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1-adrenoceptor signaling in adult rat cardiac
myocytes
Myocardial Biology Unit, Cardiovascular Division, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, 02118
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We recently reported that
1-adrenoceptor (1-AR) stimulation induces
hypertrophy via activation of the mitogen/extracellular signal-regulated kinase (MEK) 1/2-extracellular signal-regulated kinase
(ERK) 1/2 pathway and generates reactive oxygen species (ROS) in adult
rat ventricular myocytes (ARVM). Here we investigate the intracellular
source of ROS in ARVM and the mechanism by which ROS activate
hypertrophic signaling after 1-AR stimulation.
Pretreatment of ARVM with the ROS scavenger
Mn(III)terakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP)
completely inhibited the 1-AR-stimulated activation of
Ras-MEK1/2-ERK1/2. Direct addition of H2O2 or
the superoxide generator menadione activated ERK1/2, which is also prevented by MnTMPyP pretreatment. We found that ARVM express gp91phox, p22phox, p67phox, and
p47phox, four major components of NAD(P)H oxidase, and that
1-AR-stimulated ERK1/2 activation was blocked by four
structurally unrelated inhibitors of NAD(P)H oxidase
[diphenyleneiodonium, phenylarsine oxide,
4-(2-aminoethyl)benzenesulfonyl fluoride, and cadmium].
Conversely, inhibitors for other potential ROS-producing systems,
including mitochondrial electron transport chain, nitric oxide
synthase, xanthine oxidase, and cyclooxygenase, had no effect on
1-AR-stimulated ERK1/2 activation. Taken together, our
results show that ventricular myocytes express components of an NAD(P)H
oxidase that appear to be involved in 1-AR-stimulated hypertrophic signaling via ROS-mediated activation of
Ras-MEK1/2-ERK1/2.
myocardial hypertrophy; norepinephrine; mitogen/extracellular signal-regulated kinase 1/2-extracellular signal-regulated kinase 1/2; Ras; NAP(P)H oxidase expression
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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REACTIVE OXYGEN
SPECIES (ROS) are now recognized as critical regulators of
intracellular signaling cascades (14, 20) and play a role
in mediating growth of cardiac myocytes (2, 27, 32, 42).
We recently reported that 1-adrenergic receptor (1-AR) activation causes generation of ROS and
hypertrophy in adult rat ventricular myocytes (ARVM; see Ref.
2). We have further shown that
1-AR-stimulated hypertrophy in ARVM is mediated via
activation of the Ras, mitogen/extracellular signal-regulated kinase
(MEK) 1/2, and extracellular signal-regulated kinase (ERK) 1/2
signaling pathway (40). Kinase cascades in the
mitogen-activated protein kinase (MAPK) family have emerged as central
intracellular signaling intermediates regulating myocyte hypertrophy
(15), and the sensitivity of these signaling cascades to
ROS (6) makes it likely that they play a role in
ROS-mediated myocyte hypertrophy (1). In particular, the
ERK1/2 pathway can be activated by sources of ROS in the intact heart
and isolated myocytes (42). The ROS-dependent activation
of myocyte hypertrophy after 1-AR stimulation might
therefore occur through this ROS-sensitive system.
The enzymatic source of ROS involved in triggering hypertrophic growth
in ventricular myocytes remains unknown. Several ROS-producing systems,
including NAD(P)H oxidase (17), mitochondrial electron transport chain (12), xanthine oxidase (13),
nitric oxide synthase (NOS; see Ref. 39), and
cyclooxygenase (Cox; see Ref. 38) have been identified in
many cell types, and each of these has been implicated in the
activation of intracellular signaling cascades leading to changes in
cell structure and function (37). The NAD(P)H oxidases are
membrane-associated, multisubunit enzyme complexes that catalyze the
single-electron reduction of oxygen using NADH or NAD(P)H as the
electron donor (18). NAD(P)H oxidase consists of at least
the following four major subunits: p22phox,
gp91phox [or nox-1 in vascular smooth muscle cells
(VSMC)], p67phox, and p47phox. These enzymes
have been well studied in phagocytes and appear to be the primary
source of ROS responsible for ANG-stimulated proliferation and
hypertrophy in VSMC and endothelial cells, where cytosolic
O
The purpose of this study was to determine 1) whether
1-AR stimulation in ARVM induces ROS-dependent
activation of the Ras-Raf-MEK1/2-ERK1/2 pathway and 2)
whether NAD(P)H oxidase might be the source of ROS involved in
1-AR activation of hypertrophic signaling. We show that
1-AR activation of Ras-Raf-MEK1/2-ERK1/2 in ventricular myocytes is ROS dependent. We further show that transcripts of several
components of the NAD(P)H oxidase complex are expressed in ARVM and
that inhibitors of NAD(P)H oxidase, but not other known ROS-generating
systems, prevent 1-AR stimulation of ERK1/2, thereby
implicating the NAD(P)H oxidase in cardiac myocyte hypertrophic signaling.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials.
L-Norepinephrine (NE), DL-propranolol (Pro),
N
-nitro-L-arginine, myxothiazol,
thenoyltrifluoroacetone (TTFA), CdSO4, CdCl2, H2O2, nimesulide, NS-398, indomethacin,
aspirin, allopurinol, oxypurinol, BSA, L-carnitine,
creatine, taurine, and Ponceau S solution were from Sigma (St. Louis,
MO). Mn(III)terakis(1-methyl-4-pyridyl)porphyrin pentachloride
(MnTMPyP), nitro-L-arginine methyl ester,
NG-monomethyl-L-arginine,
diphenyleneiodonium (DPI) and phenylarsine oxide (PAO),
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), rotenone, DIDS,
antimycin A, and menadione were from Calbiochem (San Diego, CA). Euk-8
was from Eukarion (Bedford, MA). The Ras activation assay kit was
purchased from Upstate Biotechnology (Lake Placid, NY). The p44/42 MAP
kinase assay kit and anti-phospho-specific antibodies for ERK1/2 and
MEK1/2 kinases were purchased from New England Biolabs (Beverly, MA).
The cDNA probe for rabbit p67phox was kindly provided by
Dr. Patrick J. Pagano at Henry Fort Hospital (Detroit, MI). The
antibody for p67phox was purchased from BD Transduction
Laboratories (Lexington, KY). SuperScript II reverse transcriptase and
Taq DNA polymerase were purchased from GIBCO-BRL (Rockville,
MD). The TOPO TA cloning kit was purchased from Invitrogen (Carlsbad, CA).
Cell isolation and culture. The animal protocol used in this study was approved by the Institutional Animal Care and Use Committee at Boston University Medical Center. Ventricular myocytes were isolated from hearts of adult male Sprague-Dawley rats (250-275 g) as previously described (2, 40). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and heparinized (1,000 USP/kg iv). Hearts were removed and perfused retrograde with Krebs-Henseleit bicarbonate (KHB) buffer for 5 min. The perfusion buffer was changed to nominally Ca2+-free KHB buffer for 2-3 min until spontaneous beating stopped. Hearts were then perfused with KHB buffer containing 0.04% collagenase type II for 20 min. After removing atria and great vessels, the hearts were minced in the same buffer containing trypsin (0.02 mg/ml) and DNA (0.02 mg/ml). The cell mixture was filtered, and the cells were sedimented two times through a 6% BSA cushion to remove nonmyocyte cells. The cell pellet was resuspended and plated in DMEM, supplemented with BSA (2 g/l), L-carnitine (2 mM), creatine (5 mM), taurine (5 mM), and 0.1% penicillin-streptomycin. Cells were plated on laminin (1 µg/cm2)-coated dishes at a density of 100 cells/mm2 and kept at 37°C for 24 h before treatment. There were ~95% rod-shaped cells at the treatment time. ARVM were kept in the above-defined serum-free medium throughout all experiments.
Western blot analysis. The method is similar to that described previously (40). Briefly, ARVM were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.4 mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40) and sonicated for 2 s to shear DNA. Soluble lysates were separated by microcentrifugation, and volumes representing equal amounts of proteins with Laemmli sample buffer (Bio-Rad) were boiled at 95°C for 5 min before being resolved by 10% SDS-PAGE. The proteins were transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), blocked with 5% nonfat dry milk for 1 h at room temperature, and then incubated with primary antibody at 4°C overnight. Protein was detected with horseradish peroxidase-conjugated secondary antibody (Santa Cruz) and SuperSignal chemiluminescent substrate solution (Pierce). The protein loading of each sample was verified by staining the membrane with 0.1% Ponceau S solution.
Ras activation assay. Activated Ras was detected by a Ras activation assay kit according to the manufacture's instruction. Briefly, cell lysates (500 µg) were immunoprecipitated at 4°C overnight with an agarose conjugate Ras-GTP affinity probe [Raf-1 Ras-binding domain (RBD)] corresponding to the human RBD of Raf-1. The immunoprecipitates were resolved by 10% SDS-PAGE and detected by Western blot analysis using anti-Ras antibody.
ERK1/2 kinase assay. ERK1/2 kinase assays were conducted according to the manufacturer's instruction using a p44/42 MAP kinase assay kit. Briefly, cell lysates (200 µg) were immunoprecipitated with immobilized phospho-p44/42 MAP kinase monoclonal antibody at 4°C overnight. The immunoprecipitates were washed two times with lysis buffer and kinase buffer before being resuspended in kinase assay buffer containing the specific ERK substrate Elk-1 fusion proteins (2 µg). The reaction was carried out at 30°C for 30 min in the presence of 200 µM ATP in vitro. The reaction was terminated by addition of SDS sample buffer. Phosphorylation of Elk-1 fusion proteins was analyzed by 10% SDS-PAGE and Western blot analysis using phospho-Elk-1 specific antibody.
Amplification of partial cDNA fragments of rat NAD(P)H oxidase
from ARVM by RT-PCR.
The cDNA sequences of the cloned rat p22phox and
p47phox and mouse gp91phox were used as the
basis for the designing of the upstream or sense and downstream or
antisense oligonucleotide primers (Table
1). Each pair of primers complementary to
the cDNA sequence of the cloned NAD(P)H oxidase subunit was then used
to amplify the counterpart cDNA fragment from total RNA isolated from
ARVM. The RT and PCR reactions were performed according to the
manufacturer's protocol (GIBCO-BRL) using SuperScript II reverse
transcriptase and Taq DNA polymerase. The RT reaction was
performed under the following conditions: 10 min at 70°C, 52 min at
42°C, and 15 min at 70°C using oligo(dT) and total RNA isolated
from cultured ARVM. The cDNA fragments were amplified by PCR using the
above primers under the following conditions: 3 min at 94°C; followed
by 40 s at 94°C, 1 min at 60°C, and 1 min at 72°C, for 35 cycles; and a final incubation for 10 min at 72°C. To confirm the
sequence identities of the above RT-PCR products, the amplified cDNA
fragments were then purified and inserted in TOPO TA cloning vector
pCRII-TOPO (Invitrogen) for cycle sequencing with M-13 forward and
reverse primers. Sequences were determined by use of a DNA Sequencer
(ABI model 373; Applied Biosystems, Foster City, CA). Sequences were
validated by sequencing RT-PCR products from three separate RT-PCR
reactions.
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Northern blot analysis.
Total RNA was isolated from ARVM by the method of Chomczynski and
Sacchi (4) as previously described (40, 41).
Approximately 15 µg of total RNA were separated in 1%
formaldehyde/agarose gel and transferred to a nylon membrane
(Genescreen Plus; NEN). After sequencing to confirm identity, the above
cloned cDNA fragments of p22phox, gp91phox, and
p47phox were labeled with the [-32P]dCTP
as oligonucleotide probes by the random hexamer priming method. A
previously reported cDNA probe (714 bp) for rabbit p67phox
(30) was also labeled with the [-32P]dCTP
by the same method. Hybridization was carried out with the
-32P-labeled probes in 5× SSC, 5× Denhardt's
solution, 50% formamide, 1% SDS, and 100 µg/ml of salmon sperm DNA
at 42°C for 18 h. The membrane was washed two times for 15 min
at 42°C in 0.2× SSC and 0.1% SDS followed by two additional washes
for 15 min at 68°C in 0.1× SSC and 0.1% SDS. The blots were
analyzed by autoradiography and were developed after exposure at
70°C for 1-2 days. All blots were reprobed with an
[-32P]CTP-labeled oligonucleotide complimentary to 18S
rRNA. All mRNA levels were normalized to the level of 18S rRNA to
correct for potential variations in the amount of RNA loaded or transferred.
Statistical analysis. All data are reported as means ± SE. Significance tests (Student's unpaired t-test or ANOVA) were performed using the InStat program (GraphPAD Software). A P value <0.05 was considered to be significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ROS dependence of 1-AR stimulated activation of
Ras-MEK1/2-ERK1/2 in ARVM.
As we have reported (40), 1-AR stimulation
with NE (1 µM) in the presence of propranolol (2 µM) caused a
marked activation of Ras-MEK1/2-ERK1/2, which is evident within 5 min
and remains activated for up to 48 h (ERK1/2). Also, as we have
previously shown (40), Ras-MEK1/2-ERK1/2 activation was
completely abolished by the 1-AR-selective antagonist
prazosin (100 nM, 30 min; Fig. 1).
Pretreatment with the superoxide dismutase and catalase mimetic (8) MnTMPyP (50 µM, 30 min), which we have found
prevents 1-AR induced hypertrophy in ARVM
(2), completely prevented the
1-AR-stimulated activation of Ras, MEK1/2, and ERK1/2
and phosphorylation of the ERK1/2 substrate Elk-1 (Fig. 1, A
and B). A similar result was obtained using another
structurally unrelated ROS scavenger, Euk-8. Euk-8 (100 µM, 30 min)
pretreatment significantly prevented the 1-AR-stimulated
ERK1/2 activation (data not shown) and hypertrophy in ARVM
(2). These results suggested that ROS in the
1-AR stimulated activation of the Ras-MEK1/2-ERK1/2
pathway.
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1-AR-stimulated ERK1/2 activation (Fig.
2). Pretreatment of ARVM with MnTMPyP (50 µM, 30 min) completely prevented the activation of ERK1/2 by
H2O2 or a lower concentration of the superoxide
generator menadione (0.2 and 2 µM) and partially inhibited the ERK1/2
activation by menadione at a higher concentration (20 µM, 39 ± 6% inhibition; Fig. 2). This result demonstrates the ability of ROS to
activate the ERK1/2 pathway in ARVM and confirms that MnTMPyP acts as
an ROS scavenger in this system.
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Evidence of NAD(P)H oxidase expression in ARVM.
NAD(P)H oxidase is a membrane-bound enzyme complex that is a source of
cytosolic O
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NAD(P)H oxidase inhibitors prevented 1-AR-stimulated
activation of ERK1/2 in ARVM.
To test the potential role of NAD(P)H oxidase in the
1-AR signaling pathway, four structurally unrelated
pharmacological inhibitors of NAD(P)H oxidase (DPI, PAO, AEBSF, and
cadmium) were used. DPI concentration dependently inhibited
1-AR-stimulated activation of ERK1/2 with complete
(94 ± 8%, P < 0.01, n = 3) inhibition at 50 µM (Fig.
4A). PAO
(1 µM) also completely (93 ± 8% inhibition, P < 0.01, n = 3) prevented ERK1/2 activation induced by
1-AR stimulation (Fig. 4B). Both AEBSF and
cadmium ions (CdSO4 and CdCl2) concentration
dependently inhibited 1-AR-stimulated activation of
ERK1/2, with 63 ± 9% (P < 0.01, n = 3) inhibition by 2 mM AEBSF (Fig. 4B)
and 83 ± 8% (P < 0.01, n = 3)
inhibition by 1 mM cadmium ions (both CdSO4 and
CdCl2; Fig. 4C).
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1-AR-stimulated activation of ERK1/2.
Five structurally unrelated compounds that act at distinct sites to
inhibit the electron transport chain were used, including 10 µM
rotenone, 50 µM TTFA, 10 µM antimycin A, and 4 µM myxothiazol
(12, 36). DIDS (200 µM), an inhibitor of the
mitochondrial outer member anion channel, was also used as this has
been shown to inhibit leakage of O1-AR stimulation. In all cases,
there was no effect of mitochondrial electron transport inhibition on
1-AR-stimulated ERK1/2 activation or baseline ERK
activation (Fig. 5), suggesting that
mitochondrial ROS are not involved in the signaling pathway of
1-AR-stimulated hypertrophy.
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1-AR-stimulated ERK1/2 activation. Pretreatment of ARVM
with either allopurinol or oxypurinol (selective inhibitors of xanthine oxidase) had no inhibitory effect on 1-AR-stimulated
ERK1/2 activation (Fig. 6A).
Similarly, three L-arginine analogs used as NOS inhibitors had no inhibitory effect on 1-AR-stimulated ERK1/2
activation (Fig. 6B). Finally, neither the nonselective Cox
inhibitor indomethacin nor the Cox-2 selective inhibitors NS-398 and
nimesulide inhibited 1-AR-stimulated ERK1/2 activation
(Fig. 6C). Thus xanthine oxidase, NOS, and Cox do not appear
to be involved in 1-AR-stimulated activation of ERK1/2
and hypertrophy in ARVM.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We previously showed that ROS are involved in
1-AR-induced hypertrophy in ARVM (2).
Furthermore, we found that the 1-AR-stimulated hypertrophy in ARVM is mediated via activation of Ras-MEK1/2-ERK1/2 (40). Here we demonstrate that 1-AR
activation of the Ras-MEK1/2-ERK1/2 hypertrophic pathway is redox
sensitive, with the redox-sensitive step occurring at, or proximal to,
the level of Ras activation. Moreover, we provide molecular evidence
that an NAD(P)H oxidase system is expressed in cardiac myocytes and
pharmacological evidence that this oxidase mediates
1-AR-stimulated ERK1/2 activation.
Role of ROS in Gq-coupled receptor activation of
hypertrophy.
Recently, the role of ROS as an intracellular signaling molecule has
been recognized in many biological systems, including myocyte
hypertrophy (2, 27, 32, 42). ROS-mediated signaling has
been implicated in the hypertrophic effects of several extracellular growth signals, including ANG, endothelin, and tumor necrosis factor- in both VSMC and NRVM (27, 35). Similar to
receptors for endothelin and ANG, 1-AR are members of
the G protein-coupled receptor family that couple to Gq. In
VSMC, activation of Gq protein-coupled receptors, i.e., ANG
II receptor, stimulates ROS generation through an NAD(P)H oxidase,
leading to activation of MAPK pathways (34). Our present
1-AR signaling data in ARVM suggest that coupling via
NAD(P)H oxidase could be a common feature of Gq-mediated
growth signaling in the cardiovascular system.
Role of ROS in activation of the 1-AR-stimulated
signaling pathway.
The mechanism by which intracellular ROS activate the ERK1/2 pathway is
currently unknown. Our studies provide new information on the
relationship between ROS and the 1-AR-stimulated ERK1/2 pathway. 1-AR stimulation is known to directly activate
phospholipase C-
(PLC-) and results in formation of inositol
phosphates and diacylglycerol that cause the downstream effects
(43). We previously found that MnTMPyP completely
inhibited 1-AR-stimulated hypertrophy in ARVM but had no
effect on 1-AR-stimulated formation of total intracellular inositol phosphates (2). Thus PLC and its
upstream signaling molecules in the 1-AR signaling
pathway are not redox sensitive. Therefore, the redox-sensitive step in
this pathway is likely downstream from PLC-. The finding, in the
present study, that MnTMPyP completely inhibited the
1-AR-stimulated activation of Ras, MEK1/2, and ERK1/2
suggests that the redox-sensitive step in the 1-AR
signaling pathway in ARVM is located at, or proximal to, Ras.
subunits and
upstream of, or at the level of, Src (26). In addition,
ROS have been identified as central mediators in the signaling events
of several G protein-coupled receptors in cardiac myocytes, and the
Gi and Go subunits were identified as the
critical targets of ROS for the activation of the ERK1/2 pathway in
NRVM (28). Thus ROS appear to be important upstream intermediates in G protein-coupled receptor-stimulated ERK1/2 activation, and the target proteins of ROS are located at early steps
in these signaling pathways.
The precise location and mechanism for the redox-sensitive signaling
pathway remains to be determined. In Jurkat T cells, the reactive free
radical nitric oxide activates ERK1/2 directly by nitrosylating a
cysteine residue on the upstream enzyme Ras (23). Although
our data do not support a role for NOS/nitric oxide in
1-AR-stimulated activation of Ras, reactive free
radicals, including H2O2, can also activate Ras
(24), likely through direct modifications of
redox-sensitive cysteine residues. Other redox-dependent steps
potentially involved in mediating 1-AR-stimulated
activation of the ERK1/2 signaling pathway (9) include
Src, calcium-calmodulin, and proline-rich nonreceptor tyrosine kinase 2 (1, 16, 29).
Role of NAD(P)H oxidase in 1-AR signaling.
NAD(P)H oxidase is a membrane-associated, multisubunit enzyme complex
that was first described as an important generator of ROS and reactive
nitrogen species as part of the immune response in phagocytes
(3). The neutrophil oxidase consists of at least four
major subunits, including two membrane-spanning components (p22phox and gp91phox) and two cytosolic
components (p67phox and p47phox; see Ref.
18). This oxidase system has now been identified in other
cell types and has recently been shown to be the primary source of ROS
that mediates ANG-stimulated proliferation and hypertrophy in VSMC
(35). Although the cardiovascular NAD(P)H oxidases retain a similar enzyme complex structure to neutrophil oxidase with four
subunits, their component structures and biochemical characteristics are considerably different (18). Using RT-PCR, Northern
blot, and Western blot analysis, we have demonstrated that these four major subunits of NAD(P)H oxidase (p22phox,
gp91phox, p67phox, and p47phox) are
expressed in cardiac myocytes. These results provide the first direct
evidence for the existence of an NAD(P)H oxidase system in cardiac myocytes.
1-AR to activation of the
Ras-MEK1/2-ERK1/2 cascade. Four known inhibitors of NAD(P)H oxidases,
each structurally unique and acting on a distinct site of the NAD(P)H
oxidase, significantly inhibited 1-AR stimulated ERK1/2
activation in ARVM. As discussed below, several of these inhibitors
lack specificity for NAD(P)H oxidase. However, taken together with the
negative data using inhibitors of other oxidase enzymes, these studies
support the conclusion that an NAD(P)H oxidase is the source of ROS
linking 1-AR stimulation to ERK1/2 activation in ARVM.
DPI is a known inhibitor for NAD(P)H oxidase and exerts its effect by
blocking the binding of FAD to a flavin site of the oxidase
(7). Although DPI also inhibits the activity of other flavin-containing enzymes, we found no effect of selective inhibitors of mitochondrial respiration, xanthine oxidase, NOS, or Cox on 1-AR stimulated ERK1/2 activation. Therefore, the
inhibitory effect of DPI on 1-AR-stimulated ERK1/2
activation is not likely occurring via the blockade of these
flavin-containing enzymes.
PAO is reported to selectively bind to the -subunit
(gp91phox) of NAD(P)H oxidase and to preferentially inhibit
NAD(P)H oxidase activation at a low concentration (1 µM; 11 and 25).
Cadmium has been shown to reversibly block the proton
channel-associated NAD(P)H complex (21) and thereby
inhibit oxidase function (19, 22). AEBSF has been shown to
prevent the assembly of the NAD(P)H oxidase complex and oxidase
activity (10, 26). Like DPI, these inhibitors also have
other effects that need to be considered in interpreting these data.
PAO can inhibit tyrosine phosphatase, though at higher concentrations
than used here (IC50 = 18 µM; see Ref.
31). Moreover, PAO inhibits, rather than potentiates,
ERK1/2 signaling. Thus the tyrosine phosphatase inhibitory properties
of PAO do not appear to confound interpretation of the present data.
AEBSF is an irreversible inhibitor of serine proteases
(5). However, we found no effect of the serine protease
inhibitor phenylmethylsulfonyl fluoride on
1-AR-stimulated ERK1/2 activation (data not shown),
suggesting that the protease inhibitory action of AEBSF does not
confound the interpretation of these results.
In summary, our results show that 1) ROS mediate
1-AR-stimulated activation of the Ras-MEK1/2-ERK1/2
cascade in ARVM; 2) the redox-sensitive step in the
1-AR signaling pathway in ARVM is located at, or
proximal to, Ras; and 3) NAD(P)H oxidase is expressed in
cardiac myocytes and appears to be the intracellular source of ROS in
response to 1-AR stimulation in ARVM. These results shed
new light on the coupling between adrenergic and hypertrophic signaling
in ventricular myocytes and implicate new targets in the treatment of
heart diseases that involve myocardial hypertrophy and remodeling.
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NOTE ADDED IN PROOF |
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Since the completion and original submission of this work, a
report has appeared from Tanaka et al. (33) demonstrating
that 1-AR-stimulated hypertrophy in adult rat cardiac
myocytes is inhibited by antioxidants and that
1-AR-stimulated ROS production as measured by DCF
fluorescence is inhibited by DPI.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-07224 (L. Xiao), HL-42539 and HL-61639 (W. S. Colucci), HL-057947 (K. Singh), and HL-03878 (D. B. Sawyer); a Scientist Development Grant from the American Heart Association (AHA) National Center (L. Xiao), a Grant-in-Aid from the AHA Massachusetts Affiliate (D. B. Sawyer and K. Singh); and a Merit grant from the Department of Veterans Affairs (K. Singh).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. B. Sawyer, Cardiovascular Division, Dept. of Medicine, Boston Univ. Medical Center, X-704, 650 Albany St., Boston MA 02118 (E-mail: Douglas.sawyer{at}bmc.org).
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.
10.1152/ajpcell.00254.2001
Received 8 June 2001; accepted in final form 4 December 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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