0022-3565/06/3161-392–402$20.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
JPET 316:392–402, 2006
Vol. 316, No. 1
90597/3067154
Printed in U.S.A.
Induction of ␤3-Adrenergic Receptor Functional Expression
following Chronic Stimulation with Noradrenaline in Neonatal
Rat Cardiomyocytes
Renée Germack and John M. Dickenson
Biomedical Research Centre, School of Science, Nottingham Trent University, Nottingham, United Kingdom
Received June 6, 2005; accepted September 19, 2005
Three ␤-adrenergic receptor subtypes (␤1-, ␤2-, and ␤3-AR)
have been cloned and pharmacologically characterized
(Strosberg, 1995). These different subtypes belong to the G
protein-coupled receptor superfamily and modulate cardiac
function after stimulation by the catecholamines, noradrenaline, and adrenaline. Increases in heart rate and force of
This work was supported by the British Heart Foundation Grant FS/03/
095/16317.
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.105.090597.
and 1-(2-ethylphenoxy)-3-[[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino-(2S)-2-propanol hydrochloride (SR 59230A) (␤3selective antagonist). In addition, the level of the three subtypes
was determined by reverse transcription polymerase chain reaction and Western blotting. NOR pretreatment decreased the
activation of cAMP induced by NOR, isoprenaline, and DOB,
whereas PROC response was abolished. The inhibition of NOR
response by CGP 20712A or ICI 118551 demonstrated that ␤1and ␤2-ARs are down-regulated and that ␤2-AR functional activity was also abolished in cardiomyocytes exposed to chronic
stimulation. ␤3-AR function was observed with NOR and ISO
when ␤1-/␤2-ARs were blocked and with both ␤3-selective
agonists in NOR-treated cells only. This response was completely inhibited by SR 59230A and involved Gi protein. Furthermore, the results from functional studies agree well with
those from expression experiments. In conclusion, these data
provide strong evidence that ␤3-ARs are functionally upregulated and coupled to Gi protein in rat neonatal cardiomyocytes following chronic exposure to NOR when ␤1- and ␤2ARs are down-regulated.
contraction are mediated mainly by ␤1-ARs and to a lesser
extent by the ␤2-AR (Dzimiri, 1999; Steinberg, 1999). These
functional effects result from the sequential activation of
stimulatory Gs proteins, adenylyl cyclase, and protein kinase
A (PKA), leading to the phosphorylation of proteins involved
in cardiac contractility (troponin I, L-type Ca2⫹ channels,
and phospholamban) (Post et al., 1999). Previous studies
have shown that the ␤2-AR also couples to Gi protein in
cardiomyocytes (Xiao, 2001). In contrast to ␤1- and ␤2-AR,
activation of the ␤3-AR induces a negative inotropic effect in
different species, including human, dog, guinea pig, and rat
ABBREVIATIONS: ␤-AR, ␤-adrenergic receptor; PKA, protein kinase A; DMEM, Dulbecco’s modified Eagle’s medium; CL 316243 (CL),
5-[-2-([-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate; IGEPAL CA-630, octylphenyl-polyethylene glycol;
BRL 37344 (BRL), 4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid; ICI 118551, 1-[2,3-(dihydro-7-methyl-1H-inden-4yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride; CGP 20712A, 2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate; SR 59230A, 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride; RT-PCR, reverse transcription-polymerase chain reaction; TBS, Tris-buffered saline; NOR,
noradrenaline; ISO, isoprenaline; DOB, dobutamine; PROC, procaterol; PTX, pertussis toxin.
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ABSTRACT
This study aimed to characterize ␤3-adrenergic receptors (ARs)
in rat neonatal cardiomyocytes using the noradrenaline (NOR)
properties to modulate the expression and function of the three
␤-ARs. We assessed the effect of NOR (physiological nonselective agonist), isoprenaline (ISO, ␤-nonselective agonist), dobutamine (DOB, ␤1-selective agonist), and procaterol (PROC,
␤2-selective agonist) on cAMP accumulation using cardiomyocytes untreated or treated with 100 ␮M NOR for 24 h. The
inhibition of forskolin-stimulated cAMP accumulation was
determined using NOR, isoprenaline, and the ␤3-selective
agonists 4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344) and 5-[-2-([-2-(3chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole2,2-dicarboxylate (CL 316243). The experiments were
performed in the absence or presence of propranolol or 2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1Himidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP 20712A) and/or 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118551) to inhibit ␤1- and ␤2-AR stimulation
␤3-Adrenergic Receptor in Cardiomyocytes
Materials and Methods
Materials. Bovine serum albumin, Dulbecco’s modified Eagle’s
medium (DMEM), fetal calf serum, fibronectin, forskolin, pertussis
toxin, DL-norepinephrine hydrochloride, (⫾)-propranolol, hydrochloride, CL 316243 (CL), deoxynucleotide (dNTP) mix, IGEPAL CA-630,
octylphenoxy polyethoxyethanol, and leupeptin were all obtained
from Sigma Chemical (Poole, Dorset, UK). Trichloroacetic acid was
purchased from Calbiochem (Nottingham, UK). Procaterol hydrochloride, xamoterol hemifumarate, isoproterenol hydrochloride, BRL
37344 (BRL), ICI 118551, CGP 20712A, and SR 59230A were all
from Tocris Cookson Inc. (Bristol, UK). RQ1 RNase-free DNase,
RNasin RNase inhibitor, dithiothreitol (molecular grade), random
primers, Moloney murine leukemia virus reverse transcriptase, and
TaqDNA polymerase were all purchased from Promega (Southampton, UK). ([8-3H]Adenine was obtained from GE Healthcare (Little
Chalfont, Buckinghamshire, UK). Rat ␤1- (sc-568), ␤2- (sc-570), and
␤3 (sc-1473)-adrenergic receptor antibodies were purchased from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Culture. Neonatal ventricular myocytes were prepared from
1- to 4-day-old Wistar rats using the neonatal cardiomyocyte isolation system (Worthington Biochemicals, Lornes Laboratories, Reading, UK) as described previously (Germack and Dickenson, 2004).
The cells were preplated three times for 30 min in a humidified
incubator (95% air, 5% CO2 at 37°C) in DMEM supplemented with 2
mM L-glutamine, 10% (v/v) fetal calf serum and penicillin/streptomycin (100 U/ml) to minimize fibroblast contamination. Cardiomyocyte-rich cultures (⬎90%) were plated onto fibronectin-coated plates
at a final density of 1.25 ⫻ 105 cells/cm2 in supplemented DMEM.
For cAMP accumulation assay, the cells were plated onto 24-well
plates; and for RT-PCR and Western blotting, cells were plated onto
20-cm2 dishes. After 3 days, confluent and spontaneously beating
cells were serum starved overnight before the experiments and
treated or not treated with 100 ␮M noradrenaline.
cAMP Accumulation Assay. After serum starvation of cardiomyocytes (overnight), assays were carried out in serum-free DMEM
in a humidified incubator (95% air, 5% CO2 at 37°C). Agonists,
antagonists, and/or inhibitors according to the experiments were
added as described in the figure legends. The cells were incubated for
3 h in a humidified incubator (95% air, 5% CO2 at 37°C) with 500 ␮l
of serum-free DMEM containing [3H]adenine (2 ␮Ci/well). [3H]adenine-labeled cells were washed twice with Hanks’/HEPES buffer and
then incubated in 500 ␮l/well serum-free DMEM containing the
cyclic AMP phosphodiesterase inhibitor rolipram (10 ␮M) for 15 min
at 37°C in a humidified incubator. Agonists were added 5 min before
the incubation with 1.5 ␮M forskolin (10 min). Antagonist or inhibitors were added 30 min before agonist. Incubations were terminated
by the addition of 500 ␮l of 5% trichloroacetic acid after removing the
medium. [3H]Cyclic AMP was isolated by sequential Dowex-alumina
chromatography as described previously (Dickenson and Hill, 1998).
After elution, the levels of [3H]cyclic AMP were determined by liquid
scintillation counting.
RT-PCR. Total RNA was extracted from cardiomyocytes treated
or not treated with 100 ␮M noradrenaline using RNA isolation
reagent, RNAwiz (Ambion, Eurotech, Huntingdon, UK). Total RNA
was purified by chloroform/water extraction and isopropanol precipitation. All RNA preparations were treated with RQ1 DNase (1 U/␮l)
for 20 min at 37°C in the presence of RNasin (40 U/␮l) and dithiothreitol (100 mM).
Reverse transcription was performed with 40 ␮g of total RNA for
the synthesis of cDNA using hexadeoxynucleotide random primers
(540 ng/ml), RNasin (40 U/␮l), dNTP (5 mM), and Moloney murine
leukemia virus reverse transcriptase (200 U/ml) for 90 min at 42°C.
Polymerase chain reaction was conducted with 1 ␮l of cDNA for
␤-actin, 3 ␮l for ␤1- and ␤2-ARs, and 5 ␮l for ␤3-AR in the presence of
dNTPS (1.25 mM), 200 ng of respective primers, and 1.5 U of
TaqDNA polymerase. Following polymerase chain reaction, the samples were denatured for 5 min at 95°C. The amplification steps
involved 1-min denaturation at 94°C for ␤1- and ␤2-AR cDNA and 1.5
min for ␤3-AR and ␤-actin; 1 min annealing for ␤1-, ␤2-AR, and
␤-actin and 1.5 min for ␤3-AR; and 1 min extension at 72°C for ␤1and ␤2-AR and ␤-actin and 2 min for ␤3-AR cDNA. Annealing temperatures, sequences of the primers and amplification cycle number
are given in Table 1. The RT-PCR products were analyzed using 1.5%
agarose gel electrophoresis. ␤-Actin mRNA was used as an internal
standard. The RT-PCR products were quantified by densitometry
using GeneGenius BioImaging system (Syngene; Synoptics Ltd.,
Cambridge, UK) and normalized to the signal of ␤-actin.
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(Gauthier et al., 1998, 1999; Kitamura et al., 2000; Cheng et
al., 2001; Moniotte et al., 2001; Morimoto et al., 2004). ␤3AR-mediated decrease in cardiac contractility involves coupling to Gi/o protein(s) and activation of constitutively expressed endothelial nitric-oxide synthase and the subsequent
generation of NO (Gauthier et al., 1998; Kitamura et al.,
2000; Varghese et al., 2000; Brixius et al., 2004). Furthermore, the functional activity of ionic currents such as L-type
Ca2⫹ and potassium I(Ks) are reduced by ␤3-AR stimulation,
which also modulates cardiac contractility (Kitamura et al.,
2000; Cheng et al., 2001; Bosch et al., 2002; Morimoto et al.,
2004). Interestingly, ␤3-AR expression level increases in failing myocardium in human (Moniotte et al., 2001) and dog
(Cheng et al., 2001), contributing to the depression of cardiac
function (Morimoto et al., 2004). In contrast, ␤1-ARs are
down-regulated, and both ␤1- and ␤2-ARs are uncoupled from
Gs proteins (Dzimiri, 1999). It is widely accepted that heart
failure induces an increase in the activity of the sympathetic
nervous system, leading to an elevation in circulating catecholamines levels (Dzimiri, 1999; Steinberg, 1999). We can
assume that the consequences of elevated catecholamines are
a decrease in the positive inotropic response to ␤1- and ␤2ARs, and an increase in the negative inotropic activity induced by the ␤3-AR, resulting probably in the impairment of
the cardiac function as shown by Morimoto et al. (2004).
Furthermore, in adipocytes, chronic exposure to isoproterenol induces an increase in ␤3-AR expression and a downregulation of ␤1-AR level (Thomas et al., 1992). The ␤1-AR is
also down-regulated in transgenic mice displaying cardiacspecific overexpression of the human ␤3-AR (Kohout et al.,
2001). Similarly, in cardiomyocytes from ␤1- and ␤2-AR
knockout mice, isoproterenol induced a small decrease in
contraction rate involving presumably the ␤3-AR (Devic et
al., 2001). In marked contrast, ␤1-AR mRNA is up-regulated
in white and brown adipose tissue isolated from ␤3-AR
knockout mice (Susulic et al., 1995). Finally, Dinçer et al.
(2001) showed that ␤1-AR level decreases, whereas ␤3-AR
expression increases in hearts from diabetic rats, suggesting
that the depressed cardiac function seen in chronic diabetes
may be due to the modulation of ␤-AR subtype expression.
Overall, these observations indicate that possible compensatory interactions exist between ␤1- and ␤3-AR expression and
function. Therefore, in this study, we characterized the
␤3-AR expression and function in neonatal cardiomyocytes
using the properties of catecholamines to down-regulate ␤1/
␤2-ARs and presumably up-regulate ␤3-ARs. We demonstrate for the first time that the ␤3-AR subtype, which was
not functional in neonatal cardiomyocytes under normal
physiological conditions, is functionally up-regulated following chronic exposure to noradrenaline and inhibits cAMP
accumulation through Gi protein coupling. In marked contrast, ␤1- and ␤2-ARs were down-regulated.
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Germack and Dickenson
TABLE 1
Sequences of the primers specific for rat ␤1-AR, ␤2-AR, ␤3-AR, and ␤-actin
Primer
␤1-AR
␤2-AR
␤3-AR
␤-Actin
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
PCR Product
Annealing
Temperature
No. of
Cycles
Reference
base pairs
°C
522
60
33
Podlowski et al. (1998)
387
60
33
Podlowski et al. (1998)
204
60
40
Bensaid et al. (1993)
301
55
25
5⬘-CTCACCAACCTCTTCATCATG-3⬘
5⬘-GAAGCGGCGCTCGCAGCTGTCG-3⬘
5⬘-GAGACCCTGTGCGTGATTGC-3⬘
5⬘-CCTGCTCCACCTGGCTGAGG-3⬘
5⬘-CCAACTCTGCCTTCAACCCGCTCA-3⬘
5⬘-CCTTCATGTGGGAAATGGACGCTC-3⬘
5⬘-CGTAAAGACCTCTATGCCAA-3⬘
5⬘-GGTGTAAAACGCAGCTCAGT-3⬘
shown in Fig. 1 and Table 2, the response to noradrenaline
(NOR; physiological ␣- and ␤-adrenergic agonist) and isoprenaline (ISO; nonselective ␤-adrenergic agonist) was significantly reduced by 61 and 77%, respectively, following the
pretreatment with 100 ␮M NOR for 24 h. The desensitization
of the functional response depends mainly on the alteration
of both, ␤1- and ␤2-ARs (Dzimiri, 1999; Steinberg, 1999).
Results
Effect of Nonselective, ␤1-, and ␤2-Selective Agonists
on cAMP Accumulation in Neonatal Rat Cardiomyocytes Untreated or Treated with 100 ␮M Noradrenaline. It is well known that chronic stimulation of ␤-ARs
causes a decrease in ␤-adrenergic response by down-regulation of the ␤-ARs (Dzimiri, 1999; Steinberg, 1999). Indeed, as
Fig. 1. Activation of cAMP accumulation by nonselective ␤ agonists
noradrenaline (A) and isoprenaline (B) in neonatal rat cardiomyocytes
untreated (䡺) or treated with 100 ␮M noradrenaline (E) for 24 h. cAMP
accumulation was measured as described under Materials and Methods.
Data are expressed as percentage of the basal level of cAMP accumulation (100%). Each point represents the mean ⫾ S.E. of three to four
experiments.
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Western Blot Analysis. Cardiomyocytes treated or not treated
with 100 ␮M noradrenaline were washed with ice-cold phosphatebuffered saline and lysed in ice-cold hypotonic buffer (30 mM TrisHCl, 5 mM MgCl2, 100 mM NaCl, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, and 5 ␮g/ml leupeptin). Cell membranes were
collected by centrifugation for 30 min at 70,000g and 4°C. The resulting plasma membrane pellets were dissolved in detergent buffer
[150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% (v/v) IGEPAL
CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 1 mM
Na3VO4, 1 mM NaF, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 ␮g/ml leupeptin]. Cell membrane homogenates
were clarified by centrifugation (2 min; 12,000 rpm) using an Eppendorf microcentrifuge. Then, 100 ␮l of the supernatant was removed
and stored at ⫺20°C until required. Protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad, Hertfordshire, UK) with bovine serum albumin as the standard. Samples
were heated at 95°C in SDS-polyacrylamide gel electrophoresis sample buffer (v/v). Proteins (30 – 40 ␮g for ␤1-, 40 –50 ␮g for ␤2-, and
60 – 80 ␮g for ␤3-AR detection) were separated by SDS-polyacrylamide gel electrophoresis (12% acrylamide gel) using a Bio-Rad
Mini-Protean II system (1 h at 200 V). Proteins were transferred to
nitrocellulose membranes using a Bio-Rad Trans-Blot system (1 h at
100 V in 25 mM Tris, 192 mM glycine, and 20% MeOH). Following
transfer, the membranes were washed with Tris-buffered saline
(TBS) and blocked for 1 h at room temperature in blocking buffer
[TBS, 5% (w/v) skimmed milk powder, and 0.1% Tween 20]. Blots
were then incubated overnight at 4°C with ␤1-, ␤2-, or ␤3-AR primary
antibodies at the dilution 1/500, 1/100, and 1/50, respectively. The
primary antibody was removed, and the blot was extensively washed
three times for 5 min in TBS/Tween 20. Blots were then incubated for
1 h at room temperature with goat anti-rabbit and rabbit anti-goat
secondary antibody coupled to horseradish peroxidase (DakoCytomation Ltd., Ely, Cambridgeshire, UK) at 1:1000 and 1/3000 dilution, respectively. Following removal of the secondary antibody, blots
were extensively washed as described above and developed using the
enhanced chemiluminescence detection system (GE Healthcare) and
quantified by densitometry using GeneGenius BioImaging System
(Syngene).
Statistical Analysis. Results are expressed as means ⫾ S.E.
Concentration-response and inhibition-response curves were analyzed by computer-assisted iteration using Prism (GraphPad Software Inc., San Diego, CA). Statistical significance was determined by
Student’s t test, and P ⬍ 0.05 was considered as the limit of statistical significance.
␤3-Adrenergic Receptor in Cardiomyocytes
395
TABLE 2
Effect of nonselective and ␤1- and ␤2-selective agonists on cAMP accumulation in newborn rat cardiomyocytes treated or untreated with 100 ␮M
noradrenaline
Values are means ⫾ S.E. of three to four experiments performed in duplicate. The potencies of the agonists were evaluated by their EC50 or concentration of agonist inducing
50% of the maximal cAMP accumulation expressed in ⫺log10 EC50. Emax is the maximal response expressed in percentage over basal response. Cardiomyocytes were treated
with 100 ␮M noradrenaline for 24 h.
Emax
EC50
␤-Adrenergic Agonist
Untreated
Treated
Untreated
⫺log10 EC50
Nonselective agonist
Noradrenaline
Isoprenaline
␤1-Selective agonist
Dobutamine
␤2-Selective agonist
Procaterol
Procaterol ⫹ PTX
Treated
% over basal response
6.91 ⫾ 0.11
7.63 ⫾ 0.10
6.54 ⫾ 0.09
7.51 ⫾ 0.43
317 ⫾ 37
226 ⫾ 24
123 ⫾ 22*
51 ⫾ 16***
6.27 ⫾ 0.12
6.13 ⫾ 0.19
292 ⫾ 60
56 ⫾ 7**
7.45 ⫾ 0.10
7.18 ⫾ 0.37
NR
NR
125 ⫾ 12
134 ⫾ 19
NR
NR
NR, no response.
*P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 versus untreated cells.
Fig. 2. Activation of cAMP accumulation by dobutamine, ␤1-selective
agonist (A) and procaterol, ␤2-selective agonist (B) in neonatal rat cardiomyocytes untreated (䡺) or treated with 100 ␮M noradrenaline (E) for
24 h. cAMP accumulation was measured as described under Materials
and Methods. Data are expressed as percentage of the basal level of cAMP
accumulation (100%). Each point represents the mean ⫾ S.E. of four
experiments.
also coupled to PTX-sensitive Gi proteins in addition to the
classical Gs/cAMP/PKA pathway (Xiao, 2001). Therefore, ␤2AR-mediated Gi activation can counteract Gs-mediated
cAMP production. This dual pathway may explain the low
cAMP response to PROC stimulation in untreated cells and
the absence of stimulation in treated cardiomyocytes. To
determine whether the ␤2-AR response involves Gi coupling,
cAMP accumulation was performed in the presence of PTX in
treated and untreated cells. Pretreatment with PTX did not
modify PROC-induced cAMP production (Fig. 2B; Table 2),
showing the absence of ␤2-AR coupling to Gi proteins in
neonatal rat cardiomyocytes. In addition, these results indicate that the nonselective agonist response in NOR-treated
cardiomyocytes involves the ␤1-AR subtype rather than the
␤2-AR.
Effect of ␤1- and ␤2-Selective Antagonists on NORInduced cAMP Accumulation in Neonatal Rat Cardiomyocytes Untreated or Treated with 100 ␮M Noradrenaline. To characterize further the ␤-AR subtypes
involved in NOR-induced cAMP accumulation, we studied
the inhibition of 1 ␮M NOR response by the selective ␤1antagonist CGP 20712A and the ␤2-antagonist ICI 118551 in
cardiomyocytes untreated or treated with 100 ␮M NOR (Fig.
3). As expected, NOR pretreatment induced a decrease in 1
␮M NOR response by 72% (treated, 66 ⫾ 7% versus untreated, 239 ⫾ 11%; n ⫽ 7; P ⬍ 0.001). As shown in the Fig.
3A, CGP 20712A inhibited by 58 ⫾ 4% NOR-induced cAMP
accumulation in untreated cells. Following the pretreatment,
NOR-induced cAMP response was fully inhibited (91 ⫾ 9%
versus untreated; P ⬍ 0.01), indicating that the residual
NOR response (42%) observed in untreated cells may involve
␤2-AR stimulation. Pretreatment with 100 ␮M NOR did not
modify the CGP 20712A potency evaluated by its IC50 expressed as ⫺log10 IC50 (pIC50), treated 7.83 ⫾ 0.15 (14.7 nM;
n ⫽ 3) versus untreated 7.72 ⫾ 0.21 (19.1 nM; n ⫽ 3).
Interestingly, ICI 118551 exhibited a significant biphasic
inhibition curve in untreated cardiomyocytes (P ⬍ 0.01; Fig.
3B). The first population of receptors with a high inhibition
potency (pIC50 ⫽ 8.39 ⫾ 0.43; 4.1 nM; n ⫽ 4) accounted for
32 ⫾ 7% of the total inhibition. The second part of the curve
elicited 68 ⫾ 14% inhibition response with a pIC50 value of
5.24 ⫾ 0.30 (5.8 ␮M; n ⫽ 4). Interestingly, the inhibition
curve following NOR treatment was monophasic. The ICI
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Therefore, we determined the stimulation of cAMP induced
by dobutamine (DOB; ␤1-selective agonist) and procaterol
(PROC; ␤2-selective agonist) in untreated and treated cardiomyocytes (Fig. 2; Table 2). Pretreatment with 100 ␮M NOR
resulted in a significant decrease in DOB-induced cAMP
production by 81% without change in potency and abolished
the PROC response (Fig. 2B; Table 2). The ␤2-AR subtype is
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Fig. 3. Inhibition of 1 ␮M noradrenaline-induced cAMP accumulation by
CGP 20712A, ␤1-selective antagonist (A), and ICI 118551, ␤2-selective
antagonist (B), in neonatal rat cardiomyocytes untreated (䡺) or treated
with 100 ␮M noradrenaline (E) for 24 h. cAMP accumulation was measured as described under Materials and Methods. Data are expressed as
percentage of NOR response (100%; untreated, 239 ⫾ 11% and treated,
66 ⫾ 7%; n ⫽ 7; P ⬍ 0.001). Each point represents the mean ⫾ S.E. of
three to four experiments.
118551 potency was 5.63 ⫾ 0.30 (2.34 ␮M; n ⫽ 4) corresponding to the low-potency population with an inhibition response
of 93 ⫾ 6% (P ⬍ 0.01 versus untreated), suggesting a complete down-regulation of the ␤2-AR subtype in accordance
with PROC-induced cAMP accumulation (Fig. 2B). These
data indicate that cAMP accumulation induced by NOR involved mainly ␤1-AR subtype and weakly ␤2-ARs, which is
completely down-regulated following a chronic stimulation.
Effect of Nonselective and ␤3-Selective Agonists on
Forskolin-Induced cAMP Accumulation in Neonatal
Rat Cardiomyocytes Untreated or Treated with 100
␮M Noradrenaline. The ␤3-AR coupled to Gi/o protein is
up-regulated in heart disease where NOR level is increased
(Cheng et al., 2001; Moniotte et al., 2001) and in rat adipocytes following chronic agonist exposure (Thomas et al.,
1992). However, at present it is not known whether the
␤3-AR is up-regulated in rat heart following agonist exposure. Therefore, we determined the effect of the nonselective
agonists NOR and ISO and the selective ␤3-AR agonists BRL
and CL on forskolin-induced cAMP accumulation in untreated and treated neonatal rat cardiomyocytes with 100
␮M NOR for 24 h (Figs. 4, B and C, 5, 6, and 7).
Fig. 4. Activation of cAMP accumulation by forskolin alone (A) and the
effect of noradrenaline on forskolin-induced cAMP accumulation (B and
C) in neonatal rat cardiomyocytes untreated (䡺) or treated with 100 ␮M
noradrenaline for 24 h in the absence (E) or presence of 0.5 ␮M propranolol (F). cAMP accumulation was measured as described under Materials
and Methods. In A, data are expressed as percentage of the basal level of
cAMP accumulation (100%). The open square corresponds to the cAMP
production induced by 1.5 ␮M forskolin (245 ⫾ 27% of the basal response), the concentration used to measure the inhibition of cAMP production when receptors are coupled to Gi in the following experiments. In
B, data are expressed as the percentage of 1.5 ␮M forskolin response in
the absence of agonist (100%). C corresponds to the enlargement of the
frame in B. Each point represents the mean ⫾ S.E. of four independent
experiments.
␤3-Adrenergic Receptor in Cardiomyocytes
We used 1.5 ␮M forskolin to study ␤3-AR stimulation. This
concentration was under the maximal response of forskolininduced cAMP accumulation as illustrated in Fig. 4A. In
untreated and treated cells, no significant inhibition was
observed using both nonselective agonists. Indeed, NOR potentiated forskolin-induced cAMP accumulation (Emax of
829 ⫾ 80% over the forskolin response; n ⫽ 3) with a pEC50
value of 7.23 ⫾ 0.18 (Fig. 4, B and C). Pretreatment with 100
␮M NOR induced a significant decrease in the maximal response (Emax of 115 ⫾ 38%; n ⫽ 4; P ⬍ 0.001) without change
in the potency (7.13 ⫾ 0.19). Similarly shown in Fig. 5, ISO
stimulated a marked increase in forskolin-induced cAMP
accumulation (Emax of 1883 ⫾ 293%; n ⫽ 4), which was
significantly down-regulated after treatment with NOR
(Emax of 257 ⫾ 56%; n ⫽ 4; P ⬍ 0.01). The pretreatment did
not modify the pEC50 (treated cells, 7.23 ⫾ 0.40 versus untreated cells, 7.80 ⫾ 0.09). Interestingly, in the presence of
0.5 ␮M propranolol (nonselective ␤-AR antagonist), a concentration that inhibits ␤1/␤2-AR responses (Strosberg, 1995;
Germack et al., 1997), NOR-induced an inhibition of forskolin-stimulated cAMP accumulation (Fig. 4; Table 3). ISO also
inhibited the forskolin-induced cAMP response in treated
cardiomyocytes after incubation with 1 ␮M CGP 20712A and
ICI 118551, selective ␤1- and ␤2 antagonists, respectively
(Fig. 5; Table 3). Since the percentage of inhibition with both
nonselective agonists was similar, this would seem to exclude
an effect of ␣1B-ARs in NOR-mediated inhibition of the forskolin response. This adrenergic subtype highly expressed in
neonatal cardiomyocytes is negatively coupled to calcium
channel via Gi protein and therefore could inhibit adenylyl
cyclase activity (Deng et al., 1996, 1998). These data suggest
that the functional expression of the ␤3-AR in neonatal rat
cardiomyocytes after treatment with 100 ␮M NOR.
In untreated cells BRL potentiated forskolin-induced cAMP
accumulation (645 ⫾ 34% over forskolin response; n ⫽ 4) with a
pEC50 value of 6.03 ⫾ 0.07 (Fig. 6). In contrast, the other
selective ␤3-AR agonist CL had no effect on forskolin-induced
cAMP accumulation. Since the affinity of the ␤3-AR for BRL in
functional assays is around 1 nM (Germack et al., 1997, 2000),
the low potency would indicate the involvement ␤1/␤2-ARs. Following 24-h pretreatment with NOR, the effects of BRL on
forskolin-stimulated cAMP accumulation were biphasic. At concentrations below 0.1 ␮M, BRL inhibited forskolin-stimulated
cAMP accumulation (Fig. 6; Table 3). At concentrations above
0.1 ␮M, BRL potentiated forskolin-stimulated cAMP accumulation by 17 ⫾ 1% with a potency of 6.47 ⫾ 0.99 (n ⫽ 4). The
potency was similar to the pEC50 without treatment and the
response was decreased by 97% (P ⬍ 0.001), suggesting a downregulation of ␤1/␤2-ARs. Finally, 0.5 ␮M propranolol had no
significant effect on BRL-mediated inhibition of forskolin cAMP
accumulation (Fig. 6; Table 3). However, propranolol inhibited
BRL-induced cAMP responses in untreated cells, indicating
TABLE 3
Effect of 100 ␮M noradrenaline treatment on nonselective and ␤3-selective agonist-mediated inhibition of forskolin-induced cAMP accumulation in
newborn rat cardiomyocytes in the presence or the absence of ␤-antagonists
Values are means ⫾ S.E. of four to six experiments performed in duplicate. The potencies of the agonists were evaluated by their IC50, concentration of agonist inducing 50%
inhibition expressed in ⫺log10 IC50. Imax is the maximal percentage of inhibition. Cardiomyocytes were treated with 100 ␮M noradrenaline for 24 h. Propranolol (0.5 ␮M)
was used in the experiments with NOR and the ␤3-AR agonists. CGP 20712A and ICI 118551 (1 ␮M) were used altogether in the experiments with ISO.
Imax
IC50
␤-Adrenergic Agonist
Treated ⫹
Antagonists
Treated
⫺log10 IC50
Nonselective agonist
Noradrenaline
Isoprenaline
␤3-Selective agonist
BRL 37344
CL 316243
% of inhibition
7.95 ⫾ 0.30
8.39 ⫾ 0.34
9.52 ⫾ 0.59
7.79 ⫾ 0.48
Treated ⫹
Antagonists
Treated
9.41 ⫾ 0.32
7.85 ⫾ 0.18
28 ⫾ 5
40 ⫾ 6
25 ⫾ 1
36 ⫾ 7
30 ⫾ 2
32 ⫾ 2
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 5. Effect of isoprenaline on forskolin-induced cAMP accumulation in
neonatal rat cardiomyocytes untreated (䡺) or treated with 100 ␮M noradrenaline for 24 h in the absence (E) or presence of 1 ␮M CGP 20712A
and ICI 118551 (F). cAMP accumulation was measured as described
under Materials and Methods. Data are expressed as the percentage of
1.5 ␮M forskolin response in the absence of agonist (100%). Each point
represents the mean ⫾ S.E. of four independent experiments. B corresponds to the enlargement of the frame in A.
397
398
Germack and Dickenson
strongly an activation of ␤1/␤2-ARs. Similarly, CL decreased
forskolin-induced cAMP production in treated cells and 0.5 ␮M
propranolol did not change significantly the functional parameters (Fig. 7; Table 3). Not only the functional expression of the
␤3-AR subtype but also its coupling to Gi were strengthened by
using the selective ␤3-AR antagonist SR 59230A (Fig. 8A) and
pertussis toxin (PTX; Fig. 8B), respectively, which both abolished BRL- and CL-induced inhibition of forskolin response in
NOR-treated cardiomyocytes.
␤1-, ␤2-, and ␤3-AR mRNA and Protein Expression in
Neonatal Rat Cardiomyocytes Untreated or Treated
with 100 ␮M Noradrenaline. To confirm the functional
expression of the ␤3-AR subtype in neonatal rat cardiomyocytes and its up-regulation after 24-h incubation with 100
␮M NOR, mRNA and protein expression of the three ␤-AR
subtypes were examined by RT-PCR and Western blotting,
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 6. Effect of the selective ␤3-AR agonist BRL on forskolin-induced
cAMP accumulation in neonatal rat cardiomyocytes untreated in the
absence (䡺) or presence of 0.5 ␮M propranolol (f), or treated with 100 ␮M
noradrenaline for 24 h in the absence (E) or presence of 0.5 ␮M propranolol (F). cAMP accumulation was measured as described under Materials
and Methods. Data are expressed as the percentage of 1.5 ␮M forskolin
response in the absence of agonist (100%). Each point represents the
mean ⫾ S.E. of four independent experiments. B corresponds to the
enlargement of the frame in A. The dotted lines in B correspond to
the curves shown in A in absence of 0.5 ␮M propranolol.
Fig. 7. Effect of the selective ␤3-AR agonist Cl 316243 on forskolininduced cAMP accumulation in neonatal rat cardiomyocytes untreated
(䡺) or treated with 100 ␮M noradrenaline for 24 h in the absence (E) or
presence of 0.5 ␮M propranolol (F). cAMP accumulation was measured as
described under Materials and Methods. Data are expressed as the percentage of 1.5 ␮M forskolin response in the absence of agonist (100%).
Each point represents the mean ⫾ S.E. of six independent experiments.
Fig. 8. Effect of the selective ␤3-AR agonists CL 316243 (䡺) and BRL (E)
on forskolin-induced cAMP accumulation in neonatal rat cardiomyocytes
treated with 100 ␮M noradrenaline for 24 h in the presence of 1 ␮M SR
59230A, selective ␤3-AR antagonist (A), or 100 ng/ml PTX (B). cAMP
accumulation was measured as described under Materials and Methods.
Data are expressed as the percentage of 1.5 ␮M forskolin response in the
absence of agonist (100%). Each point represents the mean ⫾ S.E. of four
independent experiments.
␤3-Adrenergic Receptor in Cardiomyocytes
respectively (Figs. 9 and 10). Both ␤1 and ␤2-AR mRNA (n ⫽
6) and protein expression (n ⫽ 4) were significantly reduced,
following NOR treatment, by around 40 and 55% of the
control, respectively. The ␤2-ARs seemed more sensitive to
down-regulation than the ␤1-AR, which may explain the absence of the ␤2-AR response after treatment with NOR. In
contrast, ␤3-AR mRNA and protein levels increased by 156
and 169% of the control (n ⫽ 5; P ⬍ 0.05) in treated cardiomyocytes, respectively. These data are in agreement with the
functional studies showing a down-regulation of ␤1/␤2-AR
response and an up-regulation of the ␤3-AR following NOR
treatment for 24 h.
Discussion
NOR pretreatment induced a decrease in NOR (physiological
agonist) and ISO (␤-nonselective agonist) response by 61 and
77%, respectively (Fig. 1; Table 2). In addition, cAMP production induced by the ␤1-selective agonist (dobutamine) was
decreased by 81% in treated cardiomyocytes, whereas the ␤2
response using procaterol was abolished (Fig. 2; Table 2). The
inhibition study using CGP 20712A (␤1-selective antagonist)
and ICI 118551 (␤2-selective antagonist) (Fig. 3) supports the
findings obtained in agonist-induced cAMP response. Indeed,
1 ␮M NOR-induced cAMP accumulation was partially
blocked by CGP 20712A in untreated cardiomyocytes,
whereas NOR response was abolished following 24-h exposure to NOR without change in pIC50, indicating the same
population of receptors. Interestingly, the biphasic inhibition
of NOR response by ICI 118551 indicated the presence of a
minor population of receptors (32 ⫾ 7% of inhibition) with
high potency in untreated cells, which disappeared following
NOR pretreatment. These data indicate that not only ␤2-AR
is more sensitive to down-regulation than ␤1-ARs but also
␤2-AR subtype represents the minor population of ␤-ARs in
normal physiological conditions in neonatal cardiomyocytes.
This last finding is in agreement with the dose-response
curves using ␤1-and ␤2-selective agonists previously discussed. Indeed, the maximal response induced by the ␤2-
Fig. 9. Expression of ␤1-, ␤2-, and ␤3-AR mRNA obtained
from untreated (control) and treated with 100 ␮M noradrenaline for 24 h neonatal rat cardiomyocytes. Total
RNA was prepared and semiquantitative RT-PCR was
carried out as described under Materials and Methods.
Values were obtained by densitometric analysis of RTPCR products and normalized to ␤-actin signal. Data
(top) are expressed as the percentage of untreated cardiomyocytes (100%). The bottom displays 2% agarose gel
electrophoresis of ␤1-, ␤2-, and ␤3-ARs and ␤-actin; line 1,
line 2, line 3, and line 4 correspond to the ladder, untreated control cells, treated cells, and primer control
without cDNA, respectively. Each point represents the
mean ⫾ S.E. of five to six independent experiments. ⴱ,
P ⬍ 0.05 and ⴱⴱⴱ, P ⬍ 0.001 versus untreated control.
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We show for the first time in this report that the ␤3-AR
subtype coupled to Gi, is up-regulated and functional after
chronic stimulation with noradrenaline when ␤1- and ␤2-ARs
are down-regulated in neonatal rat cardiomyocytes. ␤1- and
␤2-ARs regulate cardiac function by increasing the rate and
the force of contraction after stimulation by the catecholamines (noradrenaline and adrenaline) via Gs/cAMP/
PKA signaling pathway (Dzimiri, 1999; Steinberg, 1999). It is
also well established that ␤1- and ␤2-ARs are down-regulated
after a chronic stimulation. As expected, in this study, 24-h
399
400
Germack and Dickenson
selective agonist was lower by 57% than ␤1-AR maximal
stimulation in untreated cells, indicating clearly that the
␤1-ARs are mainly expressed in neonatal cardiomyocytes in
normal physiological conditions as already shown in heart
from different species (Steinberg, 1999). Likewise, the expression of ␤1- and ␤2-ARs evaluated by RT-PCR and Western blotting (Figs. 9 and 10) revealed that ␤1-ARs are highly
expressed and resistant to a chronic NOR stimulation compared with ␤2-ARs. Susuki et al. (1992) have shown that ␤1and ␤2-ARs expressed in Chinese hamster fibroblast at the
same expression level were also differently regulated following 24-h pretreatment with isoprenaline. ␤2-ARs are also
coupled to PTX-sensitive Gi protein in addition to the classical Gs/cAMP/PKA pathway in adult rat and mouse cardiomyocytes (Xiao et al., 1995, 1999) and human atrium (Kilts et
al., 2000). However, in the present study, PTX pretreatment
did not modify procaterol response in untreated or treated
cells, suggesting that ␤2-ARs are not coupled to PTX-sensitive Gi protein in neonatal rat cardiomyocytes as already
shown in these cells (Vitalyi et al., 2003). Overall, these
results demonstrate that ␤1- and ␤2-ARs are involved in
Gs/cAMP/PKA signaling pathway and differentially downregulated in neonatal cardiomyocytes.
In contrast to ␤1- and ␤2-ARs, the ␤3-AR induces a decrease
in cardiac contractility in different species, including human,
dog, guinea pig, and rat (Gauthier et al., 1999; Kitamura et
al., 2000; Cheng et al., 2001; Morimoto et al., 2004). A coupling of ␤3-ARs to Gi/o protein and the activation of endothelial nitric-oxide synthase mediate this negative inotropic effect (Gauthier et al., 1998; Kitamura et al., 2000; Varghese et
al., 2000; Brixius et al., 2004). Therefore, the modulation of
cardiac contractility by the ␤3-ARs seems to involve the inhibition of the adenylyl cyclase and the decreased in intra-
cellular cAMP content. In contrast, this subtype controls
lipolysis in rat white adipocytes (Van Liefde et al., 1992;
Germack et al., 1997) and thermogenesis in brown adipocytes
(Atgie et al., 1997) through Gs protein activation. However, a
dual coupling of the ␤3-ARs to Gs and Gi has been reported in
white adipocytes (Chaudhry et al., 1994) and 3T3-F442A
adipocytes (Soeder et al., 1999). ␤3-ARs are also expressed in
gastrointestinal smooth muscles and mediate relaxation
through Gs protein and cAMP-independent mechanism
(Horinouchi et al., 2003). To determine the physiological significance of ␤3-ARs, we determine the effect of NOR on forskolin-induced cAMP accumulation (Fig. 4; Table 3). NOR
produced an inhibition of forskolin response in NOR-treated
cardiomyocytes only when propranolol was added and used
at the concentration, which inhibits ␤1- and ␤2-ARs. Similarly, forskolin-mediated cAMP accumulation was inhibited
by ISO in the presence of the selective ␤1- and ␤2-antagonists
following a chronic stimulation with NOR (Fig. 5; Table 3).
Gauthier et al. (1998) have also reported that NOR induced a
negative inotropic effect when ␣- and ␤1/␤2-ARs were inhibited in human heart. Similarly, ␤3-AR decreased potassium
channel activity in the presence of ␤1- and ␤2-antagonists in
guinea pig cardiomyocytes (Bosch et al., 2002). These observations indicate that ␤3-ARs are not involved in the regulation of the cardiac function in normal physiological conditions
(neonatal cardiomyocytes; this study) or that this subtype
plays a minor role in the modulation of the functional response (human and guinea pig). NOR and ISO pIC50 values
were in the same range as the corresponding potencies for
cAMP accumulation observed in rodent ␤3-ARs expressed in
Chinese hamster ovary cells (Strosberg, 1997), suggesting
the expression of ␤3-ARs in neonatal cardiomyocytes when
␤-ARs are continuously activated. The inhibition of forskolin
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 10. Expression of ␤1-, ␤2-, and ␤3-AR protein obtained
from untreated (control, C) and treated (T) with 100 ␮M
noradrenaline for 24 h neonatal rat cardiomyocytes. Cell
membrane homogenates were monitored by Western blotting
for ␤1-, ␤2-, and ␤3-ARs as described under Materials and
Methods. The immunoblots are representative of the experiments summarized in the graph. Data are expressed as the
percentage of untreated cardiomyocytes (100%). Each point
represents the mean ⫾ S.E. of four to five independent experiments. ⴱ, P ⬍ 0.05; and ⴱⴱ, P ⬍ 0.01 versus untreated
control.
␤3-Adrenergic Receptor in Cardiomyocytes
functionally up-regulated following chronic exposure to noradrenaline and inhibited cAMP accumulation through Gi
protein coupling, whereas, ␤1- and ␤2-ARs were down-regulated. The rank order of functional expression of ␤-ARs is ␤1
⬎ ␤2 in physiology conditions and ␤1 ⬎ ␤3 in chronic ␤-AR
stimulation. Neonatal rat cardiomyocytes represent a suitable in vitro model to study further the role of ␤3-AR subtype
in heart disease.
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Address correspondence to: Dr. Renée Germack, Biomedical Research Centre, School of Biomedical and Natural Sciences, Nottingham Trent University,
Clifton Lane, Nottingham NG11 8NS, UK. E-mail: [email protected]
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