Intracellular signaling leads to the hypertrophic effect of
neuropeptide Y
Yaron
Goldberg,
Gerhild
Taimor,
Hans Michael
Piper, and
Klaus-Dieter
Schlüter
Physiologisches Institut, Justus-Liebig-Universität, D-35392
Giessen, Germany
 |
ABSTRACT |
Signal transduction pathways involved in the hypertrophic effect
of neuropeptide Y (NPY) were investigated in adult cardiomyocytes. Reduction of transforming growth factor-
activity in
serum-supplemented media abolished the induction of hypertrophic
responsiveness to NPY. In responsive cells, NPY (100 nM) increased
protein synthesis, determined as incorporation of
[14C]phenylalanine, by
35 ± 15% (P < 0.05, n = 16 cultures). In these cells, NPY
activated pertussis toxin (PTx)-sensitive G proteins and
phosphatidylinositol (PI) 3-kinase. PTx and inhibition of PI 3-kinase
abolished the hypertrophic effect of NPY. NPY also activated protein
kinase C (PKC) and mitogen-activated protein (MAP) kinase. Inhibition
of these two kinases attenuated the induction of creatine kinase
(CK)-BB but not the growth response to NPY. In conclusion, NPY
stimulates protein synthesis in adult cardiomyocytes via activation of
PTx-sensitive G proteins and PI 3-kinase and it induces the fetal-type
CK-BB via activation of PKC and MAP kinase.
G proteins; phosphatidylinositol 3-kinase; p70s6k; protein kinase C; mitogen-activated protein kinase
 |
INTRODUCTION |
IN PREVIOUS STUDIES, we identified the adult
ventricular cardiomyocyte as a target cell for neuropeptide Y (NPY)
(19-21, 23). NPY is abundant in myocardial tissue, where most of
this neuropeptide is stored, and can be released from intramural nerve
endings (9, 17, 34). In cardiomyocytes, NPY exerts rapid effects on
adenylate cyclase, ion channels, and contractile performance (21). On a
longer time scale, it also causes a reduction of protein degradation, increase in protein synthesis and RNA mass, and induction of enzymes like cytosolic creatine kinase (CK; Ref. 20). These long-term effects
are hallmarks of myocardial hypertrophy. Identification of this
growth-promoting effect of NPY on cardiomyocytes seems particularly
interesting because increased plasma concentrations of NPY are found in
patients with myocardial hypertrophy or failure (12, 35).
In our first study, in which the hypertrophic effect of NPY on adult
cardiomyocytes was described, we compared the action of NPY with
effects of adrenoceptor agonists (20). NPY stimulated protein synthesis
in adult cardiomyocytes that had been precultured for 6 days in the
presence of FCS. This behavior resembles the response of adult
cardiomyocytes to
-adrenoceptor agonists, since these compounds also
stimulated protein synthesis when the cells had been precultured in
serum-containing media (22). The induction of a hypertrophic
responsiveness to
-adrenoceptor stimulation was found to depend on
the presence of transforming growth factor-
(TGF-
), a cytokine
released by adult cardiomyocytes in culture (32). Whether TGF-
is
also responsible for induction of hypertrophic responsiveness of adult
cardiomyocytes to NPY was investigated.
The previous study (20) also indicated that the effect of NPY differs
at least in part from that of
-adrenoceptor stimulation: NPY, but
not a
-adrenoceptor agonist, induces CK. Such a difference suggests
differences in intracellular signaling. The present study, therefore,
also aimed to identify key steps of intracellular signals that are
activated under NPY and cause either hypertrophic growth (protein and
RNA synthesis) or lead to induction of CK in adult cardiomyocytes
precultured in serum-containing media.
Only a few elements of the signal transduction of NPY in mammalian
cells are known. NPY can activate pertussis toxin (PTx)-sensitive G
proteins (Gi
or
Go
) (4) as well as
PTx-insensitive Gq proteins, coupled to phospholipase C and a subsequent activation of protein kinase C (PKC) (37). We investigated whether one or the other of these
signal transduction elements is involved in the hypertrophic response
to NPY.
In the same cell system, but for hypertrophic stimuli other than NPY,
phosphatidylinositol (PI) 3-kinase and its potential downstream target
p70s6k were previously identified
to be crucial steps in signaling toward regulation of protein synthesis
(25). PI 3-kinase can be activated in a PKC-dependent or in an
adenylate cyclase-dependent manner, e.g., under
-adrenoceptor
stimulation or under
-adrenoceptor stimulation, respectively. In
both cases stimulation of protein synthesis requires activation of PI
3-kinase. PI 3-kinase represents therefore a point of convergence for
various growth signals. Here, it was investigated whether PI 3-kinase
and p70s6k are also involved in
the growth response to NPY.
Induction of the fetal-type isoform of CK, CK-BB, is a common feature
of the hypertrophic response of adult cardiomyocytes stimulated with
NPY or
-adrenoceptor agonists (20). In the case of
-adrenoceptor
stimulation the induction of CK-BB is mediated through activation of
PKC and a secondary activation of mitogen-activated protein (MAP)
kinase (26). It is independent of PI 3-kinase and
p70s6k. Here, we investigated
whether NPY causes induction of CK-BB through the same pathway as does
-adrenoceptor stimulation.
 |
MATERIALS AND METHODS |
Cell culture.
Ventricular heart muscle cells were isolated from 200- to 250-g male
Wistar rats as previously described (24, 27). Isolated cells were
suspended in FCS-free culture medium and plated at a density of 1.4 × 105 elongated cells/35-mm
culture dish (Falcon type 3001). The culture dishes had been
preincubated overnight with 4% FCS in medium 199. The basic culture
medium consisted of medium 199 with Earle's salts, 5 mM creatine, 2 mM
L-carnitine, 5 mM taurine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. To prevent growth of
nonmyocytes, media were also supplemented with 10 µM cytosine
-D-arabinofuranoside.
Four hours after they were plated, cultures were washed twice with
culture medium to remove round and nonattached cells and supplied with
basic culture medium supplemented with 20% FCS, in which cells were
incubated for 6 days at 37°C. Thereafter, the culture dishes were
washed twice, and experiments were carried out in basic culture medium
without FCS on day 6 (control), with additions of the agonists at indicated concentrations. Ascorbic acid
(100 µM) was added to all cultures as an antioxidant at
1% (vol/vol). In some experiments, cardiomyocytes were
pretreated with PTx (1 µg/ml) for 6 h before experiments were
started. We have previously shown that this pretreatment is an
effective procedure to inhibit
Gi/Go
proteins in adult cardiomyocytes (13).
Incorporation of
[14C]phenylalanine and changes
in cellular protein and RNA mass.
Incorporation of phenylalanine into cells was determined by exposing
cultures to
L-[14C]phenylalanine
(0.1 µCi/ml) for 24 h and determining the incorporation of
radioactivity into acid-insoluble cell mass, as described before (22,
28). Nonradioactive phenylalanine (0.3 mM) was added to the medium to
minimize variations in the specific activity of the precursor pool
responsible for protein synthesis. In incorporation studies,
experiments were terminated by removal of the supernatant medium from
the cultures and washing three times with ice-cold PBS (composition in
mM: 1.5 KH2PO4,
137 NaCl, 2.7 KCl, and 1.0 Na2HPO4,
pH 7.4). Subsequently, ice-cold 10% (wt/vol) TCA was added. After
storage overnight at 4°C, the acid was removed from the dishes.
Radioactivity contained in this acid fraction was taken to represent
the intracellular precursor pool. The dishes were then washed twice
with ice-cold PBS. The remaining precipitate on the culture dishes was
dissolved in 1 N NaOH-0.01% (wt/vol) SDS by an incubation for 2 h at
37°C. In these samples, protein contents (3) and DNA contents (7)
were determined, and the radioactivity was counted. RNA was determined
from an aliquot of these samples after precipitation with an equal
volume of 10% (wt/vol) perchloric acid in the remaining supernatant
(18). The RNA content was also expressed relative to the DNA content of
the samples.
Analysis of CK activities.
Specific activity of the cytosolic CK was determined as described
previously (28). Cultures were first washed twice with PBS. After
addition of buffer A (composition in
mM: 5 magnesium acetate, 0.4 EDTA, 2.5 dithiothreitol, 50 Tris · HCl, and 250 sucrose, pH 6.8) to the dishes,
the cells were scraped off, homogenized, and frozen until use at
14°C. For analysis, these samples were thawed, and the
resulting suspension was sonicated and centrifuged at 12,000 g for 2 min. The supernatants were
used for enzyme analysis. The activity of CK was determined according
to Ref. 6, using standard ultraviolet methods.
Distributions of the cytosolic isoenzymes of CK, MM, MB, and BB, were
analyzed according to Ref. 14. The supernatants were applied to a 1-ml
DEAE-cellulose column that had been equilibrated with
buffer B (composition in mM: 20 NaCl,
5 magnesium acetate, 0.4 EDTA, and 100 Tris · HCl, pH
7.9). The CK-MM isoenzyme eluted directly with buffer
B, the CK-MB isoenzyme with change of NaCl concentration to 40 mM and pH to 6.4, and CK-BB with change of NaCl
concentration to 250 mM and pH to 6.4.
Determination of PI 3-kinase.
PI 3-kinase activity was determined in immunoprecipitates as described
by Whitman et al. (36). Briefly, cardiomyocytes were washed twice with
PBS, and the cells were lysed in lysis buffer [composition: 10%
(vol/vol) glycerol, 1% (vol/vol) NP-40, and 1 mM phenylmethylsulfonyl
fluoride]. After centrifugation (10 min at 10,000 g), the supernatant was used for
immunoprecipitation with an antibody against the p85
-subunit of
bovine PI 3-kinase and the immunoprecipitates were sedimented with
protein A-Sepharose. The pellets were washed with PBS, twice with
buffer A (composition: 0.5 M LiCl, 0.1 M Tris, pH 7.4) and once with buffer B
(composition: 10 mM Tris, pH 7.4, 100 mM NaCl, and 1 mM EDTA) and
resuspended in 25 µl of buffer B; 1 mg/ml PI (Sigma, Deisenhofen, Germany) was dispersed by sonification in
5 mM HEPES buffer, pH 7.4, and 20 µl of this solution were added to
the resuspended immunoprecipitates. After preincubation for 30 min at
room temperature, the phosphorylation reaction was started by addition
of 20 µCi
[
-32P]ATP in
starting buffer containing 50 µM ATP and 5 mM
MgCl2. The total volume in the
reaction tubes was 50 µl. The reaction mixture was incubated for 20 min at 25°C and terminated by addition of 100 µl of 1 M HCl.
Phospholipids were then extracted with 200 µl of
CHCl3/MeOH (1:1). The organic
phase was spotted onto a silica gel TLC plate pretreated with 1%
(wt/vol) potassium oxalate. Phosphorylated products were separated by
TLC in a CHCl3/MeOH/4 M
NH4OH (9:7:2) developing solvent
and visualized with a phosphorimager (Molecular Dynamics). To quantify
the activity of the immunoprecipitates TLC plates were scanned
densitometrically and the amount of phosphorylated PI was normalized to
the spotted radioactivity of the plates, which varied between the
reaction tubes (origin).
Determination of PKC.
PKC activity was determined in the membrane fraction of cardiomyocytes
as described previously (2, 30). Briefly, in experiments in which
activation of PKC should be measured, cultures were washed twice with
ice-cold PBS at the end of the incubation period after 15 min and the
cells were harvested in ice-cold hypotonic lysis buffer and lysed by
vortexing the cell suspension vigorously for 2 min. Nuclei and any
unlysed cells were sedimented by centrifugation at 1,000 g for 5 min at 4°C. The
postnuclear supernatant was centrifuged at 100,000 g for 10 min at 4°C, the resulting
supernatant was discarded, and the membrane sediment was suspended in
an aliquot (300 µl) of PKC assay solution. The activity of PKC in the
membrane suspension was determined by a continuous fluorescence assay
as described before (2).
Determination of p42 MAP kinase.
The determinations of p42 MAP were done as described in detail (31).
Briefly, after stimulation, cells were lysed in lysis buffer
[composition in mM: 50 Tris-Cl, pH 6.7, 2% (wt/vol) SDS, 2%
(vol/vol) mercaptoethanol, and 1 sodium orthovanadate]. Then, nucleic acids were digested with benzonase (Merck, Darmstadt, Germany).
After SDS-PAGE (100 µg protein/slot), proteins were transferred onto
reinforced nitrocellulose by semidry blotting. The sheets were
saturated with 2% (wt/vol) BSA and incubated for 2 h with rabbit
polyclonal anti-rat p42 MAP kinase (10 µg/50 ml, Santa Cruz
Biotechnology). After sheets were washed, sheep anti-rabbit alkaline
phosphatase-labeled IgG (50 mU/50 ml) was added for 2 h. Detection was
done by alkaline phosphatase activity recognized by
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. For
quantification, the blots were densitometrically scanned, and the
results were expressed as the ratio of the upper band, with retarded
gel mobility of activated and phosphorylated p42 MAP kinase, to the
total amount of p42 MAP kinase determined on the Western blots.
Accumulation of cAMP.
As an indirect indicator of adenylate cyclase activation, accumulation
of cAMP in the cultures over a period of 5 min was investigated in the
presence of a phosphodiesterase inhibitor as described previously (30).
The cells were incubated with modified Tyrode solution (pH 7.4)
containing 125 mM NaCl, 1.2 mM
KH2PO4,
2.6 mM KCl, 1.2 mM MgSO4, 1 mM
CaCl2, 10 mM glucose, 10 mM HEPES,
and 1 mM IBMX, a phosphodiesterase inhibitor and a nonselective
adenosine antagonist. All experiments were performed in the presence of
adenosine deaminase (5 U/ml), as adenosine released from the cells may
exert an antagonistic effect on adenylate cyclase by interacting with
adenosine A1 receptors (11).
Experimental incubations were terminated after 5 min by the addition of
1 ml of 1.2 M HClO4 to the
contents of the culture dishes. The cells were scraped off and
centrifuged for 1 min at 12,000 g. The
supernatant was neutralized and quickly frozen in liquid nitrogen. The
pellet was redissolved in 0.1 M NaOH, and its protein content was
determined by the method of Bradford (3) with BSA as the standard. The frozen supernatants were freeze-dried and redissolved in 100 µl of
0.1 M HEPES (pH 7.4). cAMP content of these samples was determined using a protein binding assay (Amersham-Buchler, Braunschweig, Germany).
TGF-
activity.
TGF-
activity in the medium was determined as described (1) by the
growth inhibitory effect of TGF-
on the proliferation of
microvascular endothelial cells. Isolation and cultivation of
microvascular endothelial cells were described earlier (26). Supernatants of the cell culture media were diluted and added to
subconfluent monolayers of microvascular endothelial cells plated on
96-well dishes. After 48 h, protein contents of the wells were
determined. The growth inhibitory effect was compared with a standard
curve using activated TGF-
isolated from porcine platelets.
Specificity of the inhibitory effect of medium supernatants to TGF-
was further demonstrated by the use of a neutralizing antibody to
TGF-
1, which abolished the
growth inhibitory effect.
Statistics.
Data are given as means ± SD or SE from
n different culture preparations.
Statistical comparisons were performed by one-way ANOVA and use of the
Student-Newman-Keuls test for post hoc analysis (8). Differences with
P < 0.05 were regarded as
statistically significant. All data analyses were computed using SAS
software, version 6.11 (SAS Institute, Cary, NC).
Materials.
Falcon tissue culture dishes were obtained from Becton-Dickinson
(Heidelberg, Germany). Boehringer Mannheim (Mannheim, Germany) was the
source for glutamine-free medium 199, FCS, and bisindolylmaleimide. Cytosine-
-D-arabinofuranoside,
L-carnitine, creatine, and
taurine were obtained from Sigma (Deisenhofen, Germany).
2'-Amino-3'-methoxyflavone (PD-98059) was obtained from
Calbiochem-Novabiochem (Bad Soden, Germany). All other chemicals were
of analytical grade.
 |
RESULTS |
Involvement of TGF-
in the induction of a
hypertrophic responsiveness to NPY.
As shown in a previous study (20), NPY stimulated protein synthesis in
adult cardiomyocytes only when they were cultured for several days in
the presence of FCS before stimulation. It was now investigated whether
TGF-
, released by cardiomyocytes in such cultures, is responsible
for the induction of the hypertrophic responsiveness to NPY. The
activity of TGF-
in the culture medium was determined by the growth
inhibitory effect of TGF-
on microvascular endothelial cells and
compared with that of activated TGF-
isolated from porcine
platelets. In the culture media containing 20% FCS (standard
conditions), TGF-
activity after 6 days was equivalent to 1.5 ng
TGF-
/ml (Table 1). TGF-
activity in
the culture medium was reduced either by the addition of the protease
inhibitor aprotinin (1 ng/ml), which inhibits proteolytic activation of
latent TGF-
, or by lowering the FCS concentration to 5%. In these
cultures, the TGF-
activity was equivalent to 0.8 ng/ml or 0.4 ng/ml, respectively (Table 1). It was investigated next to what extent
NPY increases protein synthesis in cultures preexposed to the various
TGF-
activities. Protein synthesis of cardiomyocytes grown for 6 days in the presence of 20% FCS before stimulation increased by 50.5% with NPY compared with untreated control cultures (Table 1). In the
presence of aprotinin during 6 days of precultivation, NPY increased
protein synthesis by only 21.0%. When the FCS concentration in the
medium was reduced to 5%, NPY was unable to increase protein synthesis. The growth-promoting effect of NPY, however, was restored in
these cultures when 1 ng/ml active TGF-
was added. In this case, NPY
increased protein synthesis by 44.5% (Table 1). In all subsequent
experiments, cardiomyocytes were cultured under standard conditions
(20% FCS for 6 days) with high TGF-
activity and used thereafter
under serum-free conditions for the subsequent 24 h.
Role of PTx-sensitive G proteins and PKC for stimulation of protein
synthesis.
Because NPY receptors may either couple to PTx-sensitive proteins
(Gi/Go)
or to Gq proteins, leading to
subsequent activation of PKC, the influence of PTx or PKC inhibition on
hypertrophic growth was studied. It was first analyzed if the
PTx-sensitive, antiadrenergic effect of NPY, known for newly isolated
cardiomyocytes (13), is preserved in cultures. The inhibitory effect of
NPY (100 nM) on isoprenaline (1 µM)-stimulated cAMP accumulation was determined. NPY attenuated the effect of the
-adrenoceptor agonist by 30% (Table 2). The attenuation was
abolished in the presence of PTx (Table 2).
After this PTx-sensitive action of NPY in cultured cardiomyocytes was
confirmed, the possibility that the NPY-stimulated increases in protein
synthesis and RNA mass were PTx sensitive was analyzed. In the absence
of PTx, NPY (100 nM) augmented protein synthesis by 34% and cellular
RNA mass by 18% (Fig. 1).
These effects of NPY were blunted when the cells were incubated for 6 h
before and during
[14C]phenylalanine
incorporation in the presence of PTx (1 µg/ml). In the same set of
experiments, stimulation of
-adrenoceptors by phenylephrine (10 µM) also increased
[14C]phenylalanine
incorporation (41 ± 12% above control,
n = 16 cultures,
P < 0.05). This increase was not
attenuated by PTx (39 ± 12% above control,
n = 16, P < 0.05 vs. control).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 1.
Protein synthesis, determined as incorporation of
[14C]phenylalanine in
cardiomyocytes (protein synthesis), and RNA mass, determined as
RNA-to-DNA ratios, of cardiomyocytes during 24-h incubations under
control conditions and in the presence of neuropeptide Y (NPY, 100 nM),
NPY and pertussis toxin (PTx, 1 µg/ml), and NPY and
bisindolylmaleimide (BIM, 5 µM). If experiments were performed in the
presence of PTx, cardiomyocytes were incubated for 6 h before and
throughout the experiments with PTx. Data are given in percentage of
the basal mean values. These correspond for
[14C]phenylalanine to
3.0 ± 0.4 dpm × 10 2/µg DNA and for RNA
content to 11.8 ± 0.4 µg RNA/µg DNA. Data are means ± SD;
n = 16. Differences from control:
* P < 0.05.
|
|
We then investigated whether NPY (100 nM) caused an increase in PKC
activity and whether the stimulation of protein synthesis by NPY is
sensitive for an inhibition of PKC activation. In the particulate
fraction, PKC activity increased by 83% within 15 min under NPY (Table
3). The effect of NPY on PKC activity was compared with the effect achieved by addition of phorbol myristate acetate (100 nM), which resulted in a 143% activation of PKC. The
presence of the PKC inhibitor bisindolylmaleimide attenuated the
activation of PKC by NPY (Table 3). The presence of bisindolylmaleimide did not alter the effects of NPY on protein synthesis and cellular RNA
mass (Fig. 1).
Roles of PI 3-kinase and p70s6k for
stimulation of protein synthesis.
PI 3-kinase and its potential downstream target
p70s6k are involved in
-adrenoceptor-mediated increase in protein synthesis. It was
investigated whether these two kinases are also involved in the
NPY-mediated growth effect in cultured adult cardiomyocytes. Activation
of PI 3-kinase by NPY was determined by quantification of the
phosphorylation of PI using immunoprecipitates of the p85 subunit of PI
3-kinase. A representative chromatogram demonstrating increased PI
3-kinase activity is shown in Fig. 2. NPY
increased PI 3-kinase activity within 15 min by 160 ± 37%
(n = 4 cultures, P < 0.01 vs. control). Activation of
PI 3-kinase by NPY was abolished in the presence of wortmannin (100 nM), a PI 3-kinase inhibitor, to 31 ± 27% above control
(n = 4 cultures, not significant vs. control).

View larger version (6K):
[in this window]
[in a new window]
|
Fig. 2.
Activation of phosphatidylinositol (PI) 3-kinase by NPY. Cardiomyocyte
PI 3-kinase was immobilized on protein G-Sepharose beads through PI
3-kinase -p85 antibodies. Immunoprecipitates were generated from
cardiomyocytes under control conditions (C), in the presence of NPY
(100 nM), or NPY plus wortmannin (WORT, 100 nM). PI 3-kinase activity
in immunoprecipitates was assayed as described in
MATERIALS AND METHODS. Lipid products
were separated by TLC as described. An autoradiogram of the TLC plate
generated on a phosphorimager is shown. Positions of spots
corresponding to
[32P]phosphatidylinositol
(PIP) and origin (ORI) are indicated.
|
|
We then analyzed if the inhibitor of PI 3-kinase, wortmannin,
influences the NPY-mediated increase in protein synthesis. Wortmannin abolished the effects of NPY on protein synthesis and greatly attenuated its effects on RNA mass (Fig.
3). In the absence of NPY, wortmannin
influenced neither protein synthesis (3.0 ± 0.4 vs. 2.9 ± 0.6 dpm × 10
2/µg DNA,
n = 4 cultures, not significant from
each other) nor RNA mass of cardiomyocytes (11.7 ± 0.2 vs. 12.0 ± 0.4 mg RNA/mg DNA, n = 4, not
significant from each other).

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 3.
Protein synthesis determined as incorporation of
[14C]phenylalanine in
cardiomyocytes and RNA mass, determined as RNA-to-DNA ratios, of
cardiomyocytes during 24-h incubations under control conditions and in
the presence of NPY (100 nM) or NPY and wortmannin (WORT, 100 nM). Data
are given in percentage of the basal mean values. These correspond for
[14C]phenylalanine to
3.1 ± 0.2 dpm × 10 2/µg DNA and for RNA
content to 11.7 ± 0.2 µg RNA/µg DNA. Data are means ± SD;
n = 16. Differences from control,
* P < 0.05.
|
|
Rapamycin (100 nM), a selective inhibitor of
p70s6k, was used to investigate
whether this kinase, known as a downstream target of PI 3-kinase,
represents another step in intracellular signaling that leads to
stimulation of protein synthesis by NPY. In the presence of rapamycin,
the stimulation of protein synthesis by NPY was reduced to a large
extent (Fig. 4). Rapamycin did not reduce
the increase in cellular RNA mass induced by NPY (100 nM) (Fig. 4). The
effects of rapamycin could not be explained by an effect of rapamycin
on its own because in the absence of NPY rapamycin did not influence
protein synthesis (3.8 ± 0.2 vs. 3.6 ± 0.3 dpm × 10
2/µg DNA)
or RNA mass (14.3 ± 0.6 vs. 14.6 ± 0.5 mg RNA/mg DNA, n = 4 cultures, not significant from
each other).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
Protein synthesis, determined as incorporation of
[14C]phenylalanine in
cardiomyocytes and RNA mass, determined as RNA-to-DNA ratios of
cardiomyocytes during 24-h incubations under control conditions and in
the presence of NPY (100 nM) or NPY and rapamycin (RAPA, 100 nM). Data
are given in percentage of the basal mean values. These correspond for
[14C]phenylalanine to
3.8 ± 0.1 dpm × 10 2/µg DNA and for RNA
content to 14.1 ± 0.7 µg RNA/µg DNA. Data are means ± SD;
n = 16. Differences from control,
* P < 0.05; + P < 0.05 from NPY.
|
|
Activation of MAP kinase and its influence on CK-BB induction.
MAP kinase activation is involved in the
-adrenoceptor-mediated
induction of CK-BB. It was therefore investigated whether this kinase
is also involved in a NPY-mediated increase in CK-BB activity. MAP
kinase activation was investigated by analysis of the reduced gel
mobility of the activated compared with nonactivated MAP kinase.
Representative Western blots are shown in Fig.
5. MAP kinase activation in the presence of
NPY was abolished by bisindolylmaleimide, indicating its dependency on
prior activation of PKC (Fig. 5A),
and PD-98059, an inhibitor of its upstream activator MAP kinase kinase
(Fig. 5B). MAP kinase activation in
the presence of NPY was not affected by PTx (Fig.
5A). The effect of NPY on MAP kinase
was compared with that of phorbol myristate acetate (100 nM). This
caused a larger activation of MAP kinase (Fig. 6).

View larger version (6K):
[in this window]
[in a new window]

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 5.
A: representative Western blot
indicating the activation of mitogen-activated protein (MAP) kinase by
NPY. MAP kinase activation was determined by reduced gel mobility of
the activated (phosphorylated) MAP kinase compared with nonactivated
(nonphosphorylated) MAP kinase. Activated and nonactivated MAP kinases
were determined by Western blotting of protein samples from
cardiomyocytes using a specific antibody against the p42 MAP kinase.
Cardiomyocytes were cultured for 15 min (control, C) alone or cultured
in the presence of NPY (100 nM), NPY and PTx (1 µg/ml), NPY and BIM
(5 µM), or phorbol myristate acetate (PMA, 100 nM).
B: representative Western blot
indicating the activation of MAP kinase by NPY. MAP kinase activation
was determined by reduced gel mobility of the activated
(phosphorylated) MAP kinase compared with nonactivated
(nonphosphorylated) MAP kinase. Activated and nonactivated MAP kinases
were determined by Western blotting of protein samples from
cardiomyocytes using a specific antibody against the p42 MAP kinase.
Cardiomyocytes were cultured for 15 min (control, C) or in the presence
of NPY (100 nM), NPY and PD-98059 (PD, 10 µM), PMA (100 nM), or PMA
(100 nM) and PD.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Activation of MAP kinase under control conditions (C) or after 15 min
in the presence of NPY (100 nM), NPY and PTx (1 µg/ml), NPY and BIM
(5 µM), or PMA (100 nM). For experiments performed in the presence of
PTx, cells were cultured for 6 h before the experiments with PTx. A
representative Western blot is given in Fig.
5A. Amount of MAP kinase activation
(MAP kinase*) is shown as the ratio of activated to total MAP kinase.
Data are means ± SD; n = 5. Differences from control, * P < 0.05; + P < 0.05 from
NPY.
|
|
As reported earlier (17), NPY induces cytosolic CK. Here, we show that
this effect is due to a selective induction of the fetal-type isoform
CK-BB (Fig. 7). PD-98059 (10 µM), which
abolished the activation of p42 MAP kinase by NPY, also abolished
induction of total CK and CK-BB by NPY (Fig. 7). PD-98059 did not
influence basal CK-BB activity in cardiomyocytes (0.94 ± 0.14 mU/mg protein vs. 1.03 ± 0.23 mU/mg protein).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
Creatine kinase (CK) activity of cardiomyocytes under control
conditions (C) or after 24-h incubations in the presence of NPY (100 nM) or in the presence of NPY and the MAP kinase kinase inhibitor
PD-98059 (PD, 10 µM). Activities of the specific isoforms (MM, MB,
and BB) were determined after ion-exchange chromatography of the
initial extract. Data are means ± SD;
n = 4. Differences from control,
* P < 0.05.
|
|
 |
DISCUSSION |
This study characterizes for the first time key steps of signal
transduction by which NPY exerts hypertrophic effects on cultured adult
ventricular cardiomyocytes. In the cardiomyocyte model investigated here, hypertrophic responsiveness to NPY is induced by the presence of
active TGF-
in culture media. In responsive cells, NPY activates two
major signal transduction pathways. The first pathway includes a
PTx-sensitive step and leads to stimulation of protein synthesis via an
activation of the PI
3-kinase/p70s6k pathway. Part of
this pathway (PI 3-kinase) is also involved in the NPY-mediated rise of
RNA mass. The second pathway, PTx insensitive, includes activation of
PKC and MAP kinase and leads to an induction of the fetal-type isoform
of CK, CK-BB. Figure 8 summarizes the
signal transduction pathways identified in this study.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 8.
Schematic view of signal transduction pathways used by NPY in adult
cardiomyocytes precultured in transforming growth factor- -containing
medium. NPY activates either PTx-sensitive G proteins
(Gi/Go)
or protein kinase C (PKC). Activation of
Gi/Go
proteins is linked to PI 3-kinase (PI3K) activation and subsequent
increase in protein synthesis. Activation of PKC is linked to
activation of MAP kinase and leads subsequently to induction of
CK-BB.
|
|
In a previous study, we showed that the hypertrophic responsiveness to
NPY requires a precultivation of cardiomyocytes in the presence of FCS
(35). The same requirement has also been described for the induction of
hypertrophic responsiveness to
2-adrenoceptor stimulation (22,
32, 38). For the latter case, we showed previously that the induction
of hypertrophic responsiveness depends on the activity of TGF-
in
the culture medium. TGF-
is released from cultured cardiomyocytes in
a latent form and must be activated. One major mechanism to activate
latent TGF-
is a proteolytic cleavage caused by the serine protease plasmin (15, 16). As shown before for other cell culture systems, the
activation of latent TGF-
can be abolished by addition of the serine
protease inhibitor aprotinin (5). Indeed, we found that presence of
aprotinin reduces TGF-
activity significantly compared with control
cultures. In cultures supplemented with aprotinin, the hypertrophic
responsiveness to NPY remained absent. Reduction of serum supplement to
the cultures also reduced the appearance of active TGF-
, most likely
due to a reduction in the proteolytic activity contained in the serum.
Under conditions with low TGF-
activity, obtained by either
reduction of the serum supplement or addition of aprotinin, the 6-day
cultured cardiomyocytes behave like newly isolated cardiomyocytes in
which NPY does not stimulate protein synthesis. The differences in the
absolute TGF-
activities seem small compared with the large
difference in the hypertrophic responsiveness. This suggests that the
induction of hypertrophic responsiveness to NPY requires exposure to a
threshold concentration of TGF-
and therefore does not follow a
linear dose-response relationship. In line with this argument is our previous observation that the dependency on serum concentration of
hypertrophic responsiveness to
-adrenoceptor stimulation does not
follow a linear dose-response curve but needs a minimum of serum (10%
FCS) (32). Taken together, we conclude that induction of hypertrophic
responsiveness to NPY in adult cardiomyocytes is caused by exposure to
active TGF-
.
In cardiomyocytes exposed to active TGF-
, NPY increases protein
synthesis in a PTx-sensitive way. In newly isolated cardiomyocytes not
exposed to TGF-
, NPY couples to PTx-sensitive G proteins exemplified
by its antiadrenergic effect on adenylate cyclase, which is inhibitable
by PTx (19). This PTx-sensitive effect is still present in cultured
cardiomyocytes exposed to TGF-
. A concentration of 100 nM NPY, which
represents the dose of NPY having maximal hypertrophic effect (20), had
a partially inhibitory effect on adenylate cyclase activation by
-adrenoceptor stimulation. The effect of NPY on protein synthesis is
also PTx sensitive but cannot be related to its inhibitory effect on
adenylate cyclase. This is because
1) in these cultures activation of
adenylate cyclase stimulates protein synthesis and NPY, which inhibits
this activation, does not antagonize this effect (20) and
2) NPY stimulates protein synthesis
in the absence of a prestimulated adenylate cyclase. The PTx-sensitive
G proteins involved in the stimulation of protein synthesis by NPY must
therefore be coupled either to signal transduction steps other than
adenylate cyclase or to another subset of NPY receptors. This point
requires further analysis.
It was also investigated in cultured adult cardiomyocytes which
intracellular signals follow the activation of PTx-sensitive G proteins
by NPY on the route to a stimulated protein synthesis. We demonstrated,
first, that NPY activates PI 3-kinase and, second, that inhibition of
PI 3-kinase (by wortmannin) blocks the stimulation of protein synthesis
under NPY. These results indicate that activation of PI 3-kinase
represents an intermediate step in the intracellular signaling toward
stimulation of protein synthesis under NPY. The intracellular targets
for PI 3-kinase have not been fully established. Various studies
suggest a downstream activation of
p70s6k, following PI 3-kinase
activation. p70s6k can be
specifically blocked by addition of rapamycin. Rapamycin reduced the
stimulatory effect of NPY on protein synthesis. In the same culture
model, rapamycin completely abolished the hypertrophic effect to
-adrenoceptor stimulation (25).
The increase in RNA mass under NPY did not strictly follow the PI
3-kinase and p70s6k pathway. This
is in contrast to the hypertrophic effects of
- or
-adrenoceptor
stimulation on cultured adult cardiomyocytes (25, 29). In the latter
cases, protein and RNA synthesis are both entirely inhibitable by
either wortmannin or rapamycin. In the case of NPY,
p70s6k inhibition by rapamycin
attenuates the effect on protein synthesis (translational activity) but
not RNA mass (translational capacity). The results indicate that the PI
3-kinase/p70s6k pathway toward
protein synthesis and control of RNA mass is subject to additional
regulatory mechanisms.
NPY activates PKC in cultured cardiomyocytes without involving a
PTx-sensitive step. This activation of PKC by NPY is not involved in
the growth induction under NPY. This represents a marked difference
between the signal transduction of NPY and
-adrenoceptor stimulation, since in the case of
-adrenoceptor stimulation the activation of PKC is causally linked to the stimulation of protein synthesis. Activation of PKC in cardiomyocytes by other hypertrophic agonists, e.g.,
-adrenoceptor agonists or parathyroid
hormone-related peptide, induce CK-BB in a MAP kinase-dependent way
(29, 31). Induction of the fetal BB isoform of CK is another feature of myocardial hypertrophy (33). NPY, like other PKC activators in adult
cardiomyocytes, activates MAP kinase and subsequently CK-BB in a MAP
kinase-dependent way. From these observations, one may hypothesize that
NPY activates only selected isoforms of PKC that are linked to
activation of MAP kinase and induction of CK-BB but not some other PKC
isoforms that are linked to the PI 3-kinase-dependent pathway toward
the regulation of protein synthesis.
In conclusion, this study shows for NPY a dissociation of intracellular
signals leading to induction of fetal-type CK-BB and increases in
protein synthesis and RNA mass. The effects of NPY on protein synthesis
and RNA mass require activation of PTx-sensitive G proteins, subsequent
PI 3-kinase activation, and, in part, activation of
p70s6k. Induction of CK-BB by NPY
requires an activation of PKC and, subsequently, of MAP kinase. The
importance of our results resides in the observation that NPY, unlike
other neurohumoral factors that are coreleased from nerve endings with
NPY, increases protein synthesis of cardiomyocytes by a mechanism
depending on PTx-sensitive G proteins. During the genesis of heart
failure, the expression of PTx-sensitive G proteins, e.g.,
Gi, is increased (13). It may be
the case, therefore, that the relative contribution of NPY to
myocardial hypertrophy increases in the failing heart.
 |
ACKNOWLEDGEMENTS |
This study was supported by the Deutsche Forschungsgemeinschaft,
project Pi 162/11-1.
 |
FOOTNOTES |
Address for reprint requests: K.-D. Schlüter, Physiologisches
Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen,
Germany.
Received 28 October 1997; accepted in final form 8 July 1998.
 |
REFERENCES |
1.
Absher, M.,
L. Baldor,
and
J. Kelly.
A rapid colorimetric bioassay for transforming growth factor
(TGF-
) using a microwell plate reader.
J. Immunol. Methods
138:
310-313,
1991.
2.
Bell, D.,
K.-D. Schlüter,
X. J. Zhou,
B. J. McDermott,
and
H. M. Piper.
Hypertrophic effects of calcitonin gene-related peptide (CGRP) and amylin on adult mammalian ventricular cardiomyocytes.
J. Mol. Cell. Cardiol.
27:
2433-2443,
1995[Medline].
3.
Bradford, M. M.
A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[Medline].
4.
Brown, N. A.,
G. McAllister,
D. Weinberg,
G. Milligan,
and
G. R. Seabrook.
Involvement of G-protein alphai1 subunits in activation of G-protein gated inward rectifying K+ channels (GIRK1) by human NPY1 receptors.
Br. J. Pharmacol.
116:
2346-2348,
1995[Abstract].
5.
Flaumenhaft, R.,
M. Abe,
P. Mignatti,
and
D. B. Rifkin.
Basic fibroblast growth factor-induced activation of latent transforming growth factor
in endothelial cells: regulation of plasminogen activator activity.
J. Cell Biol.
118:
901-909,
1992[Abstract].
6.
Gerhardt, W.
Creatine kinase.
In: Methods of Enzymatic Analysis (3rd ed.), edited by H. U. Bergmeyer. Weinheim, Germany: Verlag Chemie, 1983, vol. 3, p. 508-518.
7.
Giles, K. W.,
and
A. Myers.
An improved diphenylamine method for the estimation of deoxyribonucleic acid.
Nature
206:
93,
1965.
8.
Godfrey, K.
Comparing the means of several groups.
N. Engl. J. Med.
313:
1450-1456,
1985[Abstract].
9.
Gu, J.,
J. M. Polak,
J. M. Allen,
W. M. Huang,
M. N. Sheppard,
K. Takemoto,
and
S. R. Bloom.
High concentrations of a novel peptide, neuropeptide Y, in the innervation of mouse and rat heart.
J. Histochem. Cytochem.
32:
467-472,
1984[Abstract].
10.
Haneda, T.,
and
P. J. McDermott.
Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester.
Mol. Cell. Biochem.
104:
169-177,
1991[Medline].
11.
Henrich, M.,
H. M. Piper,
and
J. Schrader.
Evidence for cyclase coupled A1-adenosine receptors on ventricular cardiomyocytes from adult rat and dog heart.
Life Sci.
41:
2381-2388,
1987[Medline].
12.
Hulting, J.,
A. Sollevi,
B. Ullman,
A. Franco-Cereceda,
and
J. M. Lundberg.
Plasma neuropeptide Y on admission to coronary care unit: raised levels in patients with left heart failure.
Cardiovasc. Res.
24:
102-108,
1990[Medline].
13.
Kawamoto, H.,
M. Ohyanagi,
K. Nakamura,
J. Yamamoto,
and
T. Iwasaki.
Increased levels of inhibitory G protein in myocardium with heart failure.
Jpn. Circ. J.
58:
913-924,
1994[Medline].
14.
Kaye, A. M.,
N. Reiss,
A. Shaer,
M. Sluyser,
S. Iacobelli,
D. Amroch,
and
Y. Soffer.
Estrogen responsive creatine kinase in normal and neoplastic cells.
J. Steroid Biochem.
15:
69-75,
1981[Medline].
15.
Lyons, R. M.,
L. E. Gentry,
A. F. Purchio,
and
H. L. Moses.
Mechanism of activation of latent recombinant transforming growth factor
1 by plasmin.
J. Cell Biol.
110:
1361-1367,
1990[Abstract].
16.
Lyons, R. M.,
L. Keski-Oja,
and
H. L. Moses.
Proteolytic activation of latent transforming growth factors-
from fibroblast-conditioned medium.
J. Cell Biol.
106:
1659-1665,
1988[Abstract].
17.
McDermott, B. J.,
B. C. Millar,
and
H. M. Piper.
Cardiovascular effects of neuropeptide Y.
Cardiovasc. Res.
27:
893-905,
1993[Medline].
18.
McDermott, P. J.,
L. I. Rothblum,
S. D. Smith,
and
H. E. Morgan.
Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture.
J. Biol. Chem.
264:
18220-18227,
1989[Abstract/Free Full Text].
19.
Millar, B. C.,
H. M. Piper,
and
B. J. McDermott.
The antiadrenergic effect of neuropeptide Y on the ventricular cardiomyocyte.
Naunyn Schmiedebergs Arch. Pharmacol.
338:
426-429,
1988[Medline].
20.
Millar, B. C.,
K.-D. Schlüter,
X. J. Zhou,
B. J. McDermott,
and
H. M. Piper.
Neuropeptide Y stimulates hypertrophy of adult ventricular cardiomyocytes.
Am. J. Physiol.
266 (Cell Physiol. 35):
C1271-C1277,
1994[Abstract/Free Full Text].
21.
Millar, B. C.,
T. Weis,
H. M. Piper,
U. Borchard,
B. J. McDermott,
and
A. Balasubramaniam.
Positive and negative contractile effects of neuropeptide Y on ventricular cardiomyocytes.
Am. J. Physiol.
261 (Heart Circ. Physiol. 30):
H1727-H1733,
1991[Abstract/Free Full Text].
22.
Pinson, A.,
K.-D. Schlüter,
P. Schwartz,
G. Kessler-Icekson,
and
H. M. Piper.
- and
-Adrenergic stimulation of protein synthesis in cultured adult ventricular cardiomyocytes.
J. Mol. Cell. Cardiol.
25:
477-490,
1993[Medline].
23.
Piper, H. M.,
B. C. Millar,
and
B. J. McDermott.
The negative inotropic effect of neuropeptide Y on the ventricular cardiomyocyte.
Naunyn Schmiedebergs Arch. Pharmacol.
340:
333-337,
1989[Medline].
24.
Piper, H. M.,
I. Probst,
P. Schwartz,
J. F. Hütter,
and
P. G. Spieckermann.
Culturing of calcium stable adult cardiac myocytes.
J. Mol. Cell. Cardiol.
14:
397-412,
1982[Medline].
25.
Piper, H. M.,
and
K.-D. Schlüter.
Signal transduction in catecholamine-induced hypertrophy of adult cardiomyocytes (Abstract).
J. Mol. Cell. Cardiol.
29:
A26,
1997.
26.
Piper, H. M.,
R. Spahr,
S. Mertens,
A. Krützfeldt,
and
H. Watanabe.
Microvascular endothelial cells from heart.
In: Cell Culture Techniques in Heart and Vessel Research, edited by H. M. Piper. Heidelberg, Germany: Springer-Verlag, 1990, p. 158-177.
27.
Piper, H. M.,
and
A. Volz.
Adult ventricular rat heart muscle cells.
In: Cell Culture Techniques in Heart and Vessel Research, edited by H. M. Piper. Heidelberg, Germany: Springer-Verlag, 1990, p. 158-177.
28.
Schlüter, K.-D.,
and
H. M. Piper.
Trophic effects of catecholamines and parathyroid hormones on adult ventricular cardiomyocytes.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H1739-H1746,
1992[Abstract/Free Full Text].
29.
Schlüter, K.-D., M. Schäfer, G. Taimor, and H. M. Piper. Intracellular signaling of
-adrenoceptor mediated
hypertrophy (Abstract). Pflügers Arch.
Europ. J. Physiol. 433, Suppl. 33, Suppl. 1: R50, 1997.
30.
Schlüter, K.-D.,
M. Weber,
and
H. M. Piper.
Parathyroid hormone induces protein kinase C but not adenylate cyclase in adult cardiomyocytes and regulates cyclic AMP levels via protein kinase C-dependent phosphodiesterase activity.
Biochem. J.
310:
439-444,
1995[Medline].
31.
Schlüter, K.-D.,
M. Weber,
and
H. M. Piper.
Effects of PTH-rP(107-111) and PTH- rP(7-34) on adult cardiomyocytes.
J. Mol. Cell. Cardiol.
29:
3057-3065,
1997[Medline].
32.
Schlüter, K.-D.,
X. J. Zhou,
and
H. M. Piper.
Induction of hypertrophic responsiveness to isoproterenol by TGF-
in adult rat cardiomyocytes.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1311-C1316,
1995[Abstract/Free Full Text].
33.
Smith, S. H.,
M. F. Kramer,
I. Reis,
S. P. Bishop,
and
J. S. Ingwall.
Regional changes in creatine kinase and myocyte size in hypertensive and nonhypertensive cardiac hypertrophy.
Circ. Res.
67:
1334-1344,
1990[Abstract].
34.
Sternini, C.,
and
N. Brecha.
Distribution and co-localization of neuropeptide-Y and tyrosine hydroxylase-like immunreactivity in the guinea-pig heart.
Cell Tissue Res.
241:
93-102,
1985[Medline].
35.
Westfall, T. C., S. P. Han, M. Knuepfer, J. Martin, X. Chen, K. Del Valle, A. Ciarleglio, and L. Naes.
Neuropeptides in hypertension: role of neuropeptide Y and
calcitonin gene related peptide. Br. J. Pharmacol. 30, Suppl.
75S-82S, 1990.
36.
Whitman, M.,
D. R. Kaplan,
B. Schaffhausen,
L. Cantley,
and
T. M. Roberts.
Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation.
Nature
315:
239-242,
1985[Medline].
37.
Xiang, H.,
and
J. C. Brown.
Inhibitory effect of neuropeptide Y and its analogue on inositol 1,4,5-trisphosphate level in rat cardiomyocytes.
Receptors Channels
1:
315-321,
1993[Medline].
38.
Zhou, X. J.,
K.-D. Schlüter,
and
H. M. Piper.
Hypertrophic responsiveness to
2-adrenoceptor stimulation on adult ventricular cardiomyocytes.
Mol. Cell. Biochem.
163/164:
211-216,
1996.
Am J Physiol Cell Physiol 275(5):C1207-C1215
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society