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Cardiovascular Research 2000 48(2):274-284; doi:10.1016/S0008-6363(00)00170-X
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Comitogenic effect of catecholamines on rat cardiac fibroblasts in culture

Monika Leicht*, Nora Greipel and Heinz-Gerd Zimmer

Carl-Ludwig-Institute of Physiology, University of Leipzig, Liebigstr. 27, D-04103 Leipzig, Germany

* Corresponding author. Tel.: +49-341-971-5500; fax: +49-341-971-5509 leicht{at}medizin.uni-leipzig.de

Received 8 February 2000; accepted 16 June 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: We studied the ability of norepinephrine and of other catecholamines to affect the proliferation of cardiac fibroblasts isolated from adult rat hearts. Furthermore, we investigated the possible adrenergic receptor involved in this process. Methods: Norepinephrine (NE), phenylephrine (PE), isoproterenol (ISO), forskolin (FO), epidermal growth factor (EGF), platelet-derived growth factor AA (PDGF-AA) and specific inhibitors of the {alpha}1-, {alpha}2-, β1- and β2-adrenoceptors and of the protein kinase A (PKA) were applied to cardiac fibroblasts in culture. Cell number was measured by use of a Coulter Counter. Activation of the cAMP response element binding protein (CREB) was measured by Western blotting and subsequent use of a phospho-specific antibody. Activation of the p42- and the p44-mitogen activated protein kinase (p42/p44MAPK) was assessed by detection of phosphorylation shifts and by incorporation of 32P-labelled phosphate into myelin basic protein. Results: Fibroblasts isolated from hearts of adult rats were grown in 10% serum-containing media which induced an increase in cell number by 94%. After 48 h, treatment with 10 µM NE caused an even greater increase in cell number by 222%, i.e. another 128% (comitogenic effect). In contrast, NE alone had no effect on the growth of serum-deprived cells. EGF and PDGF-AA did not replace serum as the basic mitogen. After addition of NE to proliferating cells under serum conditions, there was a rapid, time-dependent significant activation of the p42/p44MAPK and of CREB for up to 60 and 120 min, respectively. In both cases, the maximum of activation was reached after 5 min. Application of FO (0.1–20 µM) caused a strong activation of CREB, while no increase in the phosphorylation of the p42/p44MAPK was detected. Treatment with 20 µM FO led to an identical increase in cell number as application of NE. Specific blockade of PKA with RpcAMPS prevented the activation of CREB and also the comitogenic effect of FO as well as of NE. The {alpha}- and β-adrenergic receptor blocker carvedilol (10 µM) normalized all NE-induced effects. Prazosin and yohimbine, inhibitors of {alpha}1- and {alpha}2-adrenoceptor activation, respectively, did not influence the NE-evoked increase in cell number. In contrast, the non-selective β-adrenoceptor blocker propranolol (1 µM) completely suppressed the comitogenic effect of NE. A similar effect was obtained with the specific β2-adrenoceptor blocker ICI 118,551 (5 µM), while the β1-adrenoceptor blocker metoprolol did not influence the increase in cell number. Conclusions: NE elicits a comitogenic effect on cultured rat cardiac fibroblasts which is prevented by β2-adrenergic blockade. The activation of CREB contributes to the increase in proliferation. The p42/p44MAPK which was also found to be activated by NE might as well be involved in the regulation of the comitogenic effect.

KEYWORDS Adrenergic (ant)agonists; Cell culture/isolation; Receptors; Protein kinases


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Earlier studies have shown that norepinephrine (NE) induced cardiac hypertrophy and various hemodynamic changes in the rat heart in vivo [1]. When the effect of NE was studied on the cellular level, the main interest had been concentrated on cardiac myocytes obtained from neonatal and adult rats [2–5,33]. In cardiac myocytes from neonatal rats, NE treatment resulted in increased RNA and protein synthesis, the induction of a fetal gene program and hypertrophy primarily via activation of {alpha}1-adrenergic receptors [6–9].

It is well known that NE also increases the expression of extracellular matrix proteins and the development of interstitial fibrosis in the rat heart. Cardiac fibroblasts constitute more than 90% of non-myocyte cells in the heart, and they are the origin of the extracellular matrix proteins. The remodelling of the matrix is an important mechanism by which the myocardium responds to stimuli leading to the development of cardiac hypertrophy [10].

Previous studies have shown that there are changes in the gene expression and in the proliferative capacity of cardiac fibroblasts under various pathophysiological conditions and after treatment with diverse regulatory factors, such as Angiotensin II, TGF-β1, bFGF and PDGF [4,11–16]. Only little information is available on the effect of NE on cultured cardiac fibroblasts. It was shown that NE increases thymidine incorporation in fibroblasts from the hearts of neonatal rats and rabbits through the activation of β-adrenergic receptors [17–19]. However, whether this increase translated into higher cell numbers was not reported.

Activation of adenylate cyclase results in the production of the second messenger cAMP. The subsequently activated cAMP dependent protein kinase A, PKA, then phosphorylates and thereby activates the transcription factor CREB, the cAMP response element binding protein, which binds to CRE, a regulatory element in the promoter region of various genes as has been shown in PC12 cells and in rat brain preparations [20,21]. Transcription mediated by CREB regulates diverse cellular responses including intermediary metabolism and cell proliferation. It can also be activated by signals which affect the intracellular levels of Ca2+ as well as by growth factors and cellular stress.

It has been shown by Yamazaki et al. [22] that NE activates both the p42- and the p44-mitogen activated protein (MAP) kinase in cardiomyocytes cultured from neonatal rats. The activation of MAP kinases plays an important role in gene regulation during cardiac hypertrophy [23,24] and in the control of transduction of mitogenic signals.

In a previous study from our laboratory it had been demonstrated that β-adrenergic stimulation of adult rats induced an increase in cardiac protein synthesis [25]. In the present study, we therefore tested the hypothesis that catecholamines might induce proliferation of cultured cardiac fibroblasts from adult rat hearts via β-adrenergic stimulation. Moreover, we investigated whether the transcription factor CREB and the p42- and the p44-MAP kinases are activated during NE treatment.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Materials
Protein molecular weight standards, myelin basic protein (MBP), p-coumaric acid, isoproterenol (ISO), phenylephrine (PE), norepinephrine (NE), propranolol, epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF-AA) were purchased from Sigma (Deisenhofen, Germany). Carvedilol was obtained from Boehringer Mannheim, prazosin from Pfizer (Karlsruhe, Germany), metoprolol from Ciba-Geigy (Wehr, Germany), ICI 118,551 (erythro-DL-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol) and yohimbine were from Tocris Cookson Ltd. (Bristol, UK), forskolin and RpcAMPS were obtained from Calbiochem (Bad Soden, Germany). Goat anti-rabbit peroxidase labeled antibodies were from Dianova (Hamburg, Germany). Benzonase was purchased from Merck (Darmstadt, Germany), Luminol was from Serva (Heidelberg, Germany), and reinforced nitrocellulose from Schleicher & Schüll (Dassel, Germany). [{gamma}-32P]ATP (300 Ci/mmol) was obtained from Hartmann Analytic (Braunschweig, Germany).

Animals used in this study were maintained in accordance 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).

2.2 Cell culture
The isolation of cardiac fibroblasts was performed according to a previously published protocol for the preparation of adult rat cardiac myocytes [26,27] with minor modifications. Four 200–250 g female Sprague–Dawley rats (Charles River, Sulzfeld) were anaesthetized with an i.p. injection of 0.1 g/kg thiopental natrium (Trapanal, Byk Gulden, Konstanz, Germany) and 6000 IU/kg heparin (Liquemin, Hoffmann–La Roche, Grenzach-Wyhlen, Germany). The hearts were excised, mounted on a Langendorff perfusion system and rinsed with Joklik's minimal essential medium (JMEM, F. Messi, Cell Culture Technologies, Zürich, Switzerland) at 37°C continuously gassed with 95% O2/5% CO2. Perfusion was continued by recirculating JMEM for 35 min, containing 29 U/ml collagenase (Worthington Biochemical Corp., New Jersey, USA). The ventricles of the hearts were then cut with tissue scissors in 1x1 mm pieces and incubated for another 10 min in 20 ml KB buffer, consisting of 70 mM KCl, 30 mM K2HPO4, 22 mM glucose, 5 mM MgSO4, 0.5 mM EGTA, 20 mM taurine, 5 mM creatine, 10 mM succinic acid, 2 mM pyruvic acid, 5 mM ATP, 2 mM butyric acid, pH 7.4, and 115 U/mg collagenase. The cell suspension was filtered through a 250-µm nylon mesh and spun down at 25xg for 3 min. The supernatant was centrifuged at 250xg for 10 min. The pellet was resuspended in Dulbecco's minimal essential medium (DMEM)/Ham's F-12 (Biochrom, Berlin, Germany)/10% fetal calf serum (FCS, Biochrom)/1% penicillin/streptomycin and plated on 58-cm2 dishes (Nunc, Wiesbaden, Germany). Four hours later, debris and nonattached cells were removed by medium change. The medium was changed the next day, and the cells were grown for 5 days in DMEM containing 10% FCS without another medium change. Confluent cells were passaged once. The culture contained more than 95% fibroblasts.

For proliferation measurements, cells were seeded at 7–8x103 cells/cm2 on 12-well plates (each well with an area of 3.5 cm2). To arrest the cells and synchronize them in G1/G0 of the cell cycle, cells were grown to confluency. Serum containing medium was then removed, and fresh DMEM/1% penicillin/streptomycin without FCS was added for 2 days. Cells were then maintained without and with NE, EGF and PDGF-AA, respectively, in serum-free (Figs. 1 and 3Go: starved–) and serum-containing (Fig. 1: starved+) medium for another 48 h. Starving cells (Figs. 1 and 3Go) had been grown to 30% confluency in the presence of serum. At the start of the experiment, serum was withdrawn and cells were maintained without and with NE, EGF and PDGF-AA, respectively, for 48 h. Proliferating cells were 30% confluent at the beginning of the experiment, 10% FCS was given throughout the incubation. All other experiments were performed with 30% confluent cells in the presence of FCS (10%).


Figure 1
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  		Fig. 1 Effect of norepinephrine (NE) on cell number under different growth conditions. Primary cultures were passaged after 6 days and seeded at 7–8x103/cm2 on 12-well plates. Starved: Cells had been grown to confluency and were then serum-deprived for 2 days. During the following experimental period, cells were kept without (starved–) and with 10% FCS (starved+) and were maintained without and with NE for 48 h. Starving: Cells had been grown to 30% confluency in the presence of serum. Then, serum was withdrawn, and cells were maintained without and with NE for 48 h. Starving cells did not receive any FCS during the experimental procedure. Proliferating: Cells were 30% confluent at the beginning of the experiment, 10% FCS was given throughout the incubation without and with NE for 48 h. Open bars: control; filled bars: 104 nM NE. After 48 h, cells were trypsinized, and cell number was measured by use of a Coulter Counter Channelyzer. The cell number which fibroblasts had reached at the start of the experiment was set to 100% for each treatment. Each point represents the mean±S.E.M. of five separate experiments. Measurements were done in duplicate. *P<0.05 vs. only FCS-treated, time-matched controls by analysis of Student's t-test.

 

Figure 3
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  		Fig. 3 Dose–response curves for norepinephrine (NE)-, isoproterenol (ISO)- and phenylephrine (PE)-stimulated increase in the number of cardiac fibroblasts. When cells had reached 30% confluency in medium containing 10% fetal calf serum, the concentrations of the substances indicated on the abscissa were applied for 48 h. Cells were trypsinized, and cell number was measured by use of a Coulter Counter Channelyzer. Each point represents the mean±S.E.M. of five separate experiments, duplicate determinations using cells from the eight isolations. The cell number which nontreated fibroblasts (0) had reached at the end of the experiment (after 48 h) was set at 100% for each treatment. *P<0.05 vs. nontreated cells by analysis of Student's t-test.

 
2.3 Determination of cell number
Cells were grown on 12-well plates. At the time points given in the figures, cells were washed with PBS, detached from the dish by treatment with 0.5% trypsin-EDTA and suspended in 1 ml PBS. The number of viable cells was determined 48 h after the addition of the agents using a Coulter Counter Channelyzer (Coulter Electronics, Krefeld, Germany). Measurements were done in triplicate in volumes of 200 µl each. The orifice tube had an aperture size of 100 µm.

2.4 Gel electrophoresis and immunostaining
SDS–polyacrylamide gel electrophoresis was performed essentially as described by [28] using 10% acrylamide gels (37.5/1 w/w) acrylamide/bisacrylamide). Cells were lysed in 50 mM Tris–Cl, pH 6.7, 2% sodium dodecyl sulfate (SDS), 2% mercaptoethanol, and 1 mM sodium orthovanadate (Laemmli buffer) followed by digestion of nucleic acids with benzonase. The lysates were heated for 10 min at 95°C, and the protein content of the supernatant was quantified according to Henkel and Bieger [29].

For electrophoresis, minigels of 0.75 mm thickness were used (Bio-Rad Mini Protean). After SDS–PAGE (10 µg/slot), proteins were transferred onto reinforced nitrocellulose by semidry blotting using 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), pH 10.0, 1 mM 3-mercaptopropionic acid, and 10% methanol as blotting buffer for 1 h at 6 V. Saturation of the nitrocellulose membrane was achieved for 1 h with 2% bovine serum albumin (BSA) in 50 mM Tris–Cl, pH 7.5, 150 mM NaCl, 0.2% Triton X-100 (TS-buffer). The sheets were incubated for 1 h with the antibodies against p42/p44MAPK (0.25 µg/ml, Santa Cruz K-23, Heidelberg, Germany), phospho-CREB and CREB (1 µg/ml, New England Biolabs, Schwalbach, Germany) in TS-buffer containing 2% BSA and 0.03% NaN3. After washing six times with TS containing 0.5% BSA (washing buffer), the sheets were incubated for 1 h with goat-anti-rabbit IgG antibodies labeled with horseradish peroxidase at a dilution of 1/5000 in TS-buffer without NaN3. The antibody was removed by washing six times with washing buffer. The detection was done by chemoluminescence, using luminol (2.5 mM) and p-coumaric acid (400 µM) as enhancer [30].

2.5 Immunoprecipitation and determination of myelin basic protein (MBP) phosphorylation activity
Cells were grown on 58-cm2 dishes. When cells had reached 30% confluency, they were treated with the respective agents for the times indicated in the figures. Cells were washed with cold PBS and lysed in 0.5 ml cold RIPA buffer (50 mM β-glycerophosphate, pH 7.5, 150 mM NaCl, 1% NP 40, 0.1% SDS, 2 mM EGTA, 1% deoxycholate, 1 mM DTT, 0.1 mM phenylmethylsulfonyl difluoride, and 10 µg/ml leupeptin). After 15 min, cells were scraped off the plates, and the lysates were centrifuged for 10 min at 13 000 rpm at 4°C. Two µg of p42/p44MAPK antibody were added to the supernatant containing 700 µg protein. After 90 min, 30 µl sepharose-protein A in RIPA buffer were given for 90 min for precipitation of the conjugates. The individual immunoprecipitates were washed twice with RIPA and twice with kinase assay buffer containing 50 mM β-glycerophosphate, pH 7.4, 10 mM MgCl2, and 1 mM DTT.

The immunoprecipitates were incubated with 0.5 mg MBP, 30 µM ATP, and 5 µCi [{gamma}-32P]ATP in a total volume of 40 µl of kinase buffer for 30 min. The reaction was terminated by addition of 10 µl 0.5 M phosphoric acid. Aliquots of the reaction mixture were spotted onto phosphocellulose, and quantification was performed using a phosphoimager (Molecular Dynamics, Krefeld, Germany).

2.6 Statistical analysis
The data were analyzed and expressed as mean±S.E.M. For evaluation of statistical significance, Student's t-test (P<0.05) was used to demonstrate statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Measurement of cell number
Fig. 1 shows that in the absence of serum, a 48-h treatment of cardiac fibroblasts with 104 nM NE had no effect on cell number. No differences to time-matched controls were measured when NE was added either to confluent cells that had been serum starved for 48 h (starved–) or to 30% confluent cells at the time serum was removed (starving). When 10% FCS was given throughout the experimental procedure, cell number was increased to 194±26.4% of control after 48 h (proliferating). Application of 104 nM NE further enhanced this increase in cell number to 322±12.4% (n = 5). This is another 128% increase. When the time-matched, FCS treated control (194%) is set at 100%, NE caused an additional percent increase of 58%. Similar increases in cell number, which were not significantly different from those where cells were proliferating, were measured when cells were first serum starved and 10% FCS (199±10.7%) and 10% FCS plus 104 nM NE (306.5±18.4%), respectively, was again added for 48 h (starved+).

These results indicate that NE is a comitogen in cardiac fibroblasts from adult rats since it increased cell number only in the presence of FCS. It is obviously not of importance whether cells are proliferating at the time the treatment begins or whether they have been serum starved.

3.2 Replacement of FCS by EGF and PDGF
Since serum is an undefined mixture of various growth factors and hormones, we tried to identify the component in this mixture which acts as the main mitogen. Therefore, the above described experiments were repeated, and FCS was replaced by epidermal growth factor (EGF, 40 ng/ml) and platelet-derived growth factor AA (PDGF-AA, 40 ng/ml), respectively. The mitogenic effect of these growth factors has already been shown in many cell types. PDGF-AA has already been found to act as a potent mitogen in adult cardiac fibroblasts [16].

Fig. 2 shows that application of both EGF and PDGF-AA caused an increase in cell number by 51.2±5.5 and 39.1±4.1%, respectively, when cells were proliferating at the time the treatment was started (Fig. 2: starving). However, in the presence of both growth factors, NE did not enhance proliferation rate. While the effect of NE plus EGF (increase by 50.1%) was identical to EGF alone, NE even reduced the PDGF-AA induced increase in cell number by 56%. When EGF and PDGF-AA were applied in confluent cells that had been serum starved for 48 h (Fig. 2: starved–), the mitogenic effect of both growth factors was greater than in proliferating cells (starving). Cell number was elevated by EGF and PDGF-AA by 83.4±9.8 and 94.1±11.1%, respectively. NE did not significantly enhance this effect. Thus, we conclude that both EGF and PDGF-AA are potent mitogens in cardiac fibroblasts, however, neither of them can replace FCS as the main mitogen to which NE could add its comitogenic effect.


Figure 2
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  		Fig. 2 Effects of epidermal growth factor (EGF) and platelet-derived growth factor AA (PDGF) on cell number. Starving: Cells had been grown in the presence of 10% FCS until they were 30% confluent, FCS was then removed, and the indicated growth factors and 104 nM norepinephrine (NE) were given for 48 h. Starved–: Cells had been grown to confluency and were then serum-deprived for 2 days. For the following experimental period, cells were maintained with the indicated growth factors and 104 nM NE in the absence of FCS. EGF and PDGF-AA were given at 40 ng/ml each. After 48 h, cells were trypsinized, and cell number was measured by use of a Coulter Counter Channelyzer. The cell number which fibroblasts had reached at the start of the experiment was set to 100% for each treatment. Each point represents the mean±S.E.M. of five separate experiments. Measurements were done in duplicate. *P<0.05 vs. nontreated, time-matched controls by analysis of Student's t-test.

 
3.3 Comparison of norepinephrine, isoproterenol, phenylephrine
The comitogenic effect of NE occurred concentration-dependently (Fig. 3). The greatest effect was obtained with the concentration of 104 nM. With higher concentrations the increase in cell number was less pronounced. Similar results were obtained with the β-adrenoceptor agonist isoproterenol (ISO). The {alpha}1-adrenoceptor agonist phenylephrine (PE) led to smaller increases in cell number at all concentrations tested with a maximal increase of 36±5.3% at 104 nM.

3.4 NE effect on MAPK and CREB
The comparable effects of NE and ISO on cell number indicate an involvement of β-adrenergic receptors. The transcription factor CREB is activated after β-adrenergic stimulation. CREB as well as the p42/p44MAPK are signalling proteins which are known for their contribution to the regulation of proliferative processes. To determine whether these signalling proteins are activated by NE treatment in cardiac fibroblasts and involved in the NE-evoked increase in cell number, these two proteins were analysed.

Fig. 4A demonstrates that NE stimulation resulted in the activation of CREB already after 5 min (12.2±2.3-fold, n = 6). After 240 min, CREB was still phosphorylated to a higher degree than control (0). Phosphorylation shifts of numerous protein kinases are tightly associated with their activation. As indicated by the appearance of a band with reduced electrophoretic mobility, the p42/p44MAPK was also highly phosphorylated after the addition of NE (Fig. 4A). After 5 min, a significant phosphorylation, and therefore activation, was observed that was maintained for 60 min. To ascertain that an activation of p42/p44MAPK after NE treatment was indeed evoked, the activity of this protein kinase was measured by MBP phosphorylation after stimulation with NE and subsequent immunoprecipitation of the protein. After 5 and 10 min, we obtained a significant increase in MBP kinase activity of 92 and 88%, respectively, over that of the unstimulated control (Fig. 4B).


Figure 4
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  		Fig. 4 Time course of activation of CREB and p42/p44MAP kinase by norepinephrine (NE). Cardiac fibroblasts were grown in DMEM supplemented with 10% fetal calf serum until 30% confluency was reached. (A) 104 nM NE was added, and cells were lysed by addition of a SDS-containing buffer after the indicated time intervals. Aliquots were electrophoresed and blotted onto nitrocellulose. Detection of individual protein kinases was done using enhanced chemoluminescence. The antibody against p44MAPK recognizes both the p42 and the p44 isoenzyme. Results are representative of data from three separate experiments. (B) Cells were treated as above. After stimulation with 104 nM NE for the indicated times, p42/p44MAP kinase activities were measured as described in Methods by immunoprecipitation followed by MBP kinase activity determination. The data are means of three experiments (graph). Below: Result of an individual representative test. *P<0.05 vs. nontreated cells by analysis of Student's t-test.

 
3.5 Effect of forskolin
Since NE led to the activation of CREB, we now investigated whether activation of the adenylate cyclase is involved in the regulation of proliferation. Forskolin, a specific activator of the adenylate cyclase, was therefore applied. After 48 h, in the presence of 2x104 nM forskolin, cell number was increased by 56% compared to time-matched FCS treated controls (Fig. 5). This increase was similar to that found with 104 nM NE. A 20.2±2.7-fold (n = 4) increase in phosphorylation of CREB was detected by Western blotting after 5 min with 104 nM forskolin (Fig. 6). At the concentration that induced maximal increase in cell number (2x104 nM), the strongest phosphorylation of CREB was detected. The phosphorylation state of the p42/p44MAPK was unaffected by forskolin treatment (Fig. 6).


Figure 5
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  		Fig. 5 Dose–response curve for forskolin-stimulated increase in the number of cardiac fibroblasts. When cells had reached 30% confluency in medium containing 10% fetal calf serum, forskolin was applied in the concentrations indicated on the abscissa for 48 h. Cell number was determined using a Coulter Counter Channelyzer. Each point represents the mean±S.E.M. of five separate experiments measured in duplicate. The cell number which nontreated fibroblasts (0) had reached at the end of the experiment (after 48 h) was set at 100%. *P<0.05 vs. nontreated cells by analysis of Student's t-test.

 

Figure 6
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  		Fig. 6 Effect of forskolin on CREB and p42/p44MAP kinase. Indicated amounts of forskolin were added to 30% confluent cells in DMEM supplemented with 10% FCS. Cells were lysed after 5 min incubation with forskolin, the extent of phosphorylation was visualized using Western blotting. Results are representative for data from three separate experiments.

 
3.6 Involvement of PKA
To clarify a possible role of PKA in the regulation of the comitogenic effect of NE, cells were pretreated with the specific PKA inhibitor RpcAMPS. The increase in cell number induced by forskolin (FO) as well as by NE was inhibited by RpcAMPS in a concentration-dependent manner (Fig. 7).


Figure 7
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  		Fig. 7 Effect of RpcAMPS on the norepinephrine (NE)- and forskolin (FO)-induced increase in cell number. Indicated amounts of RpcAMPS were added to 30% confluent cells in DMEM supplemented with 10% FCS 30 min prior to the addition of 104 nM forskolin and 104 nM NE, respectively. Cell number was determined after 48 h using a Coulter Counter Channelyzer. Each point represents the mean±S.E.M. of three separate experiments in duplicate determinations using cells from each isolation. The cell number which nontreated fibroblasts had reached at the end of the experiment (after 48 h) was set at 100% for each treatment. *P<0.05 vs. NE- and FO-treated cells by analysis of Student's t-test.

 
Investigation of the phosphorylation status of CREB and of the p42/p44MAPK showed that RpcAMPS also prevented the NE induced phosphorylation of CREB in a concentration-dependent manner (Fig. 8A). However, the enhanced phosphorylation and the MBP phosphorylation activity of the p42/p44MAPK was unaffected by treatment with RpcAMPS (103 nM) (Fig. 8A and B). RpcAMPS alone did not affect cell number and the activity of CREB and MAPK (data not shown).


Figure 8
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  		Fig. 8 Effect of RpcAMPS on the norepinephrine (NE)-evoked activation of CREB and p42/p44MAP kinase. Cardiac fibroblasts reaching 30% confluency in DMEM supplemented with 10% FCS were used. RpcAMPS was added at the indicated concentrations 30 min prior to the addition of 104 nM NE. Nontreated cells are marked as Co. (A) Cells were lysed after 5 min incubation, the extent of phosphorylation was visualized using Western blotting. Results are representative for data from three separate experiments. (B) Cells were treated as above. After stimulation with 104 nM NE and NE+103 nM RpcAMPS for 5 min, respectively, p42/p44MAP kinase activities were measured. Data are means of three experiments. *P<0.05 vs. nontreated cells (Co) by analysis of Student's t-test.

 
3.7 Adrenoceptors
To determine which receptor might be involved in the transduction of comitogenic signals, cells were pretreated with various adrenergic antagonists before NE application. Carvedilol (104 nM), an {alpha}- and β-adrenoceptor antagonist, prevented the NE-induced increase in cell number in a concentration-dependent fashion (Fig. 9). In parallel, CREB and MAPK phosphorylation were inhibited. The phosphorylation of CREB was prevented by carvedilol already at 103 nM to a major degree and was completely suppressed at 104 nM (Fig. 10A). After pretreatment with 104 nM carvedilol, the phosphorylation of MAPK was totally suppressed. The increase in MBP phosphorylation activity was also entirely suppressed by pretreatment with 104 nM carvedilol (Fig. 10B).


Figure 9
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  		Fig. 9 Influence of the {alpha}- and β-adrenoceptor blocker carvedilol on the norepinephrine (NE)-induced increase in cell number. To 30% confluent cells in DMEM supplemented with 10% FCS, carvedilol was added at the indicated concentrations 30 min prior to the addition of 104 nM NE. Cell number was determined after 48 h using a Coulter Counter Channelyzer. Each point represents the mean±S.E.M. of five separate experiments in duplicate determinations using cells from each isolation. The cell number which nontreated fibroblasts had reached at the end of the experiment was set at 100% for each treatment. *P<0.05 vs. NE-treated cells by analysis of Student's t-test.

 

Figure 10
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  		Fig. 10 Effect of carvedilol on the norepinephrine (NE)-induced activation of CREB and p42/p44MAPK. Cardiac fibroblasts reaching 30% confluency in DMEM supplemented with 10% FCS were used. Carvedilol was added at the indicated concentrations 30 min prior to the addition of 104 nM NE. Nontreated cells are marked as Co. (A) Cells were lysed after 5 min incubation, the extent of phosphorylation was visualized using Western blotting. Results are representative for data from three separate experiments. (B) Cells were treated as above. After stimulation with 104 nM NE and NE+104 nM carvedilol (Carv) for 5 min, respectively, p42/p44MAP kinase activities were measured. Data are means of three experiments. *P<0.05 vs. untreated, +P<0.05 vs. NE-treated cells by analysis of Student's t-test.

 
To further identify the adrenergic receptor involved, cells were pretreated with the respective antagonists (Fig. 11). The antagonists of the {alpha}1- and {alpha}2-adrenoceptors, prazosin and yohimbine, respectively, did not affect the comitogenic effect of NE. Prazosin and yohimbine had no effect when given alone (data not shown). Thus, the NE-induced signal is not transduced via {alpha}1- and {alpha}2-adrenoceptors.


Figure 11
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  		Fig. 11 Effect of the various adrenergic antagonists on the norepinephrine (NE)-induced increase in cell number. Cardiac fibroblasts were kept in culture until 30% confluency was reached in DMEM supplemented with 10% FCS. Prazosin (Praz, open circles), yohimbine (Yoh, open triangles), metoprolol (Met, open squares), propranolol (Propr, filled triangles) and ICI 118,551 (filled circles), respectively, were added at the indicated concentrations 30 min prior to the addition of 104 nM NE. Cell number was determined after 48 h using a Coulter Counter Channelyzer. Each point represents the mean±S.E.M. of five separate experiments. Measurements were done in duplicate. The cell number which nontreated fibroblasts had reached at the end of the experiment (after 48 h) was set at 100% for each treatment. *P<0.05 vs. NE-treated cells by analysis of Student's t-test.

 
In contrast, the β-adrenoceptor antagonist propranolol prevented the NE-induced increase in cell number at 103 nM (Fig. 11). The β1-adrenoceptor blocker metoprolol did not cause any change in cell number. However, ICI 118,551, a highly specific inhibitor of the β2-adrenoceptor, completely suppressed the comitogenic effect of NE with an IC50 of 290 nM (Fig. 11). Addition of propranolol, metoprolol and ICI 118,551 alone was without effect on cell number and on the activity of CREB and MAPK (data not shown).

The NE-induced phosphorylation of CREB was suppressed by ICI 118,551 at 103 nM to a major degree (Fig. 12A). The phosphorylation of MAPK was also prevented by ICI 118,551 in a concentration-dependent manner (Fig. 12A). Pretreatment with 5x103 nM ICI 118,551 completely suppressed the activation of MAPK. The increase in MBP phosphorylation activity was reduced by 67% after addition of 5x103 nM ICI 118,551 (Fig. 12B).


Figure 12
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  		Fig. 12 Effect of the β2-adrenergic antagonist ICI 118,551 on the norepinephrine (NE)-induced activation of CREB and p42/p44MAPK. When cardiac fibroblasts had attained 30% confluency in DMEM supplemented with 10% FCS, ICI 118,551 was added at the indicated concentrations 30 min prior to the addition of 104 nM NE. Nontreated cells are marked as Co. (A) Cells were lysed after 5 min incubation, the extent of phosphorylation was visualized using Western blotting. Results are representative for data from three separate experiments. (B) Cells were treated as above. After stimulation with 104 nM NE and NE+5x103 nM ICI 118,551 for 5 min, respectively, p42/p44MAP kinase activities were measured. Data are means of three experiments. *P<0.05 vs. untreated, +P<0.05 vs. NE-treated cells by analysis of Student's t-test.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Fibrosis: an integral component of cardiac remodelling
Myocardial fibrosis is an integral component of remodelling in many models of experimental cardiac hypertrophy. We have shown recently that interstitial fibrosis had developed in pressure-induced hypertrophy of rat hearts which was prevented by angiotensin II receptor blockade [31]. In NE-induced hypertrophy, the DNA content was increased indicating that non-myocytes had increased in number [32]. The present study demonstrates that all catecholamines tested promote the proliferation of cultured cardiac fibroblasts (Figs. 1 and 3Go). NE and ISO had by far stronger effects on cell number than the {alpha}-adrenoceptor agonist PE. Since cardiac fibroblasts are responsible for synthesis and deposition of fibrillar collagens, proliferation of these cells can be considered to be an important mechanism that adds to the remodelling process.

Other authors have shown that NE increases the capacity of cultured cardiac fibroblasts from neonatal rats and rabbits to proliferate [17–19]. However, to our knowledge, our present study is the first to demonstrate that NE actually increased the number of cardiac fibroblasts in culture. Since we measured an increase in cell number only when NE was added to serum-containing media, NE had a comitogenic effect. As to the component in the serum that might act as the basic mitogen we have demonstrated that it is neither EGF nor PDGF-AA (Fig. 2).

4.2 Involvement of β2-adrenergic receptors in proliferation of cultured cardiac fibroblasts
As to the type of the adrenergic receptor by which NE induced proliferation of cardiac fibroblasts, we have found that the β2-adrenergic receptor blocker ICI 118,551 prevented the comitogenic effect (Fig. 11). We have also shown that after NE treatment CREB and MAPK are activated (Fig. 4), and that the signals leading to their activation are transduced via the β2-adrenoceptor (Figs. 11 and 12Go).

NE was reported to induce hypertrophy of cardiac myocytes from neonatal rats via {alpha}1-adrenoceptor stimulation. Furthermore, MAPK activity was induced by angiotensin II, agonists of Gq protein-coupled receptors, such as PE, NE, and endothelin-1, but not by agonists of Gs protein-coupled receptors, such as ISO [6,33]. In another study, however, the β-adrenergic agonist ISO activated the MEK/MAPK pathway for which elevation of intracellular Ca2+ rather than cAMP appeared important [34].

On the other hand, a study by Yamazaki et al. [22] demonstrated that NE induced activation of MAPK through both {alpha}1- and β-adrenoceptors, and that signalling pathways from the two receptors synergistically induced hypertrophy of neonatal rat cardiac myocytes. Furthermore, in isolated myocytes from the hearts of adult rats, β-adrenergic agonists stimulated protein synthesis [35]. Finally, Pinson et al. [2] reported that both {alpha}1- and β-adrenoceptor agonists induced a pronounced increase in protein synthesis. This is in agreement with our earlier in vivo experiments in which NE caused hypertrophy of the rat heart by activating {alpha}1- as well as β-adrenoceptors [32].

Other authors have investigated in more detail how signals from the receptors are transduced to the MAPK: Hawes et al. [36] reported for human embryonic kidney 293-cells that Gβ{gamma} was responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G{alpha} mediated Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. This study as well as others cited above have shown that the raf-1 kinase/MAP kinase cascade can be activated by the {alpha}1 as well as by the β-adrenoceptor.

Similar results were obtained by Crespo et al. [37] in COS-7 cells expressing the β{gamma} subunit of the β-adrenoceptor. These authors found that activation of β-adrenoceptors by ISO led to the activation of ras and MAPK through the β{gamma} subunit. A more recent work by Zou et al. [38] on cardiac fibroblasts showed that angiotensin II stimulated a pathway including the Gβ{gamma} subunit of the Gi protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase leading to the activation of MAPKs.

Our results on the involvement of MAPK in the comitogenic response to NE are also in agreement with studies describing the effect of MAPK in the induction of proliferation after treatment of various cell types with different mitogens. Activation of the classical MAPK pathway also leads to the proliferation of Chinese hamster lung fibroblasts [39]. In human cardiac fibroblasts, blockade of MAPK reduced PDGF-evoked increases in DNA synthesis to a great degree [40].

4.3 Signalling downstream of the β2-adrenoceptor
The β2-adrenergic receptor is linked to the stimulation of the adenylate cyclase [41]. In the present study we have shown that NE induced activation of MAPK, while forskolin had no influence on the activity of MAPK. Forskolin is a rather specific activator of the adenylate cyclase classically leading to the activation of PKA via an increase in intracellular cAMP levels which finally results in the subsequent phosphorylation of CREB. The effects of cAMP on cell proliferation are cell type specific. Elevation of intracellular cAMP has been found to cause growth inhibition in many cell types [42–44]. However, our data contribute to a number of studies describing the induction of proliferation by forskolin and other cAMP raising agents in cells such as Swiss 3T3 and various cell types in primary culture [45–47]. A hypertrophic responsiveness to β2-adrenoceptor stimulation was also shown by Zhou et al. [3] in adult ventricular myocytes. They found that the hypertrophic effect of ISO occurred via elevation of cAMP and activation of PKA.

We have shown that in cardiac fibroblasts, the enhancement in CREB phosphorylation and the comitogenic effect were blocked by the PKA inhibitor RpcAMPS in the case of stimulation with NE as well as with forskolin (Figs. 7 and 8Go). Therefore, we suggest that both forskolin and NE induce a comitogenic effect by activating PKA and subsequently CREB. PKA activity, however, did not affect MAPK activity since there was no effect of forskolin and RpcAMPS on MAPK activity (Figs. 6 and 8Go). The NE-induced activation of MAPK obviously occurs via signalling proteins other than PKA.

EGF and PDGF-AA act as potent mitogens in cardiac fibroblasts. However, the effect of FCS was not replaced by either of these growth factors when applied in combination with NE (Fig. 2). We do therefore conclude that NE might evoke the activation of similar pathways since there was no synergistic effect. In addition, exposure of the cells to other components supplied by application of FCS induces some kind of ‘competence’ which makes the cells capable of proliferating by a greater rate. It does not matter whether cells are already proliferating or whether they had been serum starved before reinduction of proliferation by readdition of FCS. We suggest that NE further elevates the activational state of only a limited set of proteins that transduce the mitogenic signal evoked by FCS, and that it probably stimulates additional signalling cascades. It might thereby add to the mitogenic effect of FCS, however, might not exert signals that are sufficient for mitogenicity without the basic activations induced by FCS. FCS might therefore create the basis of mitogenic signalling events to which the NE-induced signals must be added to enhance proliferation of cultured cardiac fibroblasts.

Time for primary review 27 days.


   Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft (Zi 199/10-1, Zi 199/10-3). The excellent technical assistance of Grit Marx is gratefully acknowledged.


   References
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 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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