, deleterious effects of different oxidants
and oxyradicals may reflect increased formation of lipid peroxides (8,
9, 12, 17, 25, 26), and thus it is possible that the HOCl-induced
depression may be due to the observed increase in the MDA content of
the SL membranes. Treatment of SL membranes with
L-methionine, which prevented
the HOCl-induced changes in MDA and SH group content due to its
antioxidant property, partially prevented (~70% protection) the
HOCl-induced depression in
Na+-K+-ATPase
activity. Furthermore, DTT, which prevents the oxidation of SH groups,
completely attenuated the HOCl-induced increase in MDA content and
partially prevented the HOCl-induced decrease in
Na+-K+-ATPase
activity (~80% protection). Thus both elevated levels of MDA and
depressed content of SH groups may be involved in the decreased
Na+-K+-ATPase
activity in HOCl-treated membranes. Inactivation of
Na+-K+-ATPase
by HOCl in cardiac membrane (13) and uncoupling of
Na+ pump by HOCl in coronary
artery (6) are also postulated mechanisms for the HOCl-induced changes
in
Na+-K+-ATPase.
The depression in SL
Na+-K+-ATPase
activity by HOCl was associated with a decrease in
Vmax of the
enzymatic reaction when determined in the presence of different
concentrations of MgATP or Na+.
Because HOCl-treated membranes exhibited a decrease in the activity of
K+-pNPPase and this effect was
prevented by L-methionine or
DTT, it is possible that the observed depression in
Na+-K+-ATPase
activity reflects a depressant effect of HOCl on the catalytic sites of
the enzyme. However, this may not be the case because the affinity for
MgATP (1/Km)
was unaltered and the affinity for Na+
(1/Ka) was in
fact increased on treatment of membranes with HOCl. Furthermore,
neither the binding of ouabain nor the formation of acylphosphate was
affected by HOCl. It should be mentioned that
Na+,
K+, ATP, and ouabain binding occur
at the catalytic sites represented by
-subunits of
Na+-K+-ATPase
(20, 30). Because no change in the content of the
1- and
2-subunits of the enzyme was
seen on HOCl treatment, the observed depression in
Na+-K+-ATPase
activity by HOCl may not be due to its effect on the catalytic sites of
the enzyme.
Unlike the
1- and
2-subunits of SL
Na+-K+-ATPase,
the content of
-subunit was decreased by HOCl treatment. This
observation suggests that the
-subunit of
Na+-K+-ATPase
may be more sensitive to HOCl than the
1- and
2-subunits of the enzyme.
Differential effects of various oxidants on
1-,
2- and
3-subunits of
Na+-K+-ATPase
have been observed by other investigators (8, 16, 32). Our lack of
effect of HOCl on the
1- and
2-subunits of the porcine heart
Na+-K+-ATPase
may be due to species differences (3) or to the techniques used in this
study. Nonetheless, it should be pointed out that the depressed
activity of porcine kidney
Na+-K+-ATPase
in hypothyroidism is associated with a decrease in the abundance of
-subunit without any change in the
-subunit (18). Furthermore,
inactivation of
Na+-K+-ATPase
by a high concentration of 2-mercaptoethanol at high temperature is
also associated with deterioration of the
-subunit without any
effect on the
-subunits (10). The observed effect of HOCl on the
-subunit of
Na+-K+-ATPase,
unlike the
-subunits, may be due to the presence of SH
groups and disulfide bonds in the
-subunit (11). Earlier studies
have shown that the
-subunit is required for the enzymatic activity
of
Na+-K+-ATPase
because it cannot be separated from the
-subunits without an
irreversible loss of enzyme function (18, 30). It should also be noted
that SL
Na+-K+-ATPase
activity is decreased on incubation with antibody for the
-subunit
(24). Thus it appears that the observed depression in SL
Na+-K+-ATPase
activity by HOCl may be mainly due to the malfunction of
-subunit of
the enzyme.
 |
ACKNOWLEDGEMENTS |
This work was supported by a grant from the Medical Research
Council (MRC) of Canada (MRC Group in Experimental Cardiology).
 |
FOOTNOTES |
K. Kato was a postdoctoral fellow of the Heart and Stroke Foundation of
Canada, and A. Lukas was the Myles Robinson Heart Scholar during the
tenure of this study.
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: N. S. Dhalla, Institute of Cardiovascular
Sciences, St. Boniface Hospital Research Centre, 351 Tache Ave.,
Winnipeg, Manitoba, Canada R2H 2A6.
Received 19 March 1998; accepted in final form 8 June 1998.
 |
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