(Received for publication, September 17, 1995)
From the
In response to hormones and mechanical stretch, neonatal rat ventricular myocytes exhibit a hypertrophic response that is characterized by induction of cardiac-specific genes and increased myocardial cell size. Hypertrophic stimuli also activate mitogen-activated protein kinase (MAPK), an enzyme thought to play a central role in the regulation of cell growth and differentiation. To determine if MAPK activation is sufficient for acquisition of the molecular and morphological features of cardiac hypertrophy we compared four agonists that stimulate G protein-coupled receptors. Whereas phenylephrine and endothelin transactivate cardiac-specific promoter/luciferase reporter genes, increase atrial natriuretic factor (ANF) expression, and promote myofilament organization, neither carbachol nor ATP induces these responses. Interestingly, all four agonists activate both the p42 and the p44 isoforms of MAPK. Furthermore, the kinetics of MAPK activation are not different for the hypertrophic agonist phenylephrine and the nonhypertrophic agonist carbachol. Transient transfection of myocytes with dominant-interfering mutants of p42 and p44 MAPK failed to block phenylephrine-induced ANF expression, although Ras-induced gene expression was inhibited by expression of the mutant MAPK constructs. Moreover, PD 098059, an inhibitor of MAPK kinase, blocked phenylephrine-stimulated MAPK activity but not ANF reporter gene expression. Thus, MAPK activation is not sufficient for G protein receptor-mediated induction of cardiac cell growth and gene expression and is apparently not required for transcriptional activation of the ANF gene.
The mitogen-activated protein kinases (MAPK) ()are a
family of serine/threonine protein kinases thought to play a central
role in cell proliferation and differentiation. There are several
isoforms of MAPK(1) , the best characterized of which are the
p42 MAPK (Erk2) and p44 MAPK (Erk1) isoforms. Growth factors, phorbol
esters, and hormones regulate MAPK activity through a series of
phosphorylation events(2) . Upon activation, MAPK translocates
to the nucleus and can phosphorylate a variety of nuclear transcription
factors, suggesting that MAPK plays an important role in transducing
cytoplasmic signals to nuclear responses (3, 4, 5, 6) . For receptor tyrosine
kinases, the signaling pathways leading to MAPK activation and the
requirement for MAPK in various cellular responses have been well
documented. In contrast, the mechanisms by which G protein-coupled
receptors activate the MAPK pathway and the involvement of MAPK in G
protein receptor signaling is far less clear. Additionally, while MAPK
has been shown to be important in responses such as neuronal
differentiation in PC12 pheochromocytoma cells and mitogenesis in
fibroblasts(7, 8, 9) , the role of MAPK in
terminally differentiated cells has not been well studied.
In nondividing ventricular myocytes, the molecular and phenotypic changes associated with cardiac hypertrophy can be induced by treatment with phenylephrine (PE) or endothelin (ET). These agonists interact with seven transmembrane-spanning receptors to activate phospholipase C and subsequently protein kinase C(10, 11, 12) . Other agonists that interact with receptor tyrosine kinases, such as bFGF, are also effective at inducing cardiac hypertrophy(13) . Therefore, both G protein-linked and tyrosine kinase receptors stimulate pathways that elicit changes associated with hypertrophy. In neonatal myocytes PE, ET, angiotensin II and bFGF have been shown to activate two closely related MAPK isoforms, p42 MAPK and p44 MAPK(14, 15, 16, 17) . It has been proposed that the signaling pathways utilized by G protein-coupled receptors and tyrosine kinase receptors converge at MAPK and that MAPK plays a central role in cardiac hypertrophy(15) . Recently, an interfering mutant of MAPK was shown to block PE-induced ANF promoter activation(16) , further implicating MAPK in the signaling pathway that leads to cardiac hypertrophy.
In this study we examined four agonists known to activate different G protein-coupled receptors and compared their abilities to induce various features of the hypertrophic phenotype and to activate MAPK in neonatal ventricular myocytes. We demonstrate that ET, PE, carbachol (CCh), and ATP all activate the p42 and p44 isoforms of MAPK, but that only PE and ET induce ANF and MLC-2 expression and organization of myofilaments. We further demonstrate that the kinetics of MAPK activation are not different for the hypertrophic agonist PE and the nonhypertrophic agonist CCh. Finally, we demonstrate that neither transient expression of dominant interfering mutants of MAPK nor pharmacological blockade of MAPK activation inhibits PE-induced ANF promoter activity. We conclude from our studies that MAPK activation may not be necessary and is clearly not a sufficient signal for induction of cardiac gene expression and the changes in morphology associated with in vitro hypertrophy of cardiomyocytes.
In some experiments
an alternative protocol developed by Sprenkle et al.(23) was used to examine ANF expression following short
term agonist treatment. Briefly, freshly isolated ventricular myocytes
at a density of 1 10
/ml of medium were transfected
by electroporation with 10 µg of the ANF/luciferase reporter gene
and 3 µg of cytomegalovirus/
-galactosidase. After a
12-14-h post-electroporation recovery in serum-containing medium,
myocytes were washed and medium replaced with serum-free medium.
Twenty-four hours later, the medium was again replaced with serum-free
medium and cells were treated ± PE for 6 h. Cell lysates were
prepared and luciferase and
-galactosidase activities determined.
MAPK activity
was also examined using an in-gel kinase assay with modifications of
the procedure described by Kameshita and Fujisawa (27) .
Briefly, cells were treated with agonist, cell lysates prepared, and
proteins resolved in 10% SDS-polyacrylamide gels containing 0.5 mg/ml
MBP. After electrophoresis, the gels were washed with 20% 2-propanol in
50 mM HEPES, pH 7.6, then with 5 mM mercaptoethanol
in HEPES buffer. Proteins were denatured by washing the gels with 6 M urea in Tris buffer and then renatured by washing in HEPES
buffer containing 0.04% Tween 20 at 4 °C. After preincubation of
the gel for 30 min at 4 °C in 20 mM HEPES, 2 mM dithiothreitol, 10 mM MgCl, pH 7.6, in
situ phosphorylation of MBP was performed in the same buffer
containing 6.7 µCi/ml [
-
P]ATP at 30
°C for 2 h. After extensive washing in 5% trichloroacetic acid, 10%
sodium pyrophosphate, gels were dried, exposed to film, and phosphate
incorporation quantitated by radioanalytic scanning of gels.
We compared the abilities of four G protein-coupled receptor
agonists to induce promoter activities of two cardiac-specific genes in
neonatal rat ventricular myocytes. Myocytes were transiently
transfected with ANF or MLC-2 promoter/luciferase reporter genes
containing regions of the promoter previously shown to be sufficient
for PE-inducible expression(20) . Myocardial cells were then
stimulated with either ET (10 nM), the adrenergic receptor
agonist PE (100 µM plus 2 µM propranolol to
block -adrenergic effects of PE), the stable acetylcholine analog
CCh (300 µM) or ATP (50 µM) for 48 h. As
shown in Fig. 1(upper panel), CCh and ATP did not
increase ANF-luciferase activity, indicating that these agonists did
not transactivate the ANF promoter. In contrast, ET and PE markedly
increased ANF luciferase expression. Likewise, ET and PE were effective
activators of MLC-2 reporter gene expression, while CCh and ATP were
not (Fig. 1, lower panel).
Figure 1:
Differential effects of endothelin,
phenylephrine, carbachol, and ATP on ANF and MLC-2 gene expression.
Neonatal rat ventricular myocytes were transfected with an ANF
promoter/luciferase (upper panel) or an MLC-2
promoter/luciferase (lower panel) reporter gene and then
incubated for 48 h with either no drug, 10 nM ET, 100
µM phenylephrine (with 2 µM propranolol to
block -adrenergic receptors), 300 µM carbachol, or 50
µM ATP. Luciferase activity was normalized to
-galactosidase activity for each sample and agonist-induced
increases expressed as -fold stimulation relative to no drug treatment.
Values are the mean ± S.E. of data from three experiments
performed in triplicate.
The hypertrophic response
in ventricular cardiac myocytes is also characterized by increases in
myofilament organization and cell size. To assess these morphological
changes, myocytes cultured on coverslips were incubated with ET, PE,
CCh, or ATP for 48 h and subsequently fixed and immunostained for MLC-2
or ANF using specific antisera and secondary fluorescent antibodies as
described previously(22) . Myocardial cells cultured in the
presence of ET and PE showed organization of MLC-2 into contractile
units, while myofilaments in CCh- and ATP-treated cells remained
disorganized and the cells appeared smaller in size (Fig. 2, upper panel). In addition, ANF protein expression was induced
by ET and PE, but not in response to CCh or ATP (Fig. 2, lower panel). The lack of effect of ATP extends the findings
of Zheng et al.(28) , demonstrating that ATP does not
induce [C]phenylalanine incorporation into
protein or increase cell size in ventricular myocytes. Moreover, these
data corroborate results obtained from transfection experiments and
demonstrate that myofilament organization and ANF protein expression,
like transcriptional activation of the MLC-2 and ANF reporter genes,
are selectively induced by a subset of G protein-coupled receptor
agonists.
Figure 2:
Differential effects of endothelin,
phenylephrine, carbachol, and ATP on ANF protein expression and
myofilament organization. Myocytes plated on glass coverslips were
incubated in serum-free medium alone (control) or medium containing 10
nM ET, 100 µM phenylephrine, 300 µM carbachol, or 50 µM ATP for 48 h. The cells were
processed for immunofluorescent analysis using antibodies against MLC-2 (upper panels) or ANF (lower panels) as described
under ``Experimental Procedures.'' Cells were photographed
using a Zeiss Axiophot fluorescent microscope with a 63 oil
immersion Apochromat objective.
Since it has been suggested that agents which induce cardiac hypertrophy signal through a MAPK pathway(12, 14, 15, 16, 17, 29, 30) , we reasoned that MAPK might be activated by ET and PE, but not CCh and ATP. To test this hypothesis, myocytes were treated with CCh, PE, ATP, or ET and MAPK was immunoprecipitated with a p44 MAPK antibody (Santa Cruz Biotechnology). As shown in Fig. 3, (upper panel), ET induced a 6-fold increase in MAPK activity, and all other agonists increased MAPK activity 3-4-fold. Thus, the activation of p44 MAPK did not correlate with the hypertrophic potential of these G protein-coupled receptor agonists. Western analysis of immunoprecipitated proteins showed that the p44 MAPK antibody used in these studies (Fig. 3) weakly immunoprecipitates the 42-kDa form of MAPK (data not shown). Since these experiments did not assess the total MAPK activity present in cardiac myocytes (which express both the p44 and p42 isoforms of MAPK (data not shown and (14) )), we also used a p42 MAPK antibody to examine the possibility that p42 MAPK might be differentially regulated by hypertrophic and nonhypertrophic agonists. As shown in Fig. 3, (lower panel), p42 MAPK was also activated by all of the agonists. The responses to PE (2.6-fold) and ET (3.4-fold) were, on average, greater than that observed for CCh (1.5-fold); however, nearly identical increases in p42 MAPK activity were induced by PE (2.6-fold) and ATP (2.1-fold), agonists with divergent effects on hypertrophic responses. Extracts from agonist-treated neonatal ventricular myocytes were also analyzed for MAPK activity using in-gel MBP phosphorylation assays. All four agonists were also found to induce p42 and p44 MBP kinase activity in these in-gel MBP kinase assays (data not shown). These results indicate that the initial activation of specific MAPK isoforms does not differentiate the ability of agonists to induce hypertrophic responses in ventricular myocytes.
Figure 3:
Carbachol, phenylephrine, ATP, and
endothelin stimulate p44 and p42 MAPK activity. Myocytes were either
untreated or exposed to agonist for 5 min and cell lysates
immunoprecipitated with an anti-p44 MAPK antibody (upper
panel) or an anti-p42 antibody (lower panel). MAPK
activity was determined in an in vitro kinase assay using MBP
as substrate. After separation of [P]MBP by
SDS-PAGE, radioactivity was quantitated by radioanalytic scanning of
gels. Representative autoradiograms are shown. Agonist-induced
increases in [
P]MBP are expressed as -fold
stimulation relative to no drug treatment. Values are the mean ±
S.E. of data from four experiments performed in duplicate or
triplicate.
Neuronal differentiation in PC12 cells and cellular proliferation in fibroblasts have been correlated with sustained MAPK activation(8, 31) . Since changes in gene expression and morphology occur over extended times, the kinetics of PE- and CCh-induced MAPK activation in neonatal rat ventricular myocytes were determined. Agonist-induced MAPK activity was found to be maximal 5 min after the addition of agonist and remained slightly elevated until returning to control levels at 18 h (Fig. 4). Importantly, the time course of CCh-induced MAPK activity was nearly superimposable on that of PE, suggesting that sustained MAPK activation does not account for the ability of PE to elicit changes in gene expression associated with cardiac hypertrophy.
Figure 4:
Carbachol and phenylephrine show similar
kinetics of MAPK activation. Myocytes were treated with 100 µM PE () or 300 µM CCh (
) for the times
shown. Cell lysates were prepared, SDS-PAGE sample buffer was added,
and lysates were boiled prior to separation in 10% SDS-PAGE containing
0.5 mg/ml MBP. In situ phosphorylation of MBP was assayed as
described under ``Experimental Procedures.'' Results are
expressed as -fold activation relative to unstimulated cells. Values
are the mean ± S.E. of data from two experiments performed in
duplicate.
It has recently been
reported that expression of an interfering mutant of p44 MAPK prevents
PE-induced ANF promoter activity(16) . However, our results
suggesting that MAPK activation is insufficient to induce cardiac
hypertrophy led us to reexamine the requirement for MAPK in
-adrenergic receptor (
-AR)-induced
gene expression. Myocytes were transiently transfected with the cDNA
for either a dominant-interfering p42 (K52RErk2) or p44 (K71RErk1) MAPK
protein along with the ANF-luciferase reporter gene and stimulated with
PE for 48 h. As shown in Fig. 5, PE-induced ANF reporter gene
expression was not blocked by expression of either of the
dominant-interfering MAPKs. In the same experiments, we cotransfected
cells with activated Ras ([Val
]Ras), which
transactivates the ANF promoter(32) . In contrast to their
effect on PE, both mutant MAPK proteins significantly inhibited
Ras-induced ANF reporter gene expression (Fig. 5), demonstrating
that the mutant MAPK constructs were functional. Thus,
-AR-induced ANF expression can occur in a
MAPK-independent manner.
Figure 5:
Interfering mutants of MAPK block Ras- but
not PE-induced activation of the ANF promoter. Myocytes were
transfected with 6 µg of the dominant-negative p44 MAPK (K71RErk1),
dominant-negative p42 MAPK (K52RErk2), or backbone vector (pCEP4) and 2
µg of activated Ras ([Val]Ras) or its
backbone vector (pDCR) along with the ANF reporter gene. Cells were
extensively washed, then immediately stimulated with PE for 48 h (or
maintained in serum-free medium for Ras-transfected cells). Luciferase
activity was determined and normalized to protein since activated Ras
significantly increases
-galactosidase activity. The data
represent the mean ± S.E. of three experiments performed in
quadruplicate.
MAPK activation requires phosphorylation on
both threonine and tyrosine residues by the dual specificity kinase,
MAPK kinase or MEK (MAPK/Erk activating
kinase)(33, 34) . To further assess the role of MAPK
in -AR-mediated ANF induction, a cell-permeable MEK
inhibitor, PD 098059 (2-(2`-amino-3`methoxyphenyl)-oxanaphalen-4-one) (35, 36) was used to pharmacologically block MAPK
activation. Myocytes were pretreated with various concentrations of PD
098059 or Me
SO for 20 min prior to the addition of PE. Cell
lysates were prepared 5 min later and MAPK activity determined using
either immunocomplex kinase (Fig. 6A) or in-gel assays (Fig. 6B). PE-induced MAPK activation was inhibited by
about 50% using 3 µM PD 098059 and was fully inhibited by
10 µM PD 098059 (Fig. 6).
Figure 6:
The MAPK kinase inhibitor PD 098059 blocks
PE-induced MAPK activation. Serum-deprived myocytes were treated with
the indicated concentrations of PD 098059 for 20 min prior to the
addition of 100 µM PE (plus 2 µM propranolol)
for 5 min. Cells were lysed and MAPK activity was determined using
immunocomplex kinase following immunoprecipitation with a p44 MAPK
antibody (A) or in-gel kinase (B) assays as described
under ``Experimental Procedures.'' Results shown in A are the means ± S.E. of data from 3-5 experiments
performed in duplicate. Data are expressed as -fold stimulation
relative to unstimulated (0.1% MeSO) controls. PD 098059
induced a concentration dependent decrease in basal MAPK (not shown).
In B, a representative in-gel MBP kinase assay demonstrates
that PE-stimulated p42 and p44 MAPK activation is reduced to basal
levels by 10 µM PD 098059.
Two transfection protocols that differ in the duration of agonist treatment were used to determine whether inhibition of MAPK prevented PE-induced ANF reporter gene expression. In the protocol used in the experiments described above and in our previous studies, cells were transfected with ANF/luciferase reporter constructs, washed, maintained in serum-free medium or treated with PE, and luciferase activity measured in cell lysates prepared 48 h later (Fig. 7, A and B). In the second protocol, cells were transfected by electroporation with ANF/luciferase reporter constructs, serum-deprived for 24 h and then treated with PE for 6 h (Fig. 7, C and D). The effects of PD 098059 on ANF promoter activity after 6 or 48 h of PE treatment were then assessed using either the full-length (3003 base pairs, Fig. 7, B and D) or 638-base pair (Fig. 7, A and C) ANF promoter/luciferase constructs. Regardless of the construct or the duration of PE treatment, PD 098059 failed to block PE-stimulated luciferase expression, consistent with the conclusion that PE can induce ANF expression independently of MAPK activation.
Figure 7:
MAPK inhibition with PD 098059 does not
prevent PE-induced ANF reporter gene expression. Myocytes were
transiently transfected with either the 638-base pair or full-length
(3003-base pair) ANF promoter/luciferase reporter gene. Cells
preincubated with 10 µM PD 098059 or 0.1% MeSO (Ctl) for 20 min were maintained in serum-free medium without (open bars) or with PE (filled bars) for 48 h (A and B) or 6 h (C and D). In A and B, myocytes were transfected using calcium-phosphate,
then treated with MEK inhibitor (or Me
SO) ± PE. In C and D, myocytes were transfected by
electroporation, serum-deprived for an additional 24 h, and then
incubated with PD 098095 ± PE. Luciferase activity was measured
in cell lysates and normalized to protein (A and B)
or
-galactosidase activity (C and D). The data
in A, B, and D are the means ± S.E.
of triplicate samples from three experiments. The data in D are from one experiment performed in
triplicate.
As shown previously, PE (37) or ET (10) induce ANF and MLC-2 expression, myofilament organization,
and increases in cardiac myocyte size. The purinergic receptor agonist
ATP, reported not to increase protein synthesis or augment myocardial
cell size(28) , also failed to induce ANF or MLC-2 reporter
gene expression or increase myofilament organization. A stable ATP
analog, ATPS, was also ineffective (not shown), suggesting that
the lack of response to ATP is not due to hydrolysis of this nucleotide
agonist. Carbachol also did not induce ANF or MLC-2 promoter activity (22) or lead to myofilament organization in cardiomyocytes. The
failure of CCh to elicit a response through endogenous muscarinic
cholinergic receptors (mAChRs) is not likely to result from depletion
of this agonist from the medium since we have shown that CCh can induce
cardiac-specific gene expression in myocytes transfected with cDNA for
the M
mAChR(22) . Therefore, we reasoned that these
agonists, which have divergent effects on cardiac hypertrophy, must
signal through distinct pathways.
Hormones and mechanical stimuli shown to induce hypertrophic responses in neonatal ventricular myocytes (e.g. PE, ET, angiotensin II, bFGF, and stretch) have also been shown to activate MAPK(12, 14, 15, 16, 17, 29) . This observation, together with evidence demonstrating the role of MAPKs in growth regulation, suggests that MAPK could be a key mediator of cardiac hypertrophy. However, we found that MAPK activation does not correlate with the hypertrophic potential of an agonist since it is stimulated not only by PE and ET but also by CCh and ATP. Our finding that ET is a more effective activator of MAPK than PE is in agreement with an earlier study comparing these agonists on MAPK activation in ventricular myocytes(12) . However, PE is not significantly more effective than ATP as an activator of MAPK (Fig. 3). Therefore, although there are quantitative differences in the magnitude of MAPK activation by the four agonists, the extent of MAPK activation does not appear to correlate with the hypertrophic potential of these agonists.
In PC12 cells, epidermal growth factor and nerve growth factor both induce a rapid and transient activation of MAPK; however, only nerve growth factor induces neuronal differentiation and causes sustained (>6 h) MAPK activation(8, 38) . Likewise, a biphasic response with a transient, followed by a more prolonged activation phase, characterizes the response of CCL39 lung fibroblasts to the mitogenic agonist thrombin but not the nonmitogenic agonist CCh(31) . However, in cultured neonatal ventricular myocytes, MAPK activation in response to PE (Fig. 6) and ET (15) peaks at 5 min and the extent of MAPK activity at 1-18 h is the same for PE and CCh. Thus, sustained MAPK activation cannot explain the divergent effects of these agonists.
The observation that both hypertrophic and nonhypertrophic agonists
activate MAPK suggests that additional signaling pathways are required
to induce the phenotypic features of cardiac hypertrophy. Stimulation
of -AR or endothelin receptors increases
phosphoinositide hydrolysis in cardiac
myocytes(10, 11, 39) . Our previous studies
using antibodies (40) and pertussis toxin (
)to block
G protein function suggest that PE and ET regulate phosphoinositide
(PI) hydrolysis through receptor interactions with G
.
Carbachol and ATP have also been shown to increase PI turnover in
cardiac preparations(42) , but in the neonatal rat ventricular
myocyte we find that CCh (22) and ATP (data not shown) induce
only modest increases in PI hydrolysis (<2-fold) relative to PE and
ET, which markedly (15-fold) increase inositol phosphate
accumulation(11) . Thus, the hypertrophic potential of G
protein-coupled receptor agonists appears to correlate with the ability
of the agonist to activate phospholipase C through receptor
interactions with G
.
The preferential ability of
G-coupled receptors to induce cardiac-specific gene
expression and morphological changes is consistent with other data
implicating G
in hypertrophic
responses(10, 24, 41) . For example,
transient expression of a constitutively activated form of G
induces ANF and MLC-2 reporter gene expression and blockade of
endogenous G
function by microinjection of inhibitory
G
antibodies inhibits PE-induced ANF expression and
cellular hypertrophy(40) . In addition, we have shown that
cardiac specific gene expression can be stimulated through
heterologously expressed wild type and chimeric M
mAChRs
that effectively couple to G
, but not through
heterologously expressed mAChRs that couple to
G
(22) . Overall, the data suggest that either
activation of G
and the sequelae of phospholipase C
activation (i.e. Ca
release and protein
kinase C activation) or activation of other signaling pathways unique
to G
-coupled receptors are required for mediating cardiac
hypertrophy.
The question of whether MAPK activation is a necessary,
albeit an insufficient, signal may be more controversial. Our data in
this regard differ from those published by Thorburn et
al.(16) . Although we used similar methodologies for
myocyte preparation and transfection, we failed to observe significant
inhibition of ANF-reporter gene expression when myocytes were
cotransfected with the dominant-interfering p44 MAPK construct that
significantly inhibited cardiac gene expression in their studies. In
addition, we did not observe inhibition of PE-induced ANF gene
expression with cotransfection of the interfering mutant of the p42
MAPK isoform (Fig. 5) or when both p42 and p44 mutant MAPK
constructs were coexpressed (data not shown). We demonstrated that
transactivation of the ANF promoter by constitutively activated Ras was
inhibited by expression of both p42 and p44 dominant interfering MAPK
constructs, suggesting that the mutant MAPK proteins are expressed and
can functionally block Ras-mediated transcriptional responses. These
constructs also block PE-induced c-fos-luciferase reporter
gene expression (data not shown), indicating that the
dominant-interfering MAPK proteins are expressed at sufficient levels
to block agonist-induced transcriptional responses. One difference
between our studies and those published earlier is that we examined
transactivation of the 638-base pair minimal ANF promoter (20) rather than full-length ANF (3003-base pair) promoter.
When we examined the effect of interfering MAPK mutants on PE-induced
activation of the full-length ANF promoter/luciferase gene, we found
inconsistent inhibition (data not shown). Another difference is that
Thorburn et al. normalized luciferase activity to coexpressed
-galactosidase activity. When our data are expressed in the same
way, we find at most a 10% inhibition of PE-induced ANF expression by
these interfering MAPK mutants.
As a second, independent line of
investigation to assess the role of MAPK in
-AR-induced ANF expression, we used PD 098059, a newly
described cell-permeable inhibitor of the MAPK kinase (MEK) (35, 36) . This compound has been shown to block
MEK-mediated tyrosine phosphorylation and activation of MAPK, while not
affecting other tyrosine kinases (e.g. Src) or
serine/threonine kinases (e.g. protein kinase A, protein
kinase C, or Raf)(36) . When cardiac myocytes were treated with
PD 098059, a dose-dependent inhibition of PE-stimulated MAPK activation
was observed. At 10 µM PD 098059, PE-induced MAPK activity
was reduced to basal levels. There was no inhibition of PE-induced ANF
reporter gene expression, mediated through either the 638- or 3003-base
pair promoter, in response to short (6 h) or long term (48 h) agonist
treatment. These findings clearly indicate that PE-induced ANF
expression can occur independently of MAPK activation.
In summary,
the present studies demonstrate that CCh and ATP, which are not
hypertrophic agonists in this model system, activate p42 and p44 MAPK
to an extent similar to that for the hypertrophic agonists PE and ET.
Neither dominant-interfering forms of MAPK nor pharmacological
inhibition of MAPK activation prevent the activation of ANF reporter
gene expression by -AR stimulation. These results
indicate that activation of p42 and p44 MAPK can be dissociated not
only from the morphological changes associated with
-AR-mediated hypertrophy(16) , but also from G
protein-coupled receptor effects on cardiac gene expression.