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Cardiovascular Research 2002 53(4):879-887; doi:10.1016/S0008-6363(01)00517-X
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Copyright © 2002, European Society of Cardiology

Neuropeptide Y modifies the hypertrophic response of adult ventricular cardiomyocytes to norepinephrine

Marat Kanevskij, Gerhild Taimor, Matthias Schäfer, Hans Michael Piper and Klaus-Dieter Schlüter*

Physiologisches Institut, Justus-Liebig-Universität Gießen, Aulweg 129, D-35392 Gießen, Germany

klaus-dieter.schlueter{at}physiologie.med.uni-giessen.de

* Corresponding author. Tel.: +49-641-9947-212; fax: +49-641-9947-239

Received 9 May 2001; accepted 26 October 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: The hypertrophic response of adult rat cardiomyocytes to norepinephrine via {alpha}-adrenoceptor stimulation is limited by an inhibitory cross-talk of simultaneously β-adrenoceptor stimulation. On the other hand, neuropeptide Y (NPY), known to be co-secreted with norepinephrine from intramural nerve endings of the heart, exerts an anti-β-adrenergic effect. Therefore, it should be expected that NPY enhances the hypertrophic response to norepinephrine. This hypothesis was addressed in the present study. Methods: Isolated adult ventricular cardiomyocytes from rats were used. As parameters of hypertrophic growth we investigated cell volume, cross-sectional area, protein mass. Protein and RNA synthesis were determined by incorporation of [14C]phenylalanine or [14C]uridine, respectively. Results: Norepinephrine (1 µmol/l) did not significantly increase protein or RNA synthesis. In co-presence of NPY (100 nmol/l), however, norepinephrine increased protein synthesis by 44% and RNA synthesis by 18%. Under the same conditions, NPY enhanced the effect of norepinephrine on cell volume from +6.4 to +18.2%, its effect on cross-sectional area from +16 to +23%, and increased the protein/DNA ratio from 32.5 to 35.6 mg/mg. In parallel, norepinephrine caused a translocation of PKC-{alpha} and PKC-{delta} into the particular fractions and this effect of norepinephrine was also enhanced by co-presence of NPY. In contrast, NPY did not enhance ERK-activation caused by norepinephrine. Conclusion: Our study indicates the anti-β-adrenergic effect of NPY is sufficient to modulate the hypertrophic response of adult ventricular cardiomyocytes to norepinephrine. The results suggest that the hypertrophic effect of norepinephrine via {alpha}-adrenoceptor stimulation can be modulated by co-release of NPY from intramural nerve endings.

KEYWORDS Adrenergic (ant)agonists; Hypertrophy; Myocytes; Neurotransmitters; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Increased plasma levels of catecholamines are commonly found under conditions leading to myocardial hypertrophy (reviewed in Refs. [1,2]). In an experimental rat heart system of pressure induced myocardial hypertrophy, namely spontaneously hypertensive rats, myocardial norepinephrine concentrations were found to be increased from 1.93 nmol/g wet weight to 2.87 nmol/g wet weight [3]. This locally released norepinephrine is thought to act directly on ventricular cardiomyocytes by stimulation of {alpha}- and β-adrenoceptors. Selective stimulation of {alpha}1-adrenoceptors increases cellular synthesis of protein and RNA in adult ventricular cardiomyocytes, which represents hallmarks of myocardial hypertrophy [4–6]. To a lesser extent, selective stimulation of β1-adrenoceptors can also stimulate protein synthesis in adult ventricular cardiomyocytes [7]. We have recently shown that, in case of simultaneous stimulation of {alpha}1- and β1-adrenoceptors, β1-adrenoceptor stimulation attenuates the hypertrophic response evoked by {alpha}1-adrenoceptor stimulation [8]. This inhibitory receptor cross-talk is mediated by a cyclic AMP-dependent inhibition of protein kinase C (PKC) activation. For the analysis of this cross-talk we applied norepinephrine in presence of various β1-adrenoceptor antagonists, i.e., propranolol, CPG20712A, or atenolol. The effect of the β1-adrenoceptor antagonists could be mimicked by Rp-cAMPS (adenosine-3',5'-cyclic phosphorothiolate-Rp), an inhibitor of cyclic AMP-dependent protein kinase activation. These results showed that the cross-talk between β1- and {alpha}1-adrenoceptor stimulation is mediated through cyclic AMP.

Neuropeptide Y (NPY) is abundant in myocardial tissue, where most of this neuropeptide is stored and can be released from intramural nerve endings together with norepinephrine (reviewed in Ref. [9]). Significant amounts of either norepinephrine or NPY are released from the transmural nerve endings, because direct stimulation of the left stellate ganglion of guinea pig hearts resulted in an overflow of on average 180 pmol/g norepinephrine and 210 fmol/g NPY [10]. Unlike norepinephrine, which has a half-life of 1–2 min in the plasma, NPY has a half-life of 20 min. Therefore, although release of norepinephrine exceeds that of NPY, plasma concentrations are NPY significantly increased in case of excess NPY release [11]. The most consistent finding regarding NPY levels in cardiovascular disease states seems to be a correlation between plasma concentrations of the peptide and the severity of heart failure [12]. The plasma levels were found increased from on average 20–100 pmol/l.

NPY does not evoke hypertrophic effects on newly isolated cardiomyocytes, nor does it directly modify the contractile response of cardiomyocytes [13–15]. However, NPY can bind to specific receptors on cardiomyocytes and by stimulating these receptors it exerts an anti β-adrenergic effect [14]. The anti-β-adrenergic effect of NPY is mainly mediated by inhibition of the β-adrenoceptor-dependent activation of adenylate cyclase. Therefore, by local release of NPY from intramural nerve endings of the myocardium, NPY seems to represent an endogenous factor in the myocardium, released from intramural nerve endings, which is able to modify the hypertrophic response of cardiomyocytes to norepinephrine. Based on the previous arguments it should be expected that NPY enhances the hypertrophic response to norepinephrine. This hypothesis was addressed in the study presented here.

A well-defined experimental model of cultured adult ventricular cardiomyocytes from rats was used. Myocardial hypertrophy requires an induction of translational activity and translational capacity, which can be quantified in this cell culture model by incorporation of [14C]phenylalanine or [14C]uridine, respectively. Induction of [14C]uridine incorporation indicates de novo synthesis of RNA, which suggests de novo synthesis of ribosomes, although it does not discriminate between de novo synthesis of tRNA, mRNA, and rRNA. To show directly de novo formation of ribosomes, we investigated whether {alpha}-adrenoceptor stimulation activates the transcription factor IIIA (TFIIIA), which is a basal transcription factor of RNA polymerase III. It is specific for 5S rRNA gene transcription [16] and initiates assembly of the transcription factor complex by binding directly to the internal control region of the gene [17]. 5S rRNA binds to ribosomal protein L5 to form a 5S ribonucleoprotein that is believed to be a precursor for incorporation into the large subunit of ribosomes [18].

Further parameters of hypertrophy were cellular protein mass, cell volume, and cross-sectional area of the cardiomyocytes. Induction of fetal-type isoforms of creatine kinase (CK), i.e., CK-BB, is a common feature of the hypertrophic response of adult ventricular cardiomyocytes stimulated by {alpha}-adrenoceptor stimulation. In the case of {alpha}-adrenoceptor stimulation the induction of CK-BB is mediated through activation of PKC and a consecutive activation of the early response kinase [5,19]. Therefore, we investigated also how stimulation of NPY receptors affects the activation of ERK evoked by norepinephrine.

Four experimental series were performed. In the first series, it was investigated whether NPY enhances the hypertrophic effect of the natural catecholamine norepinephrine. In the second series of experiments, the effect of NPY on PKC activation caused by norepinephrine was analyzed. In the third series of experiments, we analyzed the effects of NPY on ERK activation. Finally, we investigated the effect of NPY on protein synthesis and ERK activation when {alpha}-adrenoceptor stimulation was replaced by direct stimulation of PKC with phorbol myristate acetate (PMA). The effects of NPY were compared to those achieved by pharmacological inhibition of β1-adrenoceptors with atenolol.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Cell culture
Ventricular heart muscle cells were isolated from 200- to 250-g male Wistar rats as previously described [20]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Isolated cells were suspended in fetal calf serum (FCS)-free culture medium and plated at a density of 1.4x105 elongated cells/35-mm culture dish (Falcon type 3001). The culture dishes had been pre-incubated overnight with 4% FCS in 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-β-D-arabinofuranoside.

Four hours after plating, cultures were washed twice with culture medium to remove round and nonattached cells and supplied with FCS-free experimental media, in which cells were incubated for a 24-h period at 37°C. The experiments were carried out in basic culture medium (control), with additions of the agonists at concentrations indicated. Ascorbic acid (100 µmol/l) was added to all cultures as an antioxidant.

2.2. Incorporation of phenylalanine and uridine 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 the incorporation of radioactivity into acid-insoluble cell mass was determined as described previously [5]. 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 washed three times with ice-cold phosphate-buffered saline (PBS; composition in mmol/l: 1.5 KH2PO4, 137 NaCl, 2.7 KCl, and 1.0 Na2HPO4, pH 7.4). Subsequently, ice-cold 10% (w/v) trichloroacetic acid was added. After storage overnight at 4°C, the acid was removed from the dishes. Radioactivity contained in this acid fraction was taken to present 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% (w/v) sodium dodecyl sulfate (SDS) by an incubation for 2 h at 37°C. We showed before that the incorporation of phenylalanine into cell protein is linear within the first 24 h [21]. In these samples protein contents [22] and DNA contents [23] were determined, and the radioactivity was counted. RNA was determined from an aliquot of these samples after precipitation with an equal volume of 10% (w/v) perchloric acid in the remaining supernatant [24]. Incorporation of uridine into cells was determined as described previously [24] by exposing cultures to L-[14C]uridine (0.1 µCi/ml) for 6 h and the incorporation of radioactivity into acid-insoluble cell mass was calculated. The radioactivity was determined in the aliquots used to prepare RNA as described above. We showed before that the incorporation of [14C]uridine into RNA is linear within the first 6 h [21].

2.3. Cell morphology
Cell morphology was determined as described earlier [7]. Myocytes growth was determined on phase-contrast micrographs recorded on tape using a CCD video camera. Cell volumes were calculated by the following formula: Volume=(radius)2x{pi}xlength, assuming a cylindrical cell shape. Cross-sectional area was determined by the following formula: cross-sectional area=(radius)2x{pi}.

2.4. Protein kinase C translocation
As a parameter of protein kinase C (PKC) activation its translocation into the particular fraction was investigated as described previously [8]. Briefly, cardiomyocytes were treated for 5 min with norepinephrine with or without pretreatment with NPY or atenolol. Thereafter, the cultures were washed twice with ice-cold phosphate-buffered solution (PBS), scrapped off in PBS and centrifuged for 2 min at 12 000xg. The remaining pellet was redissolved in 100 µl of lysis buffer (composition in mmol/l: Tris 20, EGTA (ethylene glycol-bis(β-amino ethyl ether)N,N,N',N'-tetraacetic acid) 10, EDTA 2, sucrose 200, PMSF (phenylmethyl sulfonyl fluoride) 0.01, pH 7.4) and stored at –20°C. Thereafter, the solution was mixed vigorously and centrifuged again for 1 h at 38 000xg. The remaining pellet was used as the particular fraction, redissolved in 100 µl lysis buffer to which Triton X-100 was added (final concentration 0.1%, v/v). The solution was incubated again for 2 h at 4°C. Thereafter, the solution was centrifuged again for 15 min with 12 000xg at 4°C. This supernatant was mixed with 20 µl Laemmli buffer. The samples were heated for 5 min at 95°C and used for gel electrophoresis. The samples were loaded on a 12.5% SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and after electrophoretic separation they were transferred on an immobilon-P membrane. The bands were immunoblotted with antibodies directed against PKC-{alpha} and PKC-{delta} and visualized via alkaline phosphatase reaction. The intensity of the final bands was analyzed densitometrically using Image-Quant software.

2.5. Determination of ERK2 activation
The determinations of ERK2 were done as described before [19]. Briefly, after stimulation cells were lysed in lysis buffer (composition: 50 mmol/l Tris–Cl, pH 6.7, 2% (w/v) sodium dodecyl sulfate, 2% (v/v) mercaptoethanol, 1 mmol/l 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% (w/v) bovine serum albumin and incubated for 2 h with rabbit polyclonal anti rat ERK2 (10 µg/50 ml, Santa Cruz Biotechnology, USA). After washing, sheep anti-rabbit IgG alkaline phosphatase-labelled (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 expressed as the ratio of the upper band, with retarded gel mobility of activated and phosphorylated ERK2, to the total amount of ERK2 determined on the Western blots.

2.6. TFIIIA activity
The binding activity of TFIIIA to the h5S rRNA gene was determined by electromobility shift assay. Nuclear extracts from isolated cardiomyocytes were prepared and incubated with 32P-labeled h5S rRNA for 30 min at 30°C in 60 mmol/l KCl, 20 mmol/l Tris (pH 7.4), 5 mmol/l MgCl2, 3 mmol/l dithiothreitol, 0.2 mmol/l phenylmethylsulfonyl fluoride, 10% (v/v) glycerin, and 0.5 µg/ml poly dIdC. Samples were loaded on 4% (w/v) native polyacrylamide gels and separated by electrophoresis. The gels were dried and exposed on a phosphor imager. Binding of TFIIIA to the 5hS rRNA promoter resulted in retardation of the protein–DNA complex in comparison to the free DNA. Shift activity was densitometrically analyzed and related to protein contents.

2.7. Creatine kinase BB activation
Specific activity of creatine kinase BB (CK-BB) was determined as described before (see Ref. [5] for details). Briefly, cell samples were generated by homogenization of cell cultures in buffer A (composition in mmol/l: 5 magnesium acetate, 0.4 EDTA, 2.5 dithiothreitol, 50 Tris, 250 sucrose, pH 6.8). The samples were applied to a 1 ml DEAE-cellulose column that had been equilibrated with buffer B (composition in mmol/l: 20 NaCl, 5 magnesium acetate, 0.4 EDTA, 100 Tris, pH 7.9). The CK-MM isoenzyme eluted directly with buffer B, the CK-MB isoenzyme with change of NaCl concentration to 40 mmol/l and pH to 6.4, and CK-BB with change of NaCl concentration to 250 NaCl and pH to 6.4. The activity was determined using standard ultraviolet methods.

2.8. Statistics
Data are given as means±S.E. from n different culture preparations. Statistical comparisons were performed by one-way analysis of variance and use of the Student–Newman–Keuls test for post hoc analysis [25]. Differences with P<0.05 were regarded as statistical significant.

2.9. Materials
Falcon tissue culture dishes were obtained from Becton–Dickinson (Heidelberg, Germany). Boehringer Mannheim (Mannheim, Germany) was the source for glutamine-free medium 199 and fetal calf serum. Cytosine-β-D-arabinofuranoside, L-carnitine, creatine, taurine, L-phenylephrine hydrochloride, and phorbol myristate acetate were obtained from Sigma (Deisenhofen, Germany). L-Norepinephrine bitartrate was purchased from Serva (Heidelberg, Germany). All other chemicals were of analytical grade.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Influence of NPY on phenylalanine incorporation in the presence of norepinephrine
As a parameter of protein synthesis, [14C]phenylalanine incorporation into cell protein was determined. Norepinephrine (1 µmol/l) did not significantly increase [14C]phenylalanine incorporation (Fig. 1). However, in co-presence with NPY, norepinephrine increased [14C]phenylalanine incorporation. This effect of NPY was concentration-dependent. At 100 nmol/l, NPY enhanced [14C]phenylalanine incorporation by 44%. A similar effect was found, when norepinephrine was applied in co-presence with the β1-adrenoceptor antagonist atenolol (10 µmol/l) (Fig. 1). On the basis of the concentration–response relationship shown in Fig. 1 an EC50 of approximately 30 nmol/l for NPY was calculated. NPY did not change the induction of [14C]phenylalanine incorporation under selective {alpha}-adrenoceptor stimulation. Phenylephrine alone increased [14C]phenylalanine incorporation from 742±84 to 1074±175 dpm/µg DNA (P<0.05 versus control) and to 1176±70 dpm/µg DNA in co-presence of NPY (P<0.05 versus control, n.s. versus phenylephrine alone, each n=4).


Figure 1
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  		Fig. 1 Incorporation of [14C]phenylalanine (Phe) of cardiomyocytes cultured for 20 h in the presence of norepinephrine (Nor, open bar), Nor plus NPY (hatched bars), or Nor plus atenolol (Ate, black bar) at concentrations as indicated. Data are expressed as percentages relative to the control values. The 100% value corresponds to 798±64 dpm/µg DNA. Data are means±S.E. from n=6 cultures. *P<0.05 versus control; #P<0.05 versus Nor.

 
We showed previously that NPY alone did not increase protein synthesis in these cell preparations but antagonizes β-adrenoceptor-dependent activation of adenylate cyclase [13,14]. In the light of our previous study [8], indicating a cAMP-dependent receptor cross-talk between {alpha}- and β-adrenoceptors, we assumed that the effect of NPY on [14C]phenylalanine incorporation in presence of norepinephrine is caused by the inhibitory effect of NPY on β-adrenoceptor-dependent adenylate cyclase activation. It is in line with these suggestions, that co-presence of Sp-cAMPS, an activator of cAMP-dependent protein kinase, blocked the effect of either NPY or atenolol on norepinephrine-dependent increases in protein synthesis (Fig. 2).


Figure 2
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  		Fig. 2 Incorporation of [14C]phenylalanine (Phe) of cardiomyocytes cultured for 20 h in the presence of norepinephrine (Nor, 1 µmol/l, open bar), Nor plus NPY (100 nmol/l, hatched bar), Nor plus Sp-cAMPS (100 µmol/l, black bar), Nor plus atenolol (Ate, 10 µmol/l, hatched bar), Nor plus atenolol and SP-cAMPS (black bar), or SP-cAMPS alone (black bar). Data are expressed as percentages relative to the control values. The 100% value corresponds to 683±34 dpm/µg DNA. Data are means±S.E. from n=6 cultures. *P<0.05 versus control.

 
3.2. Influence of NPY on the hypertrophic responses caused by norepinephrine
The effects of norepinephrine (1 µmol/l), norepinephrine plus NPY (100 nmol/l), or norepinephrine plus atenolol (10 µmol/l) on cell shapes were also investigated. Compared to untreated control cells, norepinephrine increased cell volume by 6.4%; norepinephrine plus NPY, however, increased it by 18.2% and norepinephrine plus atenolol by 36.1% (Fig. 3). Cross-sectional area of the cells was increased by 16% compared to untreated control cells in the presence of norepinephrine, by 23% in the presence of norepinephrine plus NPY, and by 46% in the presence of norepinephrine plus atenolol. Fig. 4 shows representative pictures of cardiomyocytes analyzed after 24 h cultivation without further addition, or in presence of norepinephrine, norepinephrine plus NPY, or norepinephrine plus atenolol. Protein mass of cardiomyocytes was significantly increased by norepinephrine in co-presence of either NPY or atenolol compared to norepinephrine alone (Table 1).


Figure 3
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  		Fig. 3 Cell volume and cross-sectional area of cardiomyocytes incubated for 20 h in the presence of norepinephrine (Nor, 1 µmol/l, open bar), Nor plus NPY (100 nmol/l, hatched bar), or Nor plus atenolol (Ate, 10 µmol/l, black bar). Cell pictures were recorded on a videotape, from which both cell volume and cross-sectional area were calculated. The 100% value corresponds to 19 091±1076 µm3 (cell volume) and 216.9±14.6 µm2 (cross-sectional area). Data represent means±S.E. from 76 to 92 cells. *P<0.05 versus control; #P<0.05 versus Nor.

 

Figure 4
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  		Fig. 4 Representative cell pictures of cardiomyocytes cultured for 20 h under control conditions (A), in the presence of norepinephrine (Nor, 1 µmol/l; B), Nor plus NPY (100 nmol/l; C), or Nor plus atenolol (Ate, 10 µmol/l, D). The bar indicates 100 µm.

 

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  		Table 1 Influence of NPY on norepinephrine-induced protein mass

 
The RNA synthesis in cardiomyocytes was estimated by determination of the 6 h incorporation of [14C]uridine into RNA (Table 2). In the presence of norepinephrine (1 µmol/l) [14C]uridine incorporation was not increased compared to untreated cultures. When norepinephrine was administered in the presence of NPY (100 nmol/l), the increment in [14C]uridine incorporation became enhanced by 18%, and when norepinephrine was administered in the presence of atenolol (10 µmol/l), it was enhanced by 26%. Similar results were found for selective stimulation of {alpha}-adrenoceptors by phenylephrine or direct stimulation of protein kinase C by phorbol myristate acetate (PMA). To prove that the increase in [14C]uridine incorporation reflects indeed an increase in translational capacity, we further investigated whether {alpha}-adrencoeptor stimulation activates TFIIIA. Phenylephrine (10 µmol/l) increased TFIIIA activity 1.87±0.28-fold and PMA (100 nmol/l) 1.79±0.21-fold (each n=3, P<0.05 versus control). Fig. 5 shows a representative retardation gel indicating an upper band (TFIIIA shift) representing the protein–DNA complex and a lower band representing free h5S rRNA.


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  		Table 2 Influence of NPY on norepinephrine-induced RNA synthesis

 

Figure 5
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  		Fig. 5 Representative retardation gel indicating an upper band (TFIIIA-Shift) representing the protein–DNA complex and a lower band representing free h5S rRNA.

 
3.3. Influence of NPY on PKC translocation and ERK activation caused by norepinephrine
The next series of experiments investigated whether co-presence of NPY influences the early events in {alpha}-adrenoceptor mediated hypertrophy. As illustrated in Fig. 6, atenolol and NPY augmented the norepinephrine-induced translocation of PKC-{alpha} and PKC-{delta} into the particulate fraction. Different results were obtained in case of ERK activation. As illustrated in Fig. 7, norepinephrine (1 µmol/l) activated ERK2 to a small extent. This was not changed in co-presence of NPY. However, in co-presence of atenolol, norepinephrine activated ERK2 significantly stronger. Norepinephrine (1 µmol/l) also induced creatine kinase-BB activity from 0.51±0.09 to 1.03±0.08 U/mg protein (P<0.05), but not in co-presence of PD98059 (10 µmol/l), an inhibitor of ERK activation (0.62±0.14 U/mg protein, n.s. versus control, each n=8).


Figure 6
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  		Fig. 6 Western immunoblots indicating the translocation of protein kinase C (PKC) isoforms {alpha} and {delta} into the particular fraction. Cells were incubated for 5 min (control, C), with norepinephrine (Nor, 1 µmol/l), Nor plus NPY (100 nmol/l), or Nor plus atenolol (Ate, 10 µmol/l).

 

Figure 7
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  		Fig. 7 ERK activation in cardiomyocytes cultured under control conditions (C, open bar), in the presence of norepinephrine (Nor, 1 µmol/l, hatched bar), Nor plus NPY (100 nmol/l, hatched bar), or Nor plus atenolol (Ate, 10 µmol/l, black bar). (A) Representative Western blot indicating appearance of unphosphorylated ERK (ERK2) or phosphorylated ERK (ERK2-P). Phosphorylation of ERK2 is indicated by retarded gel mobility. (B) Quantitative analysis of these experiments. ERK activation is expressed as percentage of phosphorylated ERK relative to total ERK. Data are means±S.E. from n=4 experiments. *P<0.05 versus control; #P<0.05 versus norepinephrine.

 
3.4. Influence of β1-adrenoceptor stimulation on ERK activation and protein synthesis
In the last set of experiments, it was investigated whether the different outcome of the experiments found for the effects of NPY or atenolol on norepinephrine-induced ERK activation are caused by differences in the intracellular cascade by which β-adrenoceptor stimulation cross-reacts with these pathways. In this set of experiments, {alpha}-adrenoceptor stimulation caused by norepinephrine was replaced by direct stimulation of PKC with PMA. PMA (100 nmol/l) increased [14C]phenylalanine incorporation by 58±12%. In the presence of isoprenaline, which was used to co-stimulate β1-adrenoceptors, PMA still increased [14C]phenylalanine incorporation by 53±24% (n=6, n.s. from PMA alone). In contrast, ERK activation caused by PMA was inhibited by co-presence of isoprenaline (Fig. 8).


Figure 8
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  		Fig. 8 ERK activation in cardiomyocytes cultured under control conditions, in the presence of phorbol myristate acetate (PMA, 100 nmol/l), PMA plus isoprenaline (ISO, 1 µmol/l), or PMA and ISO plus NPY (100 nmol/l). Representative Western blot indicating appearance of unphosphorylated ERK (ERK) or phosphorylated ERK (ERK-P). Phosphorylation of ERK2 is indicated by retarded gel mobility.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The central question of this study was whether NPY modulates the influence of norepinephrine on translational activity in adult ventricular cardiomyocytes. The results of the present study show that this was indeed the case. The changes described here for [14C]phenylalanine incorporation were paralleled by changes in cellular protein mass, cell volume, cross-sectional area, and RNA synthesis. Increases in protein synthesis and RNA synthesis are indicators for myocardial hypertrophy. They reflect increases in translational activity and capacity. The results of our study suggest that the hypertrophic effect of norepinephrine may be modulated by co-secretion of NPY from the intramural nerve-endings. This leads to the conclusion that the ratio of norepinephrine and its co-secreted neurotransmitter rather than the local concentration of norepinephrine limits the hypertrophic response evoked by catecholamines. The results also give a new insight into the role of NPY release for the myocardial hypertrophy and subsequently heart failure.

In a previous study we have shown that β1-adrenoceptor stimulation attenuated the hypertrophic response to {alpha}1-adrenoceptor stimulation. This inhibitory effect of β1-adrenoceptor stimulation requires an activation of the cyclic AMP-dependent protein kinase (PKA) system, because dibutyryl-cyclic AMP mimicked the inhibitory effect of β1-adrenoceptor stimulation and the PKA-inhibitor Rp-cAMPS significantly enhanced norepinephrine-evoked increase in protein synthesis [8]. The easiest explanation for the observed effect of NPY on norepinephrine-dependent increases in protein synthesis, is the inhibitory effect of NPY on adenylate cyclase stimulation caused by β1-adrenoceptor stimulation [14]. It is in line with this suggestion, that the EC50 for the inhibitory effect of NPY on adenylate cyclase activation caused by isoprenaline ({approx}50 nmol/l, Ref. [14]) is in the same order of magnitude than that found for its growth-promoting effect on norepinephrine ({approx}30 nmol/l, this study). Furthermore, stimulation of cAMP-dependent protein kinase by Sp-cAMPS blocked the effect of NPY on norepinephrine-dependent activation of protein synthesis. In some aspects, the growth-promoting effect of NPY in presence of norepinephrine was weaker than that evoked by inhibition of β-adrenoceptors with atenolol. However, NPY inhibited the β-adrenoceptor-mediated adenylate cyclase activation only to approximately 70% [14]. Therefore, one cannot expect an effect as strong as that evoked by atenolol, which completely inhibits β-adrenoceptors. The fact, that increases in cell mass and volume do not directly follow in a quantitative way increases in protein synthesis may also indicate that on the cell culture model used in this study factors influence the balance of protein synthesis and degradation independently from the effects on translational activity.

In the presence of norepinephrine, NPY and atenolol were found to increase PKC translocation into the particular fraction. This suggests again a common mechanism by which both agents enhance the hypertrophic effect of norepinephrine. Fig. 9 summarizes the supposed signaling pathways for the post-receptor cross-talk between {alpha}-, β-adrenoceptors and NPY receptors.


Figure 9
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  		Fig. 9 Schematic drawing summarizing the conclusions performed on the basis of the findings of this study. Stimulation of cardiac NPY receptors (NPY-R) leads to inhibition of cyclic AMP-dependent protein kinase (PKA) activation caused by β1-adrenoceptor (β1-AR) stimulation. PKA activation suppresses protein kinase C (PKC) activation caused by {alpha}-adrenoceptor stimulation ({alpha}1-AR). PKC activation leads to subsequent activation of PI3-kinase, p70s6k, and acceleration of protein synthesis (see Refs. [19,28]). PKC activation also leads to raf-dependent ERK activation and ERK-dependent induction of creatine kinase B (CK-B, see Refs. [5,26,27]).

 
Another aspect of cardiac hypertrophy is the re-expression of fetal-type proteins, i.e., creatine kinase B. We have recently shown for selective {alpha}-adrenoceptor stimulation, that this response depends on ERK-activation in contrast to the effects on protein synthesis [19]. {alpha}-Adrenoceptor stimulation activates ERK in a PKC-dependent way. β-Adrenoceptor inhibition or NPY enhanced PKC translocation caused by norepinephrine (this study). Therefore, one would expect an increased ERK activation, too. This was found for atenolol but not for NPY. It should be kept in mind that NPY reduces adenylate cyclase activation by 70%, but does not completely abolish it [14]. Our experiments in this study with PMA show that PKC-dependent ERK activation can be inhibited in a cAMP-dependent way distal of PKC, because isoprenaline inhibited the response to PMA. Therefore, although PKC activation by norepinephrine in co-presence of NPY is increased the remaining adenylate cyclase activation might inhibit the subsequent ERK activation. Biochemical studies with phorbol esters have recently indicated a PKC-dependent Raf-1 activation which leads to a subsequent ERK activation [26]. It should be noted that Raf-1 activation can be effectively inhibited in a cAMP-dependent way [27]. Our study would be in line with these suggestions.

The influence of differences in ERK activation between norepinephrine in co-presence with atenolol and norepinephrine in co-presence of NPY needs to be elucidated. We have shown that re-expression of CK-B requires ERK activation. However, we have also demonstrated that norepinephrine induces CK-B even at 1 µmol/l to the same extent than selective stimulation of {alpha}-adrenoceptors [5]. Strong activation of ERK seems not to be required for re-expression of CK-B. One may speculate that other genes, which are controlled by the PKC–ERK-pathway depend on stronger activation of this pathway, but such genes remain to be identified. In contrast to ERK activation, two previous studies and this study show that the same concentration of norepinephrine (1 µmol/l) does not increase ERK-independent protein synthesis to a similar extent as selective {alpha}-adrenoceptor stimulation [5,8]. One may speculate that NPY also interferes with norepinephrine-dependent activation of other members of the MAP kinase family, namely JNK or p38 MAP kinase. These might be activated by stimulation of the {alpha}- or the β-adrenoceptors. The determination of these questions was, however, out of the scope of the present study.

In summary, our study indicates that the anti-adrenergic effect of NPY is sufficient to modulate the hypertrophic response of adult ventricular cardiomyocytes to norepinephrine.

Time for primary review 27 days.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This study was supported by the Deutsche Forschungsgemeinschaft grant Pi 192/11-2.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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