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Subtype Specificity of Adenosine A2 Receptor on the Inhibition of Angiotensin IIinduced Cardiac Fibrosis
Kwanchai Bunrukchai1,*, Darawan Pinthong2 , Supachoke Mangmool1
1
Department of Pharmacology, Faculty of Pharmacy; 2Department of Pharmacology, Faculty
of Science, Mahidol University, Bangkok 10400, Thailand
*e-mail: mee_kh0945@yahoo.com
Abstract
Overstimulation of angiotensin II receptor (ATR) accelerates cardiac fibroblast into
the activated form characterized by enhancing of cell proliferation, synthesis of several
extracellular matrix (ECM) proteins and expression of alpha-smooth muscle actin (α-SMA),
resulting in cardiac fibrosis. The functions of cardiac fibroblast are regulated by several
autocrine/paracrine factors including adenosine. The previous studies showed that adenosine
can reduce the cell proliferation and secretion of inflammatory mediators in cardiac
fibroblast, supporting the cardioprotective effects of adenosine A2a and A2b receptors are
expressed mainly in cardiac fibroblasts. Pharmacological studies suggested the possible effect
of adenosine on inhibition of cardiac fibrosis appear occur predominantly via A2 receptor.
However, it is still unknown which A2 receptor subtypes involving in the inhibition of
cardiac fibrosis inducing by angiotensin II (Ang II). In this study, we used the selective A2a
receptor antagonist (SCH58261) and the selective A2b receptor antagonist (MRS1754) to
determine which subtypes are involved in the inhibition of Ang II-induced cardiac fibrosis.
Ang II-induced cell proliferation and α-SMA protein expression were assessed by MTT assay
and fluorescence microscope, respectively. Our results show that Ang II-induced cell
proliferation and α-SMA production were significantly inhibited by NECA (5′-Nethylcarboxamidoadenosine; adenosine receptor agonist), indicating activation of adenosine
receptor suppress Ang II-induced cardiac fibrosis. We also found that the action of NECA on
inhibition of Ang II-induced cell proliferation was blocked by MRS1754, but not SCH58261
in neonatal rat cardiac fibroblast. Thus, the effects of NECA on inhibition of cardiac fibrosis
are mediated though A2b receptor signaling pathway.
Keywords: Adenosine receptor, Alpha-smooth muscle actin (α-SMA), Angiotensin II,
Cardiac fibrosis, Cell proliferation
Introduction
After cardiac injury occurring in the heart (e.g., from acute coronary syndrome,
myocardial infraction, overstimulation of endothelin receptors or ATRs), these conditions
accelerate cardiac fibroblast into the activated form which characterized by increasing of cell
proliferation, synthesis of ECM proteins such as collagen I, collagen III and fibronectin (1,2).
The activation fibroblast also enhances the secretion of several growth factors and cytokines
(2).The accumulation of ECM proteins and the differentiation of fibroblast into myofibroblast
lead to the replacement of cardiac myocyte with fibrotic scar tissue, resulting in cardiac
fibrosis. Cardiac fibrosis disrupts the communicational and functional of cells in the heart,
making the abnormality of contractility and heart rhythm and also accelerates the cardiac
remodeling process which elicits the detrimental effects on the heart and increases the risk of
heart diseases.
Activation of adenosine receptor leads to the reduction of cell proliferation and
secretion of inflammatory mediators in cardiac fibroblast, supporting the cardioprotective
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effects of adenosine (3-6). In the present, there is no drug that can be used to treat cardiac
fibrosis. Thus, drugs or any active compounds that stimulate adenosine receptor signaling are
seemed to be therapeutic target for treatment of cardiac fibrosis.
Adenosine receptors exist in multiple subtypes (A1, A2a, A2b, and A3 receptors) (3).
All subtypes are members of the superfamily of G-protein-coupled receptors (GPCRs). The
A1 and A3 receptors couple to Gi/o proteins, whereas the A2a and A2b receptors couple to
Gs proteins (7,8). The functional effects of adenosine on cardiac fibroblasts include reduced
cell proliferation, reduced collagen synthesis and reduced tumor necrosis factor-alpha (TNFα) secretion (3,9-12). The researchers tried to determine which AR subtype mediated these
effects on cardiac fibroblasts. The recent study used pertussis toxin (PTX) to inhibit Gi
pathway, PTX did not alter the inhibitory effect of adenosine agonist (NECA) on Ang IImediated collagen synthesis in adult rat cardiac fibroblasts (13). Data derived from the use of
various adenosine receptor agonists and antagonists indicated that the inhibitory effects of
adenosine agonists (e.g., 2-chloroadenosine, NECA) on platelet-derived growth factor-BB
(PDGF-BB)-induced DNA synthesis, cell number, and collagen synthesis were significantly
blocked by selective A2 receptor antagonist, but not by selective A1 receptor antagonist in
adult rat cardiac fibroblasts (3-5). Thus, the effects of adenosine on reduction of cardiac
fibrosis were potentially mediated via A2 receptor.
However, it is still unknown which A2 receptor subtypes involving in the inhibition of
Ang II-induced cardiac fibrosis. The molecular mechanisms of adenosine receptor signaling
are unclear. Thus, the identification of subtype specificity and the molecular mechanism of
adenosine-mediated signaling on inhibition of cardiac fibrosis will help us to discover the
new compounds acting on stimulation of adenosine receptor for prevention of cardiac
fibrosis.
Methodology
Reagents
Ang II, NECA, SCH58261, Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and
anti-α-SMA antibody were purchased from Sigma-Aldrich. Selective adenosine A2b receptor
antagonist (MRS1754) was purchased from Calbiochem-Merck4Biosciences. Alexa Fluor
488 goat anti-mouse IgG antibody was purchased from Life technologies.
Cardiac fibroblasts isolation and culture
The animals in this study were handled according to approved protocols and animal
welfare regulations of the authors’ Institution Review Boards of Faculty of Pharmacy,
Mahidol University. A Sprague Dawley rat (pregnant) was form National Laboratory Animal
Center, Mahidol University. Primary neonatal rat cardiac fibroblast cultures were generated
form ventricular tissues of 1-or 2-day old neonatal Sprague-Dawley rats. These cells were
maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin at 37 ºC in an atmosphere of 5% CO2.
Cell proliferation assay
Cardiac fibroblast proliferation was evaluated by MTT assay, which is based on the
transformation of tetrazolium salt MTT by active mitochondria to an insoluble formazan salt.
Cardiac fibroblasts (5,000 cells/well) were plated in 96-well plates in DMEM containing 1%
FBS and incubated 24 hr. Then the cells were treated with pharmacological agents.
Antagonists were added 30 min before the addition of agonists. After 30 min of treatment
with agonists, the cells were stimulated with Ang II for 24 hr. MTT solution (concentration of
1 mg/ml) was added to each well under sterile conditions, and the plates were incubated for 3
3
hr at 37 °C. Untransformed MTT was removed by aspiration, and formazan crystals were
dissolved in dimethyl sulfoxide (100 µl/well). Formazan was quantified spectroscopically at
570 nm using microplate reader (UV scan). The cell decrease percent relative to the control
group was determined. The percentage of cell viability was calculated according to the
following equation.
The percentage of cell viability = (Absorbance of treated cells)/(Absorbance of
control cells)×100
Detection of α-SMA by fluorescence microscope
Cardiac fibroblasts (2.5x104 cells/well) were plated in 35-mm glass dishes in DMEM
containing 1% FBS. The cells were treated with pharmacological agents and stimulated with
2000 nM Ang II for 48 hours. The cells were fixed with 4% paraformaldehyde and kept at 4
ºC for 2 hours. Then 0.1% Triton-X was added and kept at room temperature for 5 minutes.
Then 1% BSA was added and kept at room temperature for 20 minutes. The cells were
incubated for 1 hour with anti-α-SMA antibody (diluted 1:500). After several washes, the
cells were incubated for 1 hour with Alexa Fluor 488 goat anti-mouse IgG antibody. The αSMA was visualized by fluorescence microscopy (Inverted microscope, Olympus IX 81).
Data analysis
Data expressed as means + SEM (standard error of mean). Some experiments, the
outcomes assessed by using the one way analysis of variance (ANOVA). Statistical analysis
by student’s paired or unpaired test t-test as appropriate, and means will be considered
significantly different when p<0.05.
Results
Stimulation of adenosine receptor inhibits Ang II-induced cell proliferation
We first examined the effect of NECA (adenosine receptor agonist) on inhibition of
Ang II-induced cardiac fibroblast proliferation. Treatment with Ang II significantly increased
the numbers of cardiac fibroblasts compared to that of control (vehicle) group (Figure 1),
indicating the induction of cell proliferation. Interestingly, pretreatment with NECA
significantly inhibited Ang II-induced cell proliferation. These results suggested that
stimulation of adenosine receptor can inhibit Ang II-induced cell proliferation in neonatal rat
cardiac fibroblast.
Figure 1. Effect of NECA on inhibition of Ang IIinduced cell proliferation. Cardiac fibroblasts were
pretreated with or without 10 µM of NECA for 30 min
before stimulation with 1000 nM Ang II for 24 hr. Cell
proliferation was quantified, expressed as percentage
relative to the non-treated cells (vehicle), and shown as
mean+SEM (n=4). #, p<0.05 versus vehicle group; *p <0.05
versus Ang II-treated group.
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Antiproliferative effect of NECA is cAMP dependent
After adenosine receptor agonist (NECA) binding, the A2 receptor can couple with 
subunit of heterotrimeric G protein (Gαs), which results in activation of adenylate cyclase
(AC), followed by elevation of cAMP levels (14,15). We investigated whether the inhibition
of Ang II-induced cell proliferation by NECA is dependent on cAMP. To demonstrate the
requirement of cAMP for NECA-mediated inhibition of cell proliferation, we used forskolin
(an AC activator) and ddA (an AC inhibitor) that is able to completely inhibit the activity of
AC. Pretreatment with forskolin leads to the reduction of cell proliferation induced by Ang II
(Figure 2). The antiproliferative activity of NECA can be inhibited by a specific AC inhibitor,
ddA (Figure 2), confirming that the activation of adenosine receptor inhibited Ang II-induced
cell proliferation through a cAMP-dependent way.
Figure 2. Effect of NECA on inhibition of Ang IIinduced cell proliferation through cAMPdependent pathway. Cardiac fibroblasts were
pretreated with ddA (AC inhibitor) 5 µM for 30
min, and then treated with either NECA or forskolin
(FSK) for 30 min before stimulation with 1000 nM
Ang II for 24 hr. Cell proliferation was quantified,
expressed as percentage relative to the non-treated
cells (vehicle), and shown as mean+SEM (n=3).
#p<0.05 versus vehicle group; *p<0.05 versus Ang
II-treated group; **p<0.05 versus Ang II+NECAtreated group.
Stimulation of adenosine A2b receptor inhibits Ang II-induced cell proliferation
We use selective adenosine A2a receptor antagonist (SCH58261) and selective
adenosine A2b receptor antagonist (MRS1754) to determine which A2 receptor subtypes are
involved in the inhibition of Ang II-induced cardiac fibroblast proliferation. We found that
selective A2b receptor antagonist, MRS1754, rescued the antiproliferative effect of NECA,
whereas selective adenosine A2a receptor antagonist, SCH58261, had no effect (Figure 3)
demonstrating that stimulation of adenosine A2 receptor by NECA attenuates Ang II-induced
cardiac fibroblast proliferation by signaling through A2b receptor subtype. These results
indicated the importance of A2b receptor stimulation on the inhibition of cardiac fibrosis.
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Figure 3. Effects of selective adenosine A2a and A2b
receptor antagonist on Ang II-induced cell
proliferation. Cardiac fibroblasts were pretreated with
either selective adenosine A2a receptor antagonist
(SCH58261) and selective adenosine A2b receptor
antagonist (MRS1754) for 30 min, before the addition
with 10 µM of NECA . After 30 min, stimulated with
1000 nM of Ang II for 24 hr. Cell proliferation was
quantified, expressed as percentage relative to the nontreated cells (vehicle), and shown as mean+SEM (n=4).
#p<0.05 versus vehicle group; *p<0.05 versus Ang IItreated group; **p<0.05 versus Ang II+NECA-treated
group.
Inhibition of Ang II-induced α-SMA expression by stimulation of adenosine receptor
We next examined the effects of NECA on Ang II-induced α-SMA expression.
Results from Fluorescence microscope revealed significantly increase of α-SMA production
in cardiac fibroblast when treated with Ang II (Figure 4). Stimulation of adenosine receptor
with NECA resulted in a decrease of α-SMA expression inducing by Ang II. Thus,
stimulation of adenosine receptor can inhibit α-SMA protein expression in response to
profibrotic agents, Ang II, that induce myofibroblasts differentiation.
A
B
Figure 4. Effects of NECA on inhibition of Ang II-induced α-SMA expression. (A and B) Cardiac
fibroblasts were pretreated with or without 20 µM of NECA for 30 min followed by 2000 nM Ang II for 48
hr. Cells were washed with PBS and incubated with anti-α-SMA antibody at 37 ºC for 1 hr followed by goat
anti-mouse antibody (Alexa Fluor 488) for 1 hr. The α-SMA was visualized by fluorescence microscopy. (A)
Cells were stained for α-SMA (green) and nuclear staining of nucleus with DAPI (blue), bar =10 µM. (B) αSMA-expressed cells were counted, expressed as the percentage of total cells and shown as mean ± SEM
(n=3). #p<0.05 versus vehicle group; * p<0.05 versus Ang II-treated group.
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Discussion and Conclusion
In the present study, we demonstrate the effects of NECA (adenosine receptor
agonist) can inhibit Ang II-induced cell proliferation and α-SMA production indicating the
potential role of adenosine receptors on inhibition of cardiac fibrosis. We also found that the
action of NECA on inhibition of Ang II-induced cell proliferation was blocked by MRS1754
(selective A2b receptor antagonist), but not SCH58261 (selective A2a receptor antagonist) in
neonatal rat cardiac fibroblast. Thus, the effects of NECA on inhibition of cardiac fibrosis are
mediated though A2b receptor signaling pathway.
Consistent with previous studies, Dubey and colleagues (4,5) have reported that
activation of A2b receptor inhibited collagen synthesis in cardiac fibroblasts. Thus, the
agents which selective agonized to adenosine A2b receptor may be considered to be potential
therapeutic target for the treatment of cardiac fibrosis. However, signaling pathway of A2b
receptor remains to be elucidated (Figure 5). Further study is necessary to clarify the
signaling pathway of adenosine A2b receptor on inhibition of cardiac fibrosis.
Figure 5. Shematic diagram representing the stimulation of A2b receptor inhibits Ang II-induced
cardiac fibrosis. Ang II can induce cardiac fibrosis by promoting cardiac fibroblasts proliferation, collagen
synthesis and α-SMA expression. NECA binding to A2b receptor activates heterotrimeric Gs protein, then
stimulates adenylate cyclase (AC) activity, leading to inhibition of Ang II-induced cardiac fibrosis.
Acknowledgements
This study was supported by the Faculty of Graduate Studies, Mahidol University.
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