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Cardiovascular Research 2001 49(4):721-730; doi:10.1016/S0008-6363(00)00291-1
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Cardiac fibroblasts are major production and target cells of adrenomedullin in the heart in vitro

Yoshio Tomodaa,b, Katsuro Kikumotoa, Yoshitaka Isumia, Takeshi Katafuchia, Akira Tanakac, Kenji Kangawaa, Kazuhiro Dohib and Naoto Minaminoa,*

aNational Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan
bFirst Department of Internal Medicine, Nara Medical University, Kashihara, Nara 634-0813, Japan
cDepartment of Pathology, Jichi Medical School, Kawachi, Tochigi 329-0498, Japan

* Corresponding author. Tel.: +81-6-6833-5012; fax: +81-6-6872-7485 minamino{at}ri.ncvc.go.jp

Received 29 May 2000; accepted 3 November 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Adrenomedullin (AM) is a potent vasodilator peptide. Plasma AM concentration is increased in patients with various heart diseases, and both myocytes (MCs) and non-myocytes (NMCs) secrete AM and express its receptors. These facts suggest that cardiac cells possess an autocrine/paracrine capability mediated by AM. Methods: MCs and NMCs were prepared from cardiac ventricles of neonatal rats. AM and endothelin-1 concentrations were measured by radioimmunoassays, and interleukin-6 level by a specific bioassay. Total nitrite/nitrate contents were measured with a fluorescence assay kit. Results: A basal secretion rate of AM from NMCs was 2.8-fold higher than that from MCs. Interleukin-1β, tumor necrosis factor-{alpha} and lipopolysaccharide stimulated AM secretion from NMCs but not from MCs. AM stimulated interleukin-6 production in the presence of these cytokines or lipopolysaccharide, which was more prominent in NMCs. In the presence of interleukin-1β, AM augmented nitric oxide synthesis 2.7-fold in NMCs, but slightly in MCs. NMCs secreted endothelin-1 at a rate nine times higher than MCs, and AM inhibited endothelin-1 secretion from NMCs. Conclusion: This in vitro study suggests that AM in the heart is mainly produced in NMCs and exerts its effects through NMCs, especially under inflammatory conditions.

KEYWORDS Cytokines; Endothelins; Myocytes; Nitric oxide

This article is referred to in the Editorial by M. Jougasaki and J.C. Burnett Jr. (pages 695–696) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Adrenomedullin (AM), a member of the calcitonin gene-related peptide (CGRP) superfamily, is a potent vasodilator originally isolated from extracts of pheochromocytoma [1]. Our studies have demonstrated that AM is secreted from endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and fibroblasts [2–4]. AM secretion rate is commonly stimulated with interleukin-1 (IL-1), tumor necrosis factor (TNF) and lipopolysaccharide (LPS). In LPS-injected rats and patients with septic shock, plasma AM concentration was highly elevated and AM gene expression was augmented in blood vessels, heart, and other tissue [5]. Furthermore, AM acts on blood vessels and reduces blood pressure through elevation of cAMP and nitric oxide (NO) concentrations [6]. Thus, AM is considered to act as an autocrine/paracrine factor causing vasodilation during septic shock and inflammation.

Among the tissues examined, the high levels of AM gene transcription and immunoreactivity were found in heart tissue, where AM receptors are found in abundance [5]. High plasma AM concentrations are observed in patients with heart disease [7]. These data suggest that AM exerts significant effects on the heart tissue. The heart consists of myocytes (MCs) and supporting non-myocytes (NMCs), and MCs make up only 30% of the total cell number in the heart [8,9]. NMCs include fibroblasts, ECs, VSMCs and macrophages, but mainly comprise fibroblasts. Interaction between MCs and NMCs has been reported to be required for maintaining cardiac function [10], but the contribution of the NMCs remains unclear. These facts lead us to surmise that the NMCs possess an important function. In this study, we separated MCs and NMCs to a high degree of purity and examined regulation of AM secretion rates and properties of AM receptors in these cells. We also cloned and sequenced full-length cDNAs encoding rat receptor-activity-modifying protein (RAMP) 1, 2 and 3. Then, the effects of AM on IL-6, NO and endothelin-1 (ET-1) secretion rates from MCs and NMCs were evaluated.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell culture of MCs and NMCs
The investigation conformed 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), and was approved by the local committee on animal experiments and care. MCs and NMCs were prepared from cardiac ventricles of 1-day-old Sprague–Dawley rats (SLC, Shizuoka, Japan) as reported with minor modification [11]. Briefly, minced ventricles were digested with collagenase II (100 U/ml, Worthington, Freehold, NJ) and DNase I (1 mg/ml, Boehringer Mannheim, Germany) in a balanced salt solution (116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4) at 37°C for 6 min. The cell suspension was mixed with fetal calf serum (FCS) and kept on ice before discontinuous centrifugation. The remaining tissue was repeatedly digested. The dispersed cells were suspended in a 58.5% Percoll solution (Pharmacia, Uppsala, Sweden) and placed under a 40.5% Percoll solution layer. After centrifugation at 3000 rpm for 30 min, MCs migrated to the interface, while NMCs were in the upper layer. MC and NMC fractions were washed twice with the balanced salt solution. The MC fraction was resuspended in Dulbecco's modified Eagle's medium (DMEM) with 10% FCS and plated on 10-cm dishes to exclude adherent NMCs. The MC suspension was collected after 30 min, and re-plated. The MC-enriched suspension was finally plated on gelatin-coated six- or 24-well plates (Iwaki, Tokyo, Japan). After 24-h incubation, the culture medium for MCs was replaced with DMEM containing 10% FCS and 100 µM 5'-bromo-2'-deoxyuridine (Sigma, St. Louis, MO). After another 72-h incubation, MCs were used for experiments. The NMC fraction was suspended in DMEM with 10% FCS and plated on 10-cm dishes. At 30 min after seeding, the culture medium was replaced to remove non-adherent cells. The culture medium for NMCs was changed to one containing acidic fibroblast growth factor (FGF, 5 ng/ml, Wako, Osaka, Japan) after 24-h incubation. After two to three passages, the NMCs were used for experiments.

2.2 Immunocytochemistry
MCs or NMCs were cultured on glass slides. The cells were washed twice with cold PBS and fixed with 3.7% formaldehyde. The cells were first reacted with antibody against sarcomeric actin (myocyte marker, alfa Sr-1, Dako A/S, Glostrup, Denmark), smooth muscle specific actin (VSMC marker, Progen, Heidelberg, Germany), factor VIII (EC marker, Cedarlane, Ontario, Canada), or vimentin (mesenchymal marker, Progen), and then stained with an AutoProbe III kit (peroxidase-based detection system, Biomeda, Foster City, CA). The MCs and NMCs were also reacted with monoclonal anti-AM antibody [12] and control ascites.

2.3 Radioimmunoassays for AM and CGRP
MCs and NMCs, grown in six-well plates, were incubated for 14 h at 37°C with DMEM containing 1% FCS and murine TNF-{alpha} (Boehringer Mannheim), murine IL-1β (Intergen, Purchase, NY), LPS (E. coli serotype O26:B6, Parsel+Lorei, Frankfurt, Germany), dexamethasone (Sigma) or human transforming growth factor β1 (TGF-β, Genzyme, Boston, MA). The media were collected and treated as reported, and levels of AM were assayed by radioimmunoassay (RIA) for AM [2,3]. Antiserum against CGRP(28–37) was prepared by immunizing rabbits with CGRP(28–37)-thyroglobulin conjugate. This antiserum showed no cross-reactivity with AM or other related peptides. Monoiodo-N-Tyr-CGRP(28–37) was used as a tracer. Details of the CGRP RIA will be reported.

2.4 Characterization of immunoreactive AM and CGRP
After 12-h incubation, the culture medium was collected and acidified with acetic acid. The peptide fraction was prepared by using a Sep-pak C18 ENV cartridge (Waters, Milford, MA), and was subjected to gel filtration on a Sephadex G-50 column (Pharmacia). The immunoreactive (IR-) AM and IR-CGRP fractions were separated by reverse phase high-performance liquid chromatography (HPLC) on a C18 column (Chemco, Osaka, Japan) with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. An aliquot of each fraction was submitted to RIAs for AM and CGRP [2,3].

2.5 Measurement of intracellular cAMP
MCs and NMCs were preincubated in 25 mM HEPES-buffered DMEM (pH 7.4) containing 0.01% BSA and 0.5 mM isobutylmethylxanthine for 1 h [4]. The media were replaced with the same buffer containing rat AM, rat CGRP, human AM(22–52) and human CGRP(8–37) (Peptide Institute, Osaka, Japan). After 10-min incubation at 37°C, the media were removed, and 70% ethanol was added. After freezing for 30 min at –80°C, the cells were suspended and centrifuged. Aliquots of the supernatants were submitted to RIA for cAMP [6].

2.6 RNA blot analysis
Partial cDNA sequences reported for rat RAMPs (GenBank accession numbers: AB028933 [GenBank] , AB028934 [GenBank] , and AB028935 [GenBank] ) were used for designing gene-specific primers (GSPs). Rat lung and kidney cDNAs were prepared using a cDNA amplification kit (Clontech, Palo Alto, CA), and RACE PCR was carried out with an adapter primer of 5'-GCTCCTCCAGACCACCAGGG-3' and GSPs of RAMP1, 5'-TTCTGCCAAGGGATTTGGG-3'; RAMP2, 5'-CATGGCCAGAAGCACATCCT-3'; and RAMP3, 5'-AACTTCATCCGGGGGGTCTT-3'. The RACE products were subcloned and their sequences were determined on both strands by the dideoxy chain termination method. By using the RACE cDNA fragments as probes, rat brain cDNA library constructed in a {lambda}ZAP II vector (Stratagene, La Jolla, CA) was screened. The positive cDNAs in the phage were transformed to the plasmid, and at least three independent clones were sequenced.

Total RNA was extracted from MCs and NMCs with RNA zol (Tel-Test, Friendswood, TX). After denaturation, total RNA (8 µg) was electrophoresed and transferred to a Zeta probe membrane, as reported [3]. cDNA fragments of rat calcitonin receptor-like receptor (CRLR, nucleotide 914–1461), RAMP1 (nucleotide 88–462), RAMP2 (nucleotide 8–482), RAMP3 (nucleotide 1–381), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, nucleotide 492–799) were used as probes. The probes were labeled with 3'-primer and Taq polymerase for ten cycles except the random primer method for the GAPDH probe.

2.7 Measurement of IL-6
The culture media of MCs and NMCs, grown in 24-well plates, were replaced with DMEM containing 1% FCS and stimulants, and the cells were incubated for 24 h at 37°C. All reagents were added at 0 h. IL-6 activities in the media were estimated by a proliferation assay of MH60.BSF-2 cell [13]. 8-Bromo-adenosine-3',5'-cyclic monophosphate (Br-cAMP) (Sigma), forskolin (Wako), AM(22–52) and CGRP(8–37) were administered to characterize the receptors and signal transduction system. The incubation time in the measurements of IL-6, NO and ET-1 was determined based on the time-course experiment, and data were obtained from at least three independent experiments.

2.8 Measurement of NO
The culture media of MCs and NMCs, grown in 24-well plates, were replaced with DMEM containing 0.5% FCS and stimulants, and the cells were incubated for 36 h at 37°C. Total nitrite and nitrate (NOx) concentrations were measured with NOx Assay Kit-F (Dojin, Kumamoto, Japan). This kit reduced nitrate to nitrite, which was then converted into a fluorescent compound.

2.9 Measurement of ET-1
The culture media of MCs and NMCs, grown in six-well plates, were replaced with DMEM containing 1% FCS and various stimulants, and the cells were incubated for 14 h at 37°C. The media were treated as in the case of AM, and submitted to RIA for ET-1 [14].

2.10 Statistical analysis
Statistical analysis of the results was performed by one-way analysis of variance (ANOVA), followed by a multiple comparison test (Dunnett's test). All data were expressed as means±S.E.M. A level of P<0.05 was considered to be statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Characterization of MCs and NMCs
In the MCs used for the present study, more than 96% of the cells were stained with anti-sarcomeric actin antibody, and little proportion of them showed positive staining with the other antibodies against smooth muscle specific actin (less than 0.2%), factor VIII (not significant) and vimentin (less than 3.5%). Almost all the MCs stained with the anti-sarcomeric actin antibody were beating. The purity of the MCs used in this study was greater than or comparable to that reported previously [15]. The NMCs were stained only with anti-vimentin antibody, and none of the other three antibodies showed positive staining. Thus, the NMCs used were confirmed to be almost exclusively composed of fibroblasts.

Both MCs and NMCs were positively stained with the monoclonal antibody against AM, and the staining in the MCs was stronger than that in the NMCs (Fig. 1). The control ascites did not show positive staining in either of these cells.


Figure 1
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  		Fig. 1 Immunostaining of MCs and NMCs with monoclonal antibody against AM. MCs (a and c) or NMCs (b and d) were cultured on glass slides. The cells were washed twice with cold PBS and fixed with 3.7% formaldehyde. The cells were first reacted with monoclonal anti-AM antibody (dilution of 1:100) (a and b) or control ascites (c and d), and then stained with the peroxidase-based detection system. Original magnification: 100x.

 
3.2 Secretion of IR-AM and IR-CGRP
The average secretion rates of IR-AM from MCs and NMCs over a 14-h period were 0.92 and 2.57 fmol/105 cells/h, respectively (Table 1). IR-AM secretion from MCs was increased only with dexamethasone. In the case of NMCs, IL-1β and dexamethasone augmented IR-AM secretion 2.6-fold. TNF-{alpha} and LPS increased it to a lesser extent, while TGF-β reduced it. All these effects were dose-dependent (data not shown). The control secretion rates of IR-CGRP averaged 0.015 and 0.027 fmol/105 cells/h for MCs and NMCs, respectively. Secretion rates of IR-CGRP were unaffected by the stimulants listed in Table 1.


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  		Table 1 Effects of TNF-{alpha}, IL-1β, LPS, dexamethasone and TGF-β on AM secretion from MCs and NMCsa

 
3.3 Characterization of IR-AM and IR-CGRP
More than 70% of the IR-AM secreted from NMCs was eluted around a relative molecular mass of 6K corresponding to AM, while the IR-CGRP was eluted after salts (Fig. 2). In the reverse phase HPLC, more than 70% of IR-AM was eluted at a retention time of AM, while IR-CGRP emerged in a wide range between 25 and 40 min. These data verified that IR-AM secreted from NMCs was a single component chromatographically identical with AM, but the IR-CGRP was low molecular weight substances cross-reacting with the antiserum. The IR-AM and IR-CGRP secreted from MCs showed properties similar to those of NMCs (data not shown).


Figure 2
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  		Fig. 2 Characterization of IR-AM and IR-CGRP secreted from NMCs. (a) Sephadex G-50 gel filtration of IR-AM (closed square) and IR-CGRP (closed triangle) from NMCs. Sample: Peptide fraction of 100-ml culture medium. Column: 19x1340 mm. Solvent: 1 M acetic acid. Fraction size: 6 ml/tube. Flow rate: 7 ml/h. (b) Reverse phase HPLC of IR-AM from NMCs. Sample: 2/3 of fractions 38–40 in (a). Column: Chemcosorb 300-5C18 (4.6x250 mm). Flow rate: 1.0 ml/min. Fraction size: 0.5 ml/tube. Solvent system: Linear gradient elution of CH3CN in 0.1% trifluoroacetic acid over 60 min. Arrows indicate elution positions of (1) BSA, (2) rat AM, (3) NaCl.

 
3.4 Cloning of RAMP1, 2 and 3
As the partially reported cDNA sequences of rat RAMPs were not probes suitable for the RNA blot analysis, we cloned and sequenced the full-length cDNA of the rat RAMP family. Fig. 3a shows the deduced amino acid sequences of RAMP1, 2 and 3, and their nucleotide sequences have been submitted to DDBJ (accession numbers: AB042887 [GenBank] , AB042888 [GenBank] and AB042889 [GenBank] ). Rat RAMP1, 2 and 3 are deduced to be 148, 182 and 147 amino acids in length, which show 71.6, 64.7, and 85.9% identity with the respective human proteins, and 93.2, 85.8, and 95.9% identity with the respective mouse proteins [16,17]. The identity observed in the rat RAMP family is slightly higher than that in the human RAMP family.


Figure 3
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  		Fig. 3 (a) Amino acid sequences of rat RAMP1, 2 and 3 aligned for comparison. The number on the right refers to the last amino acid on the line. Conserved amino acids are boxed, and putative signal peptides and transmembrane (TM) domains are indicated. (b) RNA blot analysis of RAMP1, 2, 3 and CRLR transcripts in MCs and NMCs. Total RNA (8 µg/lane) was electrophoresed. Size markers for RAMP1, 2, and 3 are on the left, and those for CRLR and GAPDH on the right.

 
3.5 Effects of AM and CGRP on intracellular cAMP levels
CRLR, RAMP1, 2, and 3 mRNA levels in MCs and NMCs were evaluated by RNA blot analysis (Fig. 3b). A single transcript of a predicted size was each detected for RAMP1, 2, and 3. The most intense band was observed in the RAMP1 lane, and RAMP2 and RAMP3 mRNAs were faintly detected in NMCs. In the case of MCs, a weak band of RAMP1 mRNA and a clear band of RAMP3 mRNA were found, but no band was detected in the RAMP2 lane. CRLR mRNA was detected in MCs and NMCs by RT-PCR (data not shown) but not by RNA blot analysis.

We next measured intracellular cAMP levels (Fig. 4). The maximal level of cAMP achieved with AM and CGRP was five times greater than basal in MCs and 200-fold basal levels in NMCs. The ED50 of CGRP in the cAMP production of MCs was about 1/100 that of AM. In the NMCs, AM and CGRP elevated the cAMP levels with an ED50 of 1.7x10–8 and 1.8x10–10 M. The dose–response curves of AM and CGRP were not shifted with AM antagonist, AM(22–52), while CGRP antagonist, CGRP(8–37), shifted them with ED50 values increasing 100-fold.


Figure 4
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  		Fig. 4 Characterization of receptors expressed in MCs and NMCs. (a) MCs were stimulated with AM (closed square) and CGRP (closed circle). (b) NMCs were stimulated with AM in the absence (closed square) or presence of 10–6 M of either AM(22–52) (closed circle) or CGRP(8–37) (closed triangle). (c) NMCs were stimulated with CGRP in the absence (open square) or presence of 10–6 M of either AM(22–52) (open circle) or CGRP(8–37) (open triangle). After 10-min incubation, intracellular cAMP level was measured by RIA. Each value represents the mean±S.E.M. of four wells.

 
3.6 Effects of AM on IL-6 secretion
The basal secretion level of IL-6 activity from NMCs was 2.7 times higher than that of MCs (Table 2). After 24-h stimulation, AM increased IL-6 activity in the media of NMCs 2.3-fold. TNF-{alpha}, IL-1β and LPS stimulated secretion of IL-6 activity from MCs and NMCs 1.9- to 300-fold. Co-administration of AM with TNF-{alpha}, IL-1β or LPS further augmented IL-6 secretion 1.5-, 1.7- and 1.5-fold from MCs, and 9.0-, 8.0- and 2.5-fold from NMCs, respectively (Fig. 5). Forskolin and Br-cAMP mimicked the effects of AM on the IL-6 secretion from NMCs. CGRP receptor antagonist dose-dependently reduced IL-6 secretion stimulated with AM and IL-1β, but AM receptor antagonist showed no effect (Fig. 6).


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  		Table 2 Effects of TNF-{alpha}, IL-1β, LPS and AM on IL-6, NOx, and ET-1 secretion from MCs and NMCsa

 

Figure 5
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  		Fig. 5 Effects of AM on TNF-{alpha}, IL-1β, or LPS-induced IL-6 secretion from MCs and NMCs. IL-6 activities in the media were measured by the MH60.BSF-2 cell proliferation assay after incubation of MCs (a) and NMCs (b) for 24 h with AM in the presence of 5 µg/l of TNF-{alpha} (filled column), 1 µg/l of IL-1β (stippled column) or 10 µg/l of LPS (open column). Each value represents the mean±S.E.M. of four wells. *P<0.05 compared with IL-6 activity stimulated only with TNF-{alpha}, IL-1β or LPS in the respective cells.

 

Figure 6
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  		Fig. 6 Effects of Br-cAMP, forskolin and receptor antagonists on IL-6 secretion from NMCs under the stimulation with IL-1β. (a) IL-6 activities in the media were measured after 24-h incubation of NMCs with AM, Br-cAMP or forskolin under the stimulation with IL-1β (1 µg/l). *P<0.05 compared with IL-6 activity stimulated only with IL-1β. (b) IL-6 activities were measured after 24-h incubation in the absence or the presence of AM(22–52) or CGRP(8–37) under the stimulation with AM (10–7 M) and IL-1β (1 µg/l). #P<0.05 compared with IL-6 activity stimulated with AM and IL-1β. Each value represents the mean±S.E.M. of four wells.

 
3.7 Effects of AM on NO synthesis
Levels of NOx in the media of the control MCs and NMCs were comparable after 36-h incubation (Table 2). NOx level in the medium of MCs was elevated by IL-1β and AM, while that of NMCs was elevated by IL-1β and LPS. IL-1β dose-dependently increased NO synthesis in both of the cells (data not shown), while LPS stimulated it only in the NMCs.

In the presence of IL-1β, AM increased NOx level in the medium of MCs only at 10–7 M, while a dose-dependent increase up to 2.7-fold was observed in NMCs (Fig. 7). This effect was not observed in the LPS-stimulated NMCs. Forskolin and Br-cAMP enhanced NO synthesis in NMCs to a level comparable to that of AM (Fig. 7b). In the receptor antagonists, CGRP(8–37) abolished AM-induced NO synthesis in NMCs, but AM(22–52) did not inhibit it (Fig. 7c).


Figure 7
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  		Fig. 7 Effects of AM on NO synthesis in MCs and NMCs under the stimulation with IL-1β. (a,b) NOx concentrations in the media of MCs (a) and NMCs (b) were measured after 36-h incubation with AM, Br-cAMP or forskolin under the stimulation with IL-1β (1 µg/l). *P<0.05 compared with control NOx concentration stimulated only with IL-1β. (c) IL-1β-induced NO synthesis was measured after 36-h incubation in the absence or the presence of AM(22–52) or CGRP(8–37) under the stimulation with AM (10–7 M) and IL-1β (1 µg/l). #P<0.05 compared with NOx concentration stimulated with AM and IL-1β. Each value represents mean±S.E.M. of four wells.

 
3.8 Effects of AM on IR-ET-1 secretion
A basal IR-ET-1 level in the media of NMCs after 14-h incubation was 9-fold higher than that of MCs (Table 2). AM, TNF-{alpha}, IL-1β and LPS did not alter IR-ET-1 secretion from MCs. In the NMCs, AM decreased IR-ET-1 secretion while TNF-{alpha} increased it, but neither IL-1β nor LPS showed any effect. AM suppressed IR-ET-1 secretion from NMCs down to 58%, but not from MCs. Forskolin and Br-cAMP inhibited IR-ET-1 secretion from NMCs. AM(22–52) did not alter IR-ET-1 secretion from NMCs, while CGRP(8–37) dose-dependently restored it to the control level (Fig. 8).


Figure 8
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  		Fig. 8 Effects of AM on ET-1 secretion from MCs and NMCs. (a,b) ET-1 concentrations in the media were measured after 14-h incubation of MCs with AM (a) and NMCs with AM, Br-cAMP or forskolin (b). *P<0.05 compared with control (without stimulation). (c) ET-1 concentrations were measured after 14-h incubation in the absence or the presence of AM(22–52) or CGRP(8–37) under the stimulation with AM (10–7 M). #P<0.05 compared with ET-1 concentration stimulated only with AM. Each value represents mean±S.E.M. of six wells.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The purity of the MCs used in this study was extremely high as compared with that reported previously [15]. Since the NMCs used were almost exclusively composed of fibroblasts, we describe NMCs as fibroblasts in this section. MCs and fibroblasts secreted IR-AM, and this IR-AM was confirmed to be the native peptide (Fig. 2). The secretion rate of IR-AM from the fibroblasts was 2.8 times higher than that from the MCs (Table 1), which was comparable to those reported by Tsuruda and co-workers [18,19]. CGRP was not significantly secreted from either MCs or fibroblasts, although the receptors on these cells were more specific for CGRP (Fig. 4).

AM-positive staining was observed in the cultured MCs and fibroblasts, but the staining intensity did not correlate with the secretion rates of AM. Øie et al. [20] recently reported that AM-like immunoreactivity was observed in the interstitium between MCs and in perivascular connective tissue in the heart of normal adult rats. Although there are differences in the experimental conditions, both of these data indicate that fibroblasts are one of the major cells secreting AM in the heart.

AM secretion from fibroblasts was stimulated with TNF-{alpha}, IL-1β, LPS and dexamethasone, but was inhibited with TGF-β in a manner similar to that of VSMCs [2]. AM secretion from MCs was only elevated by dexamethasone. It is notable that glucocorticoid is a stimulator of AM secretion in most cell types. The TNF-{alpha} and IL-1β-stimulated AM secretion reported by Horio et al. [21] is principally similar to that observed in this study. As AM synthesis in the fibroblasts is more dynamically regulated than that in the MCs by LPS and cytokine stimulation, AM secretion from the fibroblasts is deduced to be predominant under the inflammatory conditions.

As for AM receptors, McLatchie et al. [16] have demonstrated that CGRP and AM share the core receptor, CRLR, and that RAMP1, 2 and 3 determine ligand specificity, i.e. RAMP1 converts CRLR into CGRP-specific but RAMP2 and 3 turn it to be AM-specific. We use AM/CGRP receptor in this study, because it is convertible by expressing different RAMPs. Intracellular cAMP levels were measured, as they reflected biological actions of AM/CGRP receptors. Both MCs and fibroblasts express AM/CGRP receptors that possess greater affinity for CGRP. AM-induced cAMP production was only inhibited with CGRP antagonist in both of the cells (Fig. 4). These results indicate that AM action in the heart is mediated via receptors with greater affinity for CGRP. The maximal cAMP level achieved with AM or CGRP was 14 times higher in the fibroblasts than in the MCs, indicating that AM acts more efficiently on the fibroblasts.

Gene transcript levels of RAMPs (Fig. 3b) supports the specificity of AM/CGRP receptors in fibroblasts. In the fibroblasts, a high level of RAMP1 mRNA was observed, while faint levels of RAMP2 and 3 mRNAs were detected, indicating that CRLR was converted into CGRP-specific. In the MCs, a clear band of RAMP3 mRNA and a weak band of RAMP1 mRNA were found, but RAMP2 mRNA was not detected. If the relative ratio of RAMP1 to RAMP2 determines the receptor specificity, AM/CGRP receptors in the MCs are specific for CGRP as observed in the cAMP production assay (Fig. 4a). Since contribution of RAMP3 has not well been elucidated, it is hard to discuss the receptor specificity only from these data. In the previous reports, only RAMP2 mRNA was clearly observed in rat heart, while comparable levels of RAMP1, 2 and 3 mRNAs were detected in the human heart [16,20,22]. The differences observed might be derived from those in the cell conditions (in culture or in tissue) or in the developmental stages (neonate and adult). Incidentally, our determined sequences of rat RAMP1 and RAMP3 had 2 and 1 amino acid discordant to those reported by Nagae et al. [22].

We examined effects of AM on secretion of IL-6, NO and ET-1 (Table 2). Plasma levels of IL-6 have been shown to be elevated in patients with cardiovascular diseases [23]. A basal secretion rate of IL-6 from fibroblasts was greater than that from MCs, and AM increased the rates from the fibroblasts. Under the stimulation with cytokines or LPS, AM synergistically augmented IL-6 secretion rates from MCs and fibroblasts. Administration of AM with IL-1β induced fibroblast rates of IL-6 secretion 800-fold over basal, indicating that the effect of AM is more prominent in the fibroblasts. AM elicited this effect through the cAMP-mediated pathway, as Br-cAMP and forskolin induced the comparable IL-6 secretion (Fig. 6). AM is found to be a common stimulator of IL-6 production in the fibroblasts, taken together with our previous data [24].

NO elicits a variety of effects on many organs. In the heart, MCs have been recognized as the major cells synthesizing NO [15]. In this study, fibroblasts and MCs secreted NO at comparable rates, but IL-1β treatment stimulated it more potently in fibroblasts than in MCs (Table 2). LPS stimulation of NO secretion was 20-fold in fibroblasts but little in MCs. Under the stimulation of IL-1β, AM augmented NO secretion 1.1-fold from MCs and 2.7-fold from fibroblasts via the cAMP-mediated pathway. These data demonstrate that fibroblasts have a high capability for synthesizing NO in the inflammatory cardiac tissue, and AM can elicit a more predominant effect on fibroblasts.

Both fibroblasts and MCs synthesize and secrete ET-1 [10], but a secretion rate of ET-1 in MCs is only 1/10 that of fibroblasts (Table 2). AM and ET-1 may be classified into a new category of peptides being abundantly produced in fibroblasts. Among the reagents tested, only TNF-{alpha} weakly increased ET-1 secretion from fibroblasts, but AM strongly reduced it by the cAMP-mediated pathway. Thus, AM is found to be a potent inhibitor of the ET-1 production in the heart.

IL-6 and NO have been reported to induce a negative inotropic effect [25,26]. As synthesis of IL-6 and NO is stimulated with AM in the cultured heart cells, AM may be able to reduce the cardiac contractility during inflammatory diseases, such as septic shock, myocardial infarction and myocarditis. Actually, AM is reported to induce a negative inotropic effect by stimulation of NO synthesis [27]. As AM suppresses secretion of ET-1 eliciting an inotropic effect, AM may improve cardiac function like ET-1 antagonists [28,29]. NO is reported to be an anti-hypertrophic factor, while ET-1 is a hypertrophic factor [30]. AM-induced NO synthesis and inhibition of ET-1 secretion are also expected to suppress remodeling of MCs. In fact, Tsuruda and co-workers [18,19] recently reported the inhibitory effects of AM on the hypertrophy of MCs and growth of fibroblasts. Thus, the endogenously secreted AM could induce various effects on the heart tissue, if AM concentration in the intercellular space is elevated in vivo to the levels shown in Figs. 5–8GoGoGo. The present study also suggests that cardiac fibroblasts are not merely supporting cells but also exerting a wide range of biological effects.

Time for primary review 22 days.


   Acknowledgements
 
The authors are grateful to Dr K. Kitamura and Professor T. Eto of Miyazaki Medical College, T. Tsuji of Shionogi & Co. Ltd., Professor K. Yoshizaki and Dr N. Nishimoto of Osaka University, Professor K. Nakao and Dr Y. Saito of Kyoto University, for donation of antisera against AM and cAMP, monoclonal antibody against AM, MH60.BSF-2 cells, antiserum against ET-1 and preparation technique of MCs and NMCs. The authors thank M. Nakatani and M. Higuchi of this institute for technical assistance. This work was supported in part by the Special Coordination Fund for the Promotion of Science and Technology from the Science and Technology Agency, by the research grants from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, and the Health Sciences Foundation of Japan.


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

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