| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 27, 19323-19328, July 2, 1999
From the Departments of Pharmacology and Medicine, Medical College
of Ohio, Toledo, Ohio 43614
We showed before that in cardiac myocytes partial
inhibition of Na+/K+-ATPase by nontoxic
concentrations of ouabain causes hypertrophy and transcriptional
regulations of growth-related marker genes through multiple
Ca2+-dependent signal pathways many of which
involve Ras and p42/44 mitogen-activated protein kinases. The aim of
this work was to explore the roles of intracellular reactive oxygen
species (ROS) in these ouabain-initiated pathways. Ouabain caused a
rapid generation of ROS within the myocytes that was prevented by
preexposure of cells to N-acetylcysteine (NAC) or vitamin
E. These antioxidants also blocked or attenuated the following actions
of ouabain: inductions of the genes of skeletal Na+/K+-ATPase (sodium pump) catalyzes the
active transport of Na+ and K+ across the
plasma membranes of most mammalian cells (1, 2). In the heart, partial
inhibition of Na+/K+-ATPase by ouabain and
related cardiac glycosides causes a modest increase in intracellular
Na+, which in turn leads to significant increases in
intracellular Ca2+ through the
Na+/Ca2+ exchanger and in myocardial
contractility through the effects of Ca2+ on contractile
proteins (3). This effect on cardiac contractility is the basis of the
continued therapeutic use of these drugs in the management of
congestive heart failure (4, 5). Recently, we demonstrated that in
cultured cardiac myocytes the same nontoxic concentrations of ouabain
that cause partial inhibition of the sodium pump and increase
intracellular Ca2+ also stimulate hypertrophic growth and
transcriptionally regulate early- and late-response genes that are
markers of cardiac hypertrophy (6-8). This clearly indicated that the
altered activity of cardiac Na+/K+-ATPase by
cardiac glycoside drugs or by endogenous ouabain-like putative hormones
(9) must be considered as a potential signal for cardiac hypertrophy
and its transition to failure, along with other hormonal, mechanical,
and pathological stimuli of hypertrophy (10). While exploring the
mechanism of linkage between the plasma membrane sodium pump and four
marker genes (c-fos and the genes of
skACT,1 ANF, and the
ROS, such as the superoxide radical, the hydroxyl radical, and
H2O2, are continuously produced in most cells,
and their levels are regulated by a number of enzymes and physiological
antioxidants (12). It has been known for some time that excessive
generation of ROS is associated with cell injury in a variety of
pathological conditions including those of the cardiovascular system
(12). More recently, there has been rapidly growing evidence indicating that ROS also have normal roles as second messengers within several signal pathways involved in the control of gene transcription (13-15).
Because Ras has been shown to be implicated in a number of such
ROS-regulated pathways (15-18) and in view of our findings on the
central role of Ras in ouabain-induced regulation of the growth-related
genes of cardiac myocytes (11), we were prompted to determine the
possible involvement of ROS in these ouabain effects. Here we present
the results of our initial studies showing that intracellular ROS are
indeed essential mediators of the ouabain-induced hypertrophic
phenotype and that antioxidants are capable of preventing the
growth-related effects of ouabain in cultured cardiac myocytes.
Materials--
Chemicals of the highest purity available were
obtained from Sigma. TRI reagent for RNA isolation was from Molecular
Research Center, Inc. (Cincinnati, OH). Radionucleotides
(32P-labeled, about 3000 Ci/mmol) and
86Rb+ were obtained from NEN Life Science
Products. [3H]Phenylalanine and the ECL kit were from
Amersham Pharmacia Biotech. Rabbit polyclonal Anti-ACTIVE MAPK pAb and
anti-p42/44 antibodies were obtained from Promega (Madison, WI) and New
England Biolabs (Beverly, MA), respectively. Fura-2 AM and CM-DCFH
diacetate were obtained from Molecular Probes (Eugene, OR). Optitran
and Nytran membranes were obtained from Schleicher and Schuell.
Cell Preparation and Culture--
Neonatal ventricular myocytes
were prepared and cultured as described in our previous work (6).
Briefly, myocytes were isolated from ventricles of 1-day-old
Sprague-Dawley rats and purified by centrifugation on Percoll
gradients. Myocytes were then cultured in a medium containing 4 parts
of Dulbecco's modified Eagle's medium and 1 part of Medium 199 (Life
Technologies, Inc.), penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% fetal bovine serum. After 24 h of incubation at
37 °C in humidified air with 5% CO2, medium was changed
to one with the same composition as above but without the serum. All
experiments were done after 48 h of further incubation under
serum-free conditions. These cultures contain more than 95% myocytes
as assessed by immunofluorescence staining with a myosin heavy chain antibody.
Fluorescence Microscopic Measurements of Intracellular
Ca2+ Concentration and ROS Production--
Myocytes were
cultured on glass coverslips. Intracellular Ca2+ was
measured by fura-2 as we previously described (6). Fura-2 fluorescence
was recorded using an Attofluor imaging system (Atto Instruments) at an
excitation wavelength of 340/380 nm and at an emission wavelength of
505 nm. Under each experimental condition time-averaged signals were
obtained from about 40 single cells. Relative Ca2+
concentration was calculated based on the fluorescence ratio and
Ca2+ calibration curve (6). To measure intracellular ROS
production, cells were loaded with 10 µM CM-DCFH
diacetate for 15 min at room temperature in the dark. The coverslip was
affixed to a culture chamber and perfused with the same culture medium
without phenol red. During loading the acetate groups on CM-DCFH
diacetate are removed by intracellular esterase, trapping the probe
inside the myocytes. Several dihydrofluorescein derivatives have been
used for measuring intracellular ROS generation (19-21). CM-DCFH was chosen because it exhibited better retention in cells than other derivatives. Production of ROS was measured by changes in fluorescence because oxidation of CM-DCFH produced fluorescent product CM-DCF in
cardiac myocytes. Under each experimental condition about 15 single
myocytes were imaged with an Attofluor imaging system (Atto Instruments), and CM-DCF fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.
Northern Blot--
Northern blot was done as described
previously (6-8). Routinely, about 20 µg of total RNA was subjected
to gel electrophoresis, transferred to a Nytran membrane,
UV-immobilized, and hybridized to 32P-labeled probes.
Autoradiograms obtained at Measurement of Phosphorylation of p42/44 MAPKs--
Activation
of p42/44 MAPKs in cultured myocytes was determined by Western blot
using a rabbit polyclonal antibody raised against dually phosphorylated
p42/44 MAPKs (11). In brief, after cells were exposed to ouabain,
reaction was terminated by the replacement of medium with 200 µl of
ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM
Na3VO4, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 50 mM tetrasodium
pyrophosphate, 10 nM okadaic acid, 1% Triton X-100, 0.25%
sodium deoxycholate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin).
For Western blot analysis, cell lysates (60 µg/lane) were
electrophoresed on 10% SDS-polyacrylamide gels and transferred to an
Optitran membrane. The membranes were probed with Anti-ACTIVE MAPK pAb
that detects p42/44 MAPKs only when they are activated by
phosphorylation at Thr202 and Tyr204. To ensure
equal loading and protein transfer, the same blots were stripped and
probed with a polyclonal antibody recognizing both phosphorylated and
nonphosphorylated p42/44 MAPKs. These membranes were developed with
a secondary anti-rabbit antibody using the ECL method as instructed by
the manufacturer (Amersham Pharmacia Biotech). Autoradiograms were
scanned with a Bio-Rad densitometer to quantitate MAPK signals.
Assay of AP-1 Activation--
A CAT reporter gene under the
control of three copies of the AP-1 cis-element (22) was
kindly provided to us by Dr. P. A. Baeuerle. Myocytes were transfected
with 5 µg of plasmid DNA using a modified calcium phosphate method
(23). After 48 h of incubation, the transfected cells were
subjected to ouabain and other treatments. Cell lysate was prepared and
assayed for CAT as described previously (24). All transfections were
conducted in triplicate in 6.0-cm tissue culture dishes. Control
experiments with Rous sarcoma virus· Detection of NF- Preparation of Replication-defective Adenovirus Asn17
Ras and Infection of Myocytes--
The adenovirus expressing the
dominant-negative Ras was generated, amplified, and purified as
described before (11). An identical virus containing the
Protein Synthesis--
Protein synthesis in these cultured
myocytes was assayed by [3H]phenylalanine incorporation
as we previously described (7). Cells were cultured in a 12-well plate,
treated with ouabain and other agents as indicated for 44 h, and
continued for an additional 4 h in the presence of 0.5 µCi of
[3H]phenylalanine/well before total protein and
phenylalanine incorporation were assayed as indicated (7).
Assay of 86Rb+ Uptake by
Myocytes--
The initial rate of ouabain-sensitive uptake of
Rb+ through the Na+/K+-ATPase of
the intact myocytes was measured with modification of our previously
described procedures (6). Monensin (20 µM) was added to
the medium prior to the initiation of the Rb+ uptake assay
to assure that the maximal capacity of active uptake was measured
(25).
Statistics--
Data are given as mean ± S.E. Statistical
analysis was performed using the Student's t test, and
significance was accepted at p < 0.05.
Ouabain Increases Intracellular ROS in Cardiac Myocytes--
To
determine if ouabain increases ROS production in cardiac myocytes,
cells were incubated with 10 µM CM-DCFH diacetate for 15 min at room temperature in the dark, washed with fresh medium, exposed
to ouabain, and monitored for the production of the fluorescent CM-DCF.
As shown in Fig. 1, ouabain caused dose-
and time-dependent increases in fluorescence, indicating
stimulated ROS production in these cultured myocytes. The highest
ouabain concentration used was 100 µM to avoid loss of
viable cells (6). As expected, myocytes loaded with the fluorogenic
probe also exhibited increased fluorescence when exposed to
H2O2 (data not shown). The maximal fluorescence
increase obtained with ouabain in experiments of Fig. 1 was about the
same as that produced by the addition of 2 µM
H2O2. Preincubation of myocytes with the
antioxidant NAC (10 mM) suppressed the fluorescence
increases induced by ouabain (Fig. 1) and by
H2O2 (not shown). Preincubation with 0.5 mM Effects of Antioxidants on the Gene Regulatory Effects of
Ouabain--
The experiments shown in Figs.
2-6 were done to assess the relation of
the ouabain-induced generation of ROS to the regulations of the four
genes that we had previously shown to be transcriptionally regulated by
ouabain (6-8, 11). The results showed that NAC blocked, in a
dose-dependent manner, the ouabain-induced increases in
skACT and ANF mRNAs (Figs. 2 and 3)
and the ouabain-induced decrease in mRNA of the
We showed before that induction of c-fos by ouabain is
accompanied by an increase in the transcription factor AP-1, which has
been implicated in the regulation of cardiac growth-related genes (6).
In view of the lack of effect of NAC on the induction of
c-fos by ouabain (Fig. 6) but the known activation of
cardiac myocyte AP-1 by exogenous H2O2 (35), in
the experiments shown in Fig. 7 we
examined the effects of ouabain and NAC on myocytes that were
transfected with a CAT gene construct containing three AP-1 elements.
Ouabain-induced increase in CAT expression was also unaffected by NAC
(Fig. 7), providing further support for the conclusion that some gene
regulatory effects of ouabain are through ROS-independent pathways.
NF-
In control experiments, the results of which are not shown, it was
established that antioxidants, at the concentrations and for the
durations used in the experiments shown in Figs. 2-8, had no
significant effects on myocyte viability as measured by lactate dehydrogenase release (6).
The Roles of Ras and ROS in Ouabain Stimulation of Protein
Synthesis--
We showed before that nontoxic concentrations of
ouabain cause moderate hypertrophy of the myocytes as evidenced by
ouabain-induced increases in total cell protein (6) and in the rate of
[3H]phenylalanine incorporation into cell proteins (7).
Because we had shown the involvement of Ras in several signal pathways activated by ouabain (11), it was of interest to know if Ras was also
involved in the effect of ouabain on protein synthesis. Experiments
depicted in Fig. 9 showed that the
adenoviral-mediated expression of a dominant-negative Ras blocked
ouabain stimulation of [3H]phenylalanine incorporation
into myocyte proteins. Experiments of Fig.
10 showed that ouabain stimulation of
the protein synthesis was also blocked by NAC. The combined data of
Figs. 9 and 10 indicate that ouabain-induced hypertrophy, like
ouabain-induced regulations of the late-response genes (skACT, ANF, and
Different Roles of ROS in Ouabain-induced and PMA-induced Effects
on Growth-related Genes and on Protein Synthesis--
PMA is a well
established hypertrophic stimulus for the cultured neonatal myocytes
(6, 7, 26). Under the same conditions where ouabain-induced effects on
ANF and Effect of NAC on Ouabain-induced Activation of p42/44
MAPKs--
The antagonistic effects of antioxidants on ouabain-induced
actions, as noted in Figs. 2-10, occurred in experiments with
durations of several hours. On the other hand, the rapid onset of the
ouabain effect on the intracellular production of CM-DCF (Fig. 1)
suggested that increased generation of ROS may represent an early event in ouabain-initiated signal transduction pathways. Because the maximal
activation of the Ras-Raf-MEK-p42/44 MAPK cascade occurs within 5-15
min after exposure to ouabain (11), it was of interest to examine the
effect of NAC on this rapid action of ouabain. Experiments depicted in
Fig. 12 showed that ouabain activation of p42 MAPK was indeed attenuated by NAC.
NAC and Ouabain Effects on 86Rb+ Uptake and
on Intracellular Ca2+--
Because all of the hypertrophic
and gene regulatory effects of ouabain are dependent on ouabain-induced
inhibition of the sodium pump and the resulting rapid rise in
intracellular Ca2+ (6-8, 11), we examined the combined
effects of NAC and ouabain on the transport function of the intact
myocyte Na+/K+-ATPase as measured by
86Rb+ uptake, and on intracellular
Ca2+ concentrations of the cultured myocytes. In
experiments similar to those we have reported before (6), neither the
maximal inhibitory effect of 1 mM ouabain nor the partial
inhibitory effect of 100 µM ouabain on the initial rate
of 86Rb+ uptake was prevented or reduced by
preincubation of myocytes with 10 mM NAC for 15 min (data
not shown). In agreement with our previous results (6), 100 µM ouabain increased intracellular Ca2+
concentration from 89 ± 18 nM to 167 ± 24 nM. In myocytes that were pretreated with 10 mM
NAC, 100 µM ouabain raised Ca2+ concentration
from 104 ± 25 nM to 191 ± 34 nM.
The fact that ROS are second messengers within a variety of
intracellular signal transduction pathways has been indicated by a
large number of studies in cells other than cardiac myocytes (13-18).
The signaling roles of intracellularly generated ROS in cardiac
myocytes have been suggested by previous studies when myocytes were
subjected to hypoxia or hypoxia reoxygenation (21, 27, 28) and when
myocytes were exposed to tumor necrosis factor- Of particular interest are our findings on the different roles of ROS
in the ouabain-induced regulations of the four growth-related genes.
Although the regulations of three genes (those of skACT, ANF, and the
The nature and the source of the ouabain-induced ROS remain to be
determined. Although available data suggest that the fluorogenic compound used here for the detection of ROS is more sensitive to
H2O2 and the hydroxyl radical than to the
superoxide radical (21), the insufficient selectivity of the probe and
the interconversions of the generated ROS do not permit firm
conclusions on the nature of the species that act as second messengers.
In cells other than cardiac myocytes, mitochondria and NADH·NADPH
oxidase complexes, which include Rac as a component, have been
implicated as sources of signaling ROS (15-17). In cardiac myocytes,
mitochondria seem to be the source of ROS involved in hypoxia-induced
signaling (30). The existence of a cytosolic NADH oxidase system
responsible for the production of superoxide in cardiac myocytes has
also been suggested (31), and the presence of Rac in cardiac myocytes has been shown (32, 33). Because we have shown Ras activation to be
involved in all ouabain-initiated pathways (11) and because others have
implicated a signal cascade involving Ras-Rac in the development of
cardiac myocyte hypertrophy (33), it is reasonable to suspect that a
source of ouabain-induced ROS generation may indeed be a
Ras-Rac-NAD(P)H oxidase cascade. Our studies on the relative roles of
NAD(P)H complexes and mitochondria as sources of ouabain-induced ROS
are in progress.
In the ouabain-initiated signal pathways, as in most ROS-regulated
pathways identified to date, the immediate targets of ROS effects are
not known. Our data showing that ouabain-induced ROS production is
antagonized by NAC and that ouabain-induced inhibition of
Na+/K+-ATPase and the resulting rise in
intracellular Ca2+ persist in the presence of NAC clearly
show that the generated ROS must affect the ouabain-initiated pathways
beyond the point where increased intracellular Ca2+ affects
all pathways (11). One such potential target is Ras, because direct
activation of Ras by ROS has been indicated by studies on cells other
than myocytes (18). This, coupled with the likelihood of the
Ras-Rac-oxidase cascade being the source of the generated ROS, suggests
the possible existence of a signal amplification cycle within the
ouabain-initiated and ROS-regulated pathways. AP-1 and NF- Perhaps the most intriguing of our present findings is that in the
cultured myocytes it is possible to dissociate the effects of ouabain
on growth and growth-related genes from the classical effect of the
drug on the resting intracellular Ca2+. Because the latter
is thought to be responsible for the effects of ouabain and related
cardiac glycosides on cardiac contractility (3), this raises the
important question of whether or not the redox state of the myocyte or
the intact heart can alter the effects of the cardiac glycosides on
cardiac hypertrophy without affecting the positive inotropic effects of
the drug. As discussed before (6), in the context of the use of these
drugs in the management of heart failure, at this time it is not
possible to say if the hypertrophic and the gene regulatory effects of
the drugs will turn out to be among their beneficial or undesirable effects.
*
This work was supported by NHLBI National Institutes of
Health Grant HL-36573, by a grant-in-aid from the American Heart
Association, and by funds contributed in part by the American Heart
Association, Ohio-West Virginia Affiliate.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. Section
1734 solely to indicate this fact.
The abbreviations used are:
skACT, skeletal
Intracellular Reactive Oxygen Species Mediate the Linkage of
Na+/K+-ATPase to Hypertrophy and Its Marker
Genes in Cardiac Myocytes*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actin and atrial
natriuretic factor, repression of the gene of the
3-subunit of Na+/K+-ATPase,
activation of mitogen-activated protein kinases, activation of
Ras-dependent protein synthesis, and activation of
transcription factor NF-
B. Induction of c-fos and
activation of AP-1 by ouabain were not sensitive to NAC.
Ouabain-induced inhibition of active Rb+ uptake through
Na+/K+-ATPase and the resulting rise in
intracellular Ca2+ were also not prevented by NAC. A
phorbol ester that also causes myocyte hypertrophy did not increase ROS
generation, and its effects on marker genes and protein synthesis were
not affected by NAC. We conclude the following: (a) ROS are
essential second messengers within some but not all signal pathways
that are activated by the effect of ouabain on
Na+/K+-ATPase; (b) the
ROS-dependent pathways are involved in ouabain-induced hypertrophy; (c) increased ROS generation is not a common
response of the myocyte to all hypertrophic stimuli; and
(d) it may be possible to dissociate the positive inotropic
effect of ouabain from its growth-related effects by alteration of the
redox state of the cardiac myocyte.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 subunit of Na+/K+-ATPase), we
also found that ouabain interaction with the pump initiates multiple
signal transduction cascades and that many of these gene regulatory
pathways involve activations of the GTP-binding protein Ras and p42/44
MAPKs (11).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C were scanned with a Bio-Rad
densitometer. Multiple exposures were analyzed to assure that the
signals are within the linear range of the film. The relative amount of
RNA in each sample was normalized to that of glyceraldehyde-3-phosphate
dehydrogenase mRNA to correct for differences in sample loading and transfer.
-galactosidase reporter
plasmid indicated that transfection efficiency varied less than 15%
within a given experiment under our experimental conditions.
B by Immunofluorescence--
This was done as
described before (35) using an antibody against the p65 subunit of
NF-B (Santa Cruz Biotechnology).
-galactosidase gene was used as virus control. Myocytes were
transduced by the same experimental procedures used before (11). Under
these conditions viral infection had no effect on cell viability
(11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tocopherol (vitamin E) had about the same effect as
that of NAC shown in Fig. 1.
View larger version (30K):
[in a new window]
Fig. 1.
Time courses of the effects of varying
ouabain concentrations on intracellular ROS production and the
prevention of these effects by NAC. Cardiac myocytes were loaded
with the fluorogenic probe, exposed to ouabain (Ou), and
assayed for the fluorescence of the oxidized probe as indicated under
"Experimental Procedures." When NAC was used, cells were incubated
with it for 30 min prior to the addition of ouabain. Each value is the
mean ± S.E. of determinations on 40-60 cells in four independent
experiments.
3-subunit of Na+/K+-ATPase (Fig.
4). Experiments depicted in Fig.
5 showed that vitamin E and NAC similarly
antagonized the effects of ouabain on skACT, ANF, and
3-subunit genes. In contrast to the antioxidant effects on these late-response genes, induction of the early-response gene
c-fos by ouabain was not affected by either NAC (Fig.
6) or vitamin E (not shown). The combined
data of Figs. 2-6 clearly indicate that ouabain initiates
ROS-dependent and ROS-independent gene regulatory pathways
in these myocytes.
View larger version (28K):
[in a new window]
Fig. 2.
Effects of NAC on ouabain-induced expression
of the skACT gene. Cells were pretreated with NAC for 15 min,
exposed to ouabain (Ou) for 12 h, and assayed for
mRNAs of skACT and glyceraldehyde-3-phosphate dehydrogenase as
described under "Experimental Procedures." Values, normalized to
those of corresponding glyceraldehyde-3-phosphate dehydrogenase, are
mean ± S.E. of five experiments.
View larger version (32K):
[in a new window]
Fig. 3.
Effects of NAC on ouabain-induced expression
of the ANF gene. Experiments were done as in Fig. 2.
Ou, ouabain.
View larger version (38K):
[in a new window]
Fig. 4.
Effects of NAC on ouabain-induced
down-regulation of the gene of the
3-subunit of
Na+/K+-ATPase. Experiments were done as in
Fig. 2. Ou, ouabain.
View larger version (16K):
[in a new window]
Fig. 5.
Comparison of NAC and vitamin E effects on
ouabain-induced regulations of the genes of ANF, skACT, and
3-subunit of
Na+/K+-ATPase. Cells were pretreated with
10 mM NAC or 0.5 mM vitamin E for 15 min,
exposed to 100 µM ouabain for 12 h, and assayed for
mRNAs as indicated under "Experimental Procedures." A
representative Northern blot of four different experiments is shown.
Vit E, vitamin E; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
View larger version (29K):
[in a new window]
Fig. 6.
Lack of effect of NAC on ouabain-induced
expression of c-fos. Cells were pretreated with
NAC for 30 min, exposed to ouabain (Ou) for 45 min, and
assayed for mRNAs as in Fig. 2. Values are mean ± S.E. of
three experiments.
View larger version (28K):
[in a new window]
Fig. 7.
Lack of effect of NAC on ouabain-induced
activation of AP-1. Myocytes were transfected with a report CAT
gene directed by AP-1 elements, exposed to ouabain (Ou) for
16 h in the presence or absence of NAC, and assayed for CAT as
described under "Experimental Procedures." Values are mean ± S.E. of three experiments.
B is another transcription factor that, along with AP-1, is
regulated by the redox state in a variety of cell types (13-15, 34)
and is known to be activated by exogenous H2O2
in cardiac myocytes (35). Exposure of myocytes to ouabain caused the
nuclear translocation of the p65 subunit of NF-B (Fig.
8), indicative of its activation.
Preincubation of myocytes with NAC blocked this ouabain-induced
translocation (Fig. 8), suggesting a role of intracellular ROS in
ouabain-induced activation of NF-B.
View larger version (67K):
[in a new window]
Fig. 8.
Effect of NAC on ouabain-induced nuclear
translocation of NF- B. Immunostaining of
myocytes with anti-p65 antibody was done as indicated under
"Experimental Procedures." A, control myocytes;
B, myocytes treated with 100 µM ouabain for
1 h; C, myocytes pretreated with 10 mM NAC
for 30 min and then exposed to ouabain as in B.
3-subunit), involves both Ras and ROS. Interestingly,
NAC also significantly decreased the [3H]phenylalanine
incorporation in the absence of ouabain (Fig. 10), suggesting that
basal levels of ROS may be important signals for the regulation of
protein synthesis.
View larger version (37K):
[in a new window]
Fig. 9.
Prevention of ouabain effect on protein
synthesis by dominant-negative Ras. Myocytes were transduced
either with the adenoviruses expressing an Asn17
dominant-negative mutant of Ras or with the control virus containing
the -galactosidase (-gal) gene for 12 h and then
assayed for [3H]phenylalanine incorporation in the
absence or presence of 100 µM ouabain (Ou) as
indicated under "Experimental Procedures." Values are mean ± S.E. of three experiments. *, p < 0.05 against
control.
View larger version (44K):
[in a new window]
Fig. 10.
Effects of NAC on ouabain-induced and
PMA-induced activations of protein synthesis. Myocytes exposed to
the indicated concentrations of ouabain (Ou), PMA, and NAC
were assayed for [3H]phenylalanine incorporation as
described under "Experimental Procedures." Values are mean ± S.E. of three experiments. *, p < 0.05 against
control; **, p < 0.05 against 10 mM
NAC.
3-subunit genes were blocked by NAC, the
antioxidant had no effect on PMA-induced effects on the same genes
(Fig. 11). Stimulation of protein
synthesis by PMA was also not blocked by NAC, in contrast to the
antioxidant effect on ouabain-stimulated protein synthesis (Fig. 10).
In experiments similar to those shown in Fig. 1, the same
concentrations of PMA used the experiments shown in Figs. 10 and 11 did
not cause increased generation of ROS (data not shown). These results
clearly show that increased production of ROS is not a common response
of the cardiac myocytes to all hypertrophic stimuli.
View larger version (32K):
[in a new window]
Fig. 11.
Lack of effect of NAC on PMA-induced
regulations of ANF (A) and
3-subunit
(B) of Na+/K+-ATPase
genes. Control and NAC-treated cells were exposed to PMA and
assayed for mRNAs as in Fig. 2.
View larger version (34K):
[in a new window]
Fig. 12.
Effect of NAC on ouabain-induced activation
of p42 MAPK. Myocytes were pretreated with NAC for 30 min, exposed
to ouabain (Ou) for 15 min, and assayed for MAPK activation
as described under "Experimental Procedures." Values are mean ± S.E. of four experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
or angiotensin II,
two stimuli that induce cardiac myocyte hypertrophy (29). Our findings
presented here clearly establish the essential roles of ROS-regulated
signaling pathways in ouabain-induced hypertrophy, and in
transcriptional regulations of several growth-related genes of these
cardiac myocytes by ouabain. However, our findings showing that
PMA-induced hypertrophy does not involve ROS also establish that
increased intracellular generation of ROS is not a requirement for the
induction of myocyte hypertrophy per se. Clearly, the potential signaling roles of ROS need to be explored for each physiological and pharmacological stimulus of cardiac hypertrophy to
allow the determination of how the various stimuli interact to cause
pathological cardiac hypertrophy and to permit the assessment of the
roles of ROS and antioxidants in the development of cardiac hypertrophy
and failure.
3 subunit of Na+/K+-ATPase) are
dependent on ROS generated in response to ouabain, these ROS do not
seem to be involved in the induction of c-fos by ouabain. A
plausible explanation for this apparent pathway specificity is that
within the complex network of multiple pathways activated by ouabain
(11), the target of the generated ROS is not a proximal component that
is shared by all the pathways of the network. Alternatively, because
there are two branches within the ouabain-activated pathways leading to
the induction of c-fos (11), it may be that repression of a
ROS-regulated branch is accompanied by a compensatory change in the
activity of a ROS-independent branch.
B, the two
transcription factors whose activations by ROS have been shown in a
large number of studies (13-15, 34), should also be considered as
potential downstream targets for any ROS-generating stimulus. Although
we have shown before that AP-1 is indeed activated when cardiac
myocytes are exposed to H2O2 (35), our present
findings (Fig. 7) argue against a role of ouabain-generated ROS in
ouabain-induced activation of AP-1. NF-B of the cardiac myocytes,
which is also activated by exogenous H2O2 (35),
is indeed a target of ouabain-generated ROS (Fig. 8). The potential
role of this transcription factor in the gene regulatory effects of
ouabain needs further exploration.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pharmacology,
Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4182; Fax: 419-383-2871; E-mail: ZXie{at}mco.edu.
ABBREVIATIONS
-actin;
ANF, atrial natriuretic factor;
CAT, chloramphenicol
acetyltransferase;
CM-DCF, 5-(and 6)
chloromethyl-2',7'-dichlorofluorescein;
MAPK, mitogen-activated protein
kinase;
NAC, N-acetylcysteine;
PMA, phorbol 12-myristate
13-acetate;
ROS, reactive oxygen species;
MEK, mitogen-activated
protein kinase/extracellular signal-regulated kinase;
CM-DCFH, reduced
CM-DCF.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  D. Li, C. Yang, Y. Chen, J. Tian, L. Liu, Q. Dai, X. Wan, and Z. Xie Identification of a PKC{varepsilon}-dependent regulation of myocardial contraction by epicatechin-3-gallate Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H345 - H353. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Liu, X. Zhao, S. V. Pierre, and A. Askari Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1489 - C1497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Schoner and G. Scheiner-Bobis Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth Am J Physiol Cell Physiol, August 1, 2007; 293(2): C509 - C536. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Elkareh, D. J. Kennedy, B. Yashaswi, S. Vetteth, A. Shidyak, E. G. R. Kim, S. Smaili, S. M. Periyasamy, I. M. Hariri, L. Fedorova, et al. Marinobufagenin Stimulates Fibroblast Collagen Production and Causes Fibrosis in Experimental Uremic Cardiomyopathy Hypertension, January 1, 2007; 49(1): 215 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Khundmiri, M. A. Metzler, M. Ameen, V. Amin, M. J. Rane, and N. A. Delamere Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1247 - C1257. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Kennedy, S. Vetteth, M. Xie, S. M. Periyasamy, Z. Xie, C. Han, V. Basrur, K. Mutgi, V. Fedorov, D. Malhotra, et al. Ouabain decreases sarco(endo)plasmic reticulum calcium ATPase activity in rat hearts by a process involving protein oxidation Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H3003 - H3011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Sandstrom, S.-J. Zhang, J. Bruton, J. P. Silva, M. B. Reid, H. Westerblad, and A. Katz Role of reactive oxygen species in contraction-mediated glucose transport in mouse skeletal muscle J. Physiol., August 15, 2006; 575(1): 251 - 262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Frese, M. Frese-Schaper, A.-C. Andres, D. Miescher, B. Zumkehr, and R. A. Schmid Cardiac Glycosides Initiate Apo2L/TRAIL-Induced Apoptosis in Non-Small Cell Lung Cancer Cells by Up-regulation of Death Receptors 4 and 5 Cancer Res., June 1, 2006; 66(11): 5867 - 5874. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Kennedy, S Vetteth, S. Periyasamy, M Kanj, L Fedorova, S Khouri, M. Kahaleh, Z Xie, D Malhotra, N. Kolodkin, et al. Uremic Cardiomyopathy--An Endogenous Digitalis Intoxication?: Central Role for the Cardiotonic Steroid Marinobufagenin in the Pathogenesis of Experimental Uremic Cardiomyopathy. Hypertension 47: 488-495, 2006 J. Am. Soc. Nephrol., June 1, 2006; 17(6): 1493 - 1497. [Full Text] [PDF]
|
|
|
|
 |
|
 K. T. Murphy, W. A. Macdonald, M. J. McKenna, and T. Clausen Ionic mechanisms of excitation-induced regulation of Na+-K+-ATPase mRNA expression in isolated rat EDL muscle Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2006; 290(5): R1397 - R1406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Kennedy, S. Vetteth, S. M. Periyasamy, M. Kanj, L. Fedorova, S. Khouri, M. B. Kahaleh, Z. Xie, D. Malhotra, N. I. Kolodkin, et al. Central Role for the Cardiotonic Steroid Marinobufagenin in the Pathogenesis of Experimental Uremic Cardiomyopathy Hypertension, March 1, 2006; 47(3): 488 - 495. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. T. Murphy, A. C. Petersen, C. Goodman, X. Gong, J. A. Leppik, A. P. Garnham, D. Cameron-Smith, R. J. Snow, and M. J. McKenna Prolonged submaximal exercise induces isoform-specific Na+-K+-ATPase mRNA and protein responses in human skeletal muscle Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2006; 290(2): R414 - R424. [Abstract] [Full Text] [PDF]
|
|
|
|
