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INTRODUCTION |
1- and
2-Adrenergic receptors
(
1- and
2-ARs)1
coexist in the hearts of various animal species, including humans.
However, their relative amount and their respective participation in
the positive chronotropic and inotropic effects of adrenaline and noradrenaline vary depending on the cardiac tissue, the animal species,
and/or the pathophysiological state (1, 2). In the non-failing human
left ventricle,
1-ARs represent 80% of the total
-ARs but mediate about 60% only of
-adrenergic-induced ventricular contractility (3). In the human failing heart, the
1/
2-AR ratio decreases, and the
contribution of
2-AR to the contractile responses
becomes predominant over that of
1-AR, in particular at
low adrenaline concentrations (3, 4). For these reasons, the potential
role of
2-AR for improving cardiac performance has
received considerable attention. In fact, the myocardial-targeted
overexpression of
2-ARs in transgenic mice significantly
enhanced myocardial left ventricular contractility (5).
It is well documented that
1-AR and
2-AR
subtypes are coupled to adenylyl cyclase activation and that
stimulation of both receptors generally leads to an increase in
cellular cAMP (4, 6, 7). In human healthy heart,
2-ARs
are more efficiently coupled to adenylyl cyclase than
1-ARs (6-10). However, during cardiac failure,
2-AR subtypes are partially uncoupled from adenylyl cyclase (6, 7), whereas their contribution to the positive inotropic
effects of adrenaline and noradrenaline is increased to 63% (7). In
addition, studies in the rat heart (11, 12) and in the non-failing and
failing canine heart (13) have demonstrated a dissociation between the
inotropic effect of
2-AR and cellular cAMP increase.
Based on those observations, Xiao et al. (12) proposed that
unidentified signal transduction pathway(s), other than adenylyl
cyclase and cAMP, could be involved in the cardiac inotropic response
to
2-AR stimulation.
Angiotensin II (14, 15), bradykinin (16, 17), and endothelin (15, 18),
which exert positive inotropic responses, evoke AA release in heart.
Furthermore, in a recent study, we have demonstrated that glucagon
action relies not only on cAMP but also on the synergistic support of
AA, by activation of the cPLA2 which hydrolyzes the
sn-2 fatty acyl ester bonds of membranous phospholipids
(15).
The aim of the present study was to investigate the respective role of
cAMP and AA in the cardiac response to
-adrenergic agonists. We used
the model of embryonic chick ventricular cardiomyocytes that has been
widely exploited for studies on metabolism, contractile physiology,
electrophysiology, and examination of pathophysiologic states such as
ischemia (19). We show that those cells, in addition to expressing
1-AR (19, 20), also respond to
2-AR
stimulation. We compared the
1- and
2-AR-mediated effects on adenylyl cyclase, [Ca2+]i transients, cell contraction, and AA
release. Our results demonstrate that cAMP is the messenger of
1-AR responses. In contrast, cell responses to
2-AR stimulation were mediated by AA but under cAMP control.
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EXPERIMENTAL PROCEDURES |
Materials
Zinterol and CGP 20712A were kindly supplied by Squibb and
CIBA-Geigy (Basel, Switzerland), respectively. Mini-glucagon was obtained from ICN (Orsay, France). (Rp)-cAMPS, H89, HELSS,
and AACOCF3 were purchased from Biomol (Plymouth Meeting, PA).
Penicillin/streptomycin, antibiotic solution, trypsin, nucleotides,
(±)-isoproterenol, bovine serum albumin, arachidonic acid, ICI 118551, and pertussis toxin were obtained from Sigma (Saint Quentin Fallavier,
France). Fura-2/AM was from Molecular Probes (Interchim,
Montluçon, France). Fetal calf serum and phosphate-buffered
saline 2040 medium were from Life Technologies, Inc. (Cergy Pontoise,
France). M199 medium was obtained from Eurobio (Les Ulis, France).
[
-32P]ATP (30 Ci/mmol) and [8-3H]cAMP
(38-50 Ci/mmol) were from Amersham Corp. (Les Ulis, France). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (180-240
Ci/mmol) was from NEN Life Science Products (Les Ulis, France).
Methods
Primary Culture of Embryonic Chick Ventricular
Cardiomyocytes--
Fecundated eggs were obtained from the Haas farm
(Kaltenhouse, France). Primary monolayer cultured heart cells were
prepared from 13-day-old chick embryo ventricles as described
previously (15, 21, 22). Briefly, cells were dissociated by repeated cycles of trypsinization. The resulting cell suspension (5-7 × 105 cells/ml) was bubbled with 5%
CO2, 95% air, at 4 °C, and kept in buffer A
(M199 medium containing 0.1% (w/v) NaHCO3, 0.01% (w/v) L-glutamine, 0.1% penicillin/streptomycin antibiotic
solution) until used, up to 5 days.
Fura-2 Loading and [Ca2+]i
Imaging--
Cells were plated on plastic dishes, the bottom of which
was replaced by a glass coverslip coated with laminin (1 µg/ml), and
were incubated at 37 °C in humidified 5% CO2, 95% air
for 17-24 h.
Cells, attached to laminin, were bathed in 2 ml of saline buffer B (10 mM glucose, 130 mM NaCl, 5 mM KCl,
10 mM Hepes buffered at pH 7.4 with Tris base, 1 mM MgCl2, 2 mM CaCl2)
and were incubated for 20 min at 25 °C with 1.5 µM
Fura-2/AM (3 µl of 1 mM Fura-2/AM in Me2SO),
in the presence of 1 mg/ml bovine serum albumin to improve Fura-2
dispersion and facilitate cell loading. Cells were then washed with
saline buffer B (2 × 2 ml) and allowed to incubate in the same
buffer for 15 min at 25 °C to facilitate hydrolysis of intracellular
Fura-2/AM. The concentration of Fura-2 in myocytes was estimated as
described previously (15, 21, 22), according to the procedure of
Donnadieu et al. (23). Under usual loading conditions, the
average intracellular concentration of Fura-2 was 15 µM.
Ca2+ imaging, developed by A. Trautman in collaboration
with the IMSTAR (Paris, France), was essentially as described by
Sauvadet et al. (21). Field electrical stimulation (square
waves, 10-ms duration, amplitude 20% above threshold, 0.5 Hz) was
supplied through a pair of platinum electrodes connected to the output
of an HAMEG stimulator (Paris, France). Cells were perifused with
saline buffer B containing 2 mM CaCl2 and
stimulated until a steady-state level of the Ca2+
transients was achieved, before addition of drugs and peptides to the
perfusion medium.
Contractility Measurements--
Experiments were performed in
conditions similar to Ca2+ imaging, but cells were
illuminated with visible light and images transmitted through a
solid-state camera (CCD, black and white, 0.847-cm high sensitivity)
connected to the sideport of the microscope, as described previously
(15). Contractions of single stimulated (0.5 Hz) myocytes were
displayed on a video monitor, and the corresponding images (pixel × pixel) were recorded at a frequency of 9/s. Contractility measurements were determined by assessing changes in cell length using
the Morphostar II software, developed by the IMSTAR (Paris, France).
Adenylyl Cyclase Assay--
A particulate fraction of embryonic
chick ventricular cardiomyocytes was obtained from cells washed twice
in saline buffer B, disrupted by sonication, and centrifuged for 30 min
at 30,000 × g. The pellet was resuspended in 50 mM Hepes, pH 7.4, and stored in liquid nitrogen. Adenylyl
cyclase activity was measured as described previously (24). The assay
medium contained, in a final volume of 60 µl, 50 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 1 mM EDTA, 1 mM [
-32P]ATP (106
cpm), 1 mM [8-3H]cAMP (20,000 cpm), 50 µM GTP, 0.2 mM methylisobutylxanthine, 25 mM creatine phosphate, 1 mg/ml creatine kinase. The
incubation was initiated by the addition of 20-70 µg of proteins and
run at 37 °C. The reaction was terminated by adding 0.2 ml of 0.5 N HCl. Samples were boiled for 6 min and thereafter
buffered with 0.2 ml of 1.5 M imidazole.
[32P]cAMP formed was separated from
[32P]ATP by chromatography on alumina columns according
to the procedure of White (25). Results were obtained from triplicate determinations.
[3H]Arachidonic Acid Labeling--
Embryonic chick
ventricular cardiomyocytes (5 × 105 cells/ml),
suspended in buffer A, were plated in 24-well plates, left for 24 h in humidified 5% CO2, 95% air, at 37 °C, and then
incubated with 1.5 µCi/ml [3H]AA (6.75 nM).
After 24 h incubation with [3H]AA, the cells were
washed twice in saline buffer B containing 0.2% fatty acid-free bovine
serum albumin and resuspended in saline buffer B.
Measurements of [3H]Arachidonic Acid Release in
Intact Cells--
At time 0 of the experiment,
[3H]AA-labeled cells were exposed to various peptides
and/or enzymatic inhibitors and incubated for various periods at
37 °C. Incubation was terminated by the addition of ice-cold EGTA (2 mM final), and the media were immediately transferred to
microcentrifuge tubes. Centrifugation at 17,600 × g
for 20 min in a Sigma centrifuge (model 2K15) at 4 °C was performed
to pellet any cells or debris inadvertently collected with the
extracellular medium. The amount of radioactivity in the supernatant
was quantitated by liquid scintillation counting.
Analysis of the lipids released in the incubation medium was performed
as described (26). At the end of the incubation period, the reaction
mixture was acidified to pH 3.0 with HCl, and the products were
extracted twice with ethyl acetate. The dried extracts were dissolved
in ethanol/chloroform (1:2, v/v) and chromatographed on silica gel thin
layer plate (Whatman LK5) in ethyl acetate/isooctane/water/acetic acid
(11:5:10:2, v/v) as the solvent system. Standard concentrations of AA,
prostaglandins E, and hydroxyeicosatetraenoic acids were co-chromatographed and visualized by exposing the plates to ultraviolet light. The area corresponding to each visualized spot was carefully extracted, and the radioactivity was determined by liquid scintillation counting.
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RESULTS |
1- and
2-AR Stimulations Increase
[Ca2+]i Cycling and Contractility in Electrically
Stimulated Embryonic Chick Ventricular Myocytes--
The effect of
increasing concentrations of isoproterenol was examined on
[Ca2+]i cycling of electrically stimulated
embryonic chick ventricular myocytes. A dose-dependent
increase in the amplitude of [Ca2+]i transients
was observed, reaching a maximal (210 ± 9%) stimulation at 1-10
µM isoproterenol (Fig. 1).
Preincubation for 10 min with 100 nM of the selective
2-AR antagonist, ICI 118551 (7), significantly reduced
the stimulation evoked by 10 µM isoproterenol (26%
inhibition) but was poorly effective in inhibiting the effect of lower
concentrations. In contrast, 300 nM of the selective
1-AR antagonist, CGP 20712A (7), markedly blocked the
effect of low isoproterenol concentrations, leading to a
rightward-shifted dose-response curve of isoproterenol effect. CGP
20712A also reduced by 54% the maximal effect of 10 µM
isoproterenol (Fig. 1). It thus appeared that isoproterenol behaved as
a
1-AR agonist at concentrations below 100 nM, and as a mixed
1/
2-AR agonist in the micromolar range of concentrations.

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Fig. 1.
Isoproterenol increases the amplitude of
[Ca2+]i transients in electrically stimulated
cells. Embryonic chick ventricular cardiomyocytes, loaded with
Fura-2 and electrically stimulated at 0.5 Hz as described under
"Experimental Procedures," were preincubated for 10 min in the
absence or in the presence of either 300 nM of the specific
1-AR antagonist CGP 20712A or 100 nM of the
specific 2-AR antagonist ICI 118551, and perfused with
increasing concentrations of isoproterenol, each concentration being
applied for 3 min. Values are means ± S.E. of the effects
observed on 20-30 cells, obtained from three different
isolations.
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Zinterol, a specific, partial
2-AR agonist (8, 27),
elicited a dose-dependent increase in
[Ca2+]i transient amplitude of electrically
stimulated embryonic chick cardiomyocytes (Fig.
2A). A maximal, 144 ± 3%, increase was observed at 30 nM zinterol, with a
half-maximal response occurring at 10 nM zinterol. As
illustrated by the typical traces of [Ca2+]i
transients (Fig. 2B), the effect of zinterol was reversed by
100 nM of the selective
2-AR antagonist, ICI
118551, but was not affected by 300 nM of the selective
1-AR antagonist, CGP 20712A (Fig. 2B).

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Fig. 2.
Zinterol, the specific
2-AR agonist, increases the amplitude of
[Ca2+]i transients in electrically stimulated
cells. Embryonic chick ventricular cardiomyocytes, loaded with
Fura-2, were electrically stimulated at 0.5 Hz as described under
"Experimental Procedures." A, cells were perfused with
increasing concentrations of zinterol, each concentration being applied
for 3 min. Values are means ± S.E. of the effects observed on
20-30 cells, obtained from three different isolations. B,
cells were preincubated for 10 min in the absence or in the presence of
either 100 nM of the specific 2-AR
antagonist ICI 118551 or 300 nM of the specific
1-AR antagonist CGP 20712A, and perfused without or with
30 nM zinterol. The traces are representative of at least
20 cells obtained from two different isolations.
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Both
1- and
2-AR stimulatory effects on
[Ca2+]i cycling were correlated with increases in
the amplitude of cell contraction; 30 nM zinterol
(
2-AR agonist) and 100 nM isoproterenol (at
a concentration at which the agonist functioned as a
1-AR agonist) increased the amplitude of cell
contraction by 80 and 150% over basal, respectively (Fig.
3, A and B).
Furthermore, as shown in the normalized and superimposed tracings of
contraction (Fig. 3C), zinterol, like isoproterenol,
markedly accelerated the kinetics of relaxation.

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Fig. 3.
Zinterol like isoproterenol increases cell
contraction. Embryonic chick ventricular cardiomyocytes were
electrically stimulated at 0.5 Hz and perfused in the absence or in the
presence of either 30 nM zinterol (A) or 100 nM isoproterenol (B). Contractions of single
myocytes were measured as described under "Experimental
Procedures." Each trace is an average of 10 steady-state beats in a
single cell. C, the traces from A and
B have been normalized to their peak amplitude. Data are
representative of at least 5 cells obtained from two different
isolations.
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2-AR Stimulation of [Ca2+]i
Cycling Does Not Rely on Adenylyl Cyclase Activation--
The effects
of zinterol and isoproterenol on adenylyl cyclase activity were
examined in a particulate fraction of embryonic chick ventricular
myocytes. Isoproterenol, at 10 µM, elicited a maximal
1.8-fold stimulation of adenylyl cyclase activity, the half-maximal
effect being obtained at 0.15 µM isoproterenol (Fig. 4A). This effect was totally
blocked by 300 nM of the
1-AR antagonist, CGP 20712A. Under the same assay conditions, zinterol had no effect on
adenylyl cyclase activity (Fig. 4A). These results suggest that, in embryonic chick ventricular cardiomyocytes, adenylyl cyclase
is specifically coupled to
1-ARs but not to
2-ARs.

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Fig. 4.
Adenylyl cyclase activation and cyclic AMP
mediate 1-AR response but do no support the increase in
[Ca2+]i cycling triggered by
2-AR agonists. A, adenylyl
cyclase activity was assayed in a particulate fraction prepared from
embryonic chick ventricular cardiomyocytes, as described under
"Experimental Procedures," with varying concentrations of either
zinterol or isoproterenol. When the specific 1-AR
antagonist CGP 20712A was used, the enzyme fraction was preincubated
for 10 min with the compound prior to adenylyl cyclase assay. Data are
the means ± S.E. of at least 4 different experiments performed in
triplicate. B, the effects on the amplitude of
[Ca2+]i transients of 30 nM zinterol
and 100 nM fenoterol ( 2-AR agonists), 300 nM prenalterol ( 1-AR agonist), and 100 nM isoproterenol were examined, after 1 h
preincubation in the absence or in the presence of 10 µM
of the specific cAMP antagonist, (Rp)-cAMPS, in cells loaded
with Fura-2 and electrically stimulated at 0.5 Hz as described under
"Experimental Procedures." Values are means ± S.E. of the
effects observed on the number of cells indicated, obtained from three
different isolations.
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To analyze further cAMP dependence of
[Ca2+]i cycling modulations in response to
2- and
1-AR stimulations, we used the
cell-permeable selective cAMP antagonist, (Rp)-cAMPS, (28). Following preincubation for 1 h with 10 µM
(Rp)-cAMPS, the increase in amplitude of
[Ca2+]i transients in response to 300 nM prenalterol, a specific
1-AR agonist, was
reduced by 83% (from 142 ± 7 to 107 ± 1% of control
amplitude, Fig. 4B). The cAMP antagonist produced a similar reduction in the
1-AR-mediated effect of 100 nM isoproterenol (from 178 ± 9 to 118 ± 3%,
Fig. 4B). In contrast, (Rp)-cAMPS failed to
inhibit the
2-AR response to either zinterol (30 nM) or fenoterol (100 nM) (Fig. 4B).
These findings demonstrate that adenylyl cyclase and cAMP govern
1-AR agonist-induced stimulation of
[Ca2+]i cycling but are not involved in
2-AR-mediated effects.
Zinterol Stimulates [3H]AA Release from Embryonic
Chick Ventricular Cardiomyocytes--
The next series of experiments
was performed to assess the possible involvement of AA in mediating
2-AR effects. Embryonic chick ventricular myocytes were
labeled for 24 h with [3H]AA before the addition of
agonist. As shown in Fig. 5A,
zinterol evoked a dose-dependent release of
[3H]AA, which reached a maximal (147 ± 4%)
increase with 30 nM zinterol, the half-maximal effects
occurring at 5 nM zinterol (Fig. 5A). The
1-AR agonist prenalterol, as well as isoproterenol
100
nM, when functioning in the
1-AR mode, had
no effect on [3H]AA release (Fig. 5A). Only at
concentrations above 1 µM did isoproterenol, functioning
in the mixed
1/
2-AR mode, evoke a limited, 10% increase over basal in AA release.

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Fig. 5.
Zinterol-induced [3H]AA release
is dose-dependent and 2-activated
[Ca2+]i cycling is blocked by the
cPLA2 inhibitor, AACOCF3. A, embryonic
chick ventricular cardiomyocytes were labeled for 24 h with 1.5 µCi/ml [3H]AA, as described under "Experimental
Procedures." Radiolabeled cells were washed twice in saline buffer
containing 0.2% fatty acid-free bovine serum albumin and were
incubated for 30 min with varying concentrations of either zinterol,
prenalterol ( 1-AR agonist), or isoproterenol. The amount
of [3H]AA released is expressed as percentage of control
values (33 ± 2 dpm/µg). Values are the means ± S.E. of 5 different experiments, done in triplicate. B, the effects on
the amplitude of [Ca2+]i transients of 30 nM zinterol and 100 nM fenoterol
( 2-AR agonists) and 300 nM prenalterol or
100 nM isoproterenol ( 1-AR agonists) were
examined after 10 min preincubation in the absence or in the presence
of the specific cPLA2 inhibitor, AACOCF3, in cells loaded
with Fura-2 and electrically stimulated at 0.5 Hz as described under
"Experimental Procedures." Values are means ± S.E. of the
effects observed on the number of cells indicated, obtained from three
different isolations.
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Resolution on thin layer chromatography (TLC) of the
3H-labeled material, released in cell supernatants,
identified [3H]AA as the major product, both in control
and zinterol-treated cells (75 and 79%, respectively) (Table
I). Non-enzymatic degradation or
contaminants of standard [3H]AA represented 4-19% of
the total radioactivity recovered in supernatants; lipoxygenase and
cycloxygenase products represented 4-24 and 1-8%, respectively
(Table I).
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Table I
Identification of 3H-labeled metabolites in supernatants of
cells prelabeled with [3H]AA
Embryonic chick heart cells were labeled with 1.5 µCi/ml
[3H]AA as described under "Experimental Procedures."
After two washings in saline buffer containing 0.2% fatty acid-free
bovine serum albumin, [3H]AA-labeled cells were incubated for
30 min in the presence or in the absence of 30 nM zinterol,
and with or without 10 µM AACOCF3. Analysis of
the 3H-lipids released in the incubation medium was performed
following extraction and chromatography on silica gel thin layer plate
(TLC plate) as described under "Experimental Procedures." Results,
corrected for yield of extraction, are expressed in dpm of
[3H] product/104 cells and in percent of total
radioactivity recovered in the migration lane. Each sample represents
the pool of quadruplicates. Data are from a typical experiment that has
been repeated twice. Standard [3H]AA was migrated in parallel
in order to determine the nonenzymatic breakdown of AA. HETE,
hydroxyeicosatetraenoic acid.
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Since AA formation in the heart is essentially attributed to
PLA2 activity, we examined the effect of AACOCF3, a
specific inhibitor of the cPLA2. The addition of 10 µM AACOCF3 to the perfusion medium dramatically reduced
[3H]AA release evoked by zinterol (from 206 to 136% of
control [3H]AA release, Table I). This inhibitory effect
correlated with a blockade of the stimulatory effects on
[Ca2+]i transients of both specific
2-AR agonists zinterol and fenoterol (Fig.
5B). In contrast, AACOCF3 did not affect the
1-AR-mediated increase in [Ca2+]i
cycling triggered by either prenalterol or isoproterenol at 100 nM (Fig. 5B). Taken together, these findings
further supported the notion that
2-AR stimulation
elicited AA release by stimulating the cPLA2, sensitive to
AACOCF3. It may be noted that AACOCF3 completely inhibited
2-AR-mediated effects on [Ca2+]i
cycling, whereas it had only a partial effect on
2-AR-stimulated AA release (Table I). This may suggest
that the onset of the Ca2+ response requires the cellular
AA level to reach a threshold.
Importantly, exogenous application of micromolar concentrations of AA
reproduced the effect of
2-AR agonists on
[Ca2+]i transients; at 3 µM, AA
evoked a 140% increase in amplitude of
[Ca2+]i transients (Fig.
6). The activating effect of AA on [Ca2+]i cycling was potentiated by
8-Br-cAMP (Fig. 6).

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Fig. 6.
AA mimics 2-AR
action on [Ca2+]i cycling and 8-Br-cAMP
potentiates AA effect. Embryonic chick ventricular cardiomyocytes,
loaded with Fura-2, were electrically stimulated at 0.5 Hz, as
described under "Experimental Procedures," and perfused with
increasing concentrations of AA, in the absence or in the presence of
75 µM 8-Br-cAMP. Values are means ± S.E. of the
effects observed on at least 10 cells, obtained from two different
isolations.
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The
1-AR/cAMP Pathway Occludes Cell Responses to
2-AR Stimulation--
Next, we looked for a possible
cross-talk between
1- and
2-AR responses.
In a first series of experiments, cells were electrically stimulated
and exposed to 300 nM of the
1-agonist,
prenalterol. The time course of the amplitude of
[Ca2+]i transients is illustrated in Fig.
7. [Ca2+]i
transient amplitude increased for the first minutes of exposure to
prenalterol, reaching a maximal 50% increase over basal at 10 min.
After 15 min, a decline in stimulation of [Ca2+]i
cycling occurred, and after 30 min, the
1-AR-mediated effect was no more detectable (Fig. 7). Such a waning of a stimulated response in the face of continuous agonist exposure is typical of a
desensitization phenomenon (29).

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Fig. 7.
Desensitization of the
1-AR effect and blockade by
1-ARs of the 2-AR-mediated
effect on [Ca2+]i cycling. Embryonic chick
ventricular cardiomyocytes, loaded with Fura-2, were electrically
stimulated at 0.5 Hz, as described under "Experimental Procedures,"
and perfused with 300 nM prenalterol (a specific
1-AR agonist). The amplitude of
[Ca2+]i transients was examined after different
times of incubation with prenalterol. Values obtained are means ± S.E. of the effects observed on at least 15 cells obtained from two
different isolations. Insets, 30 nM zinterol was
added to the perfusion medium after 3 or 45 min incubation with
prenalterol. The traces are representative of at least 15 cells obtained from two different isolations.
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In a second series of experiments, we examined the response to
2-AR stimulation of cells under two extreme conditions:
(i) after 3 min exposition to prenalterol, when
1-ARs
are fully activated; (ii) after 45 min exposition to prenalterol, when
1-ARs are desensitized. As shown in the
insets of Fig. 7, after 3 min incubation with prenalterol,
the addition of zinterol did not produce further increase in
[Ca2+]i transient amplitude. In contrast,
zinterol added to
1-AR-desensitized cells evoked a
2-fold increase in the amplitude of [Ca2+]i
transients. Those results suggested a negative constraint exerted by
1-AR activation on the
2-AR-stimulated
[Ca2+]i cycling.
8-Br-cAMP reproduced prenalterol effect and inhibited the
2-AR-mediated effects on [Ca2+]i
cycling (Fig. 8A). In
addition, the cAMP antagonist, (Rp)-cAMPS, as well as the
PKA inhibitor, H89, blocked the inhibitory effect of the
1-AR agonist, prenalterol, on the cell response to
2-AR stimulation (Fig. 8, B and
C). Taken together, those data suggest that cAMP, via PKA
activation, exerts an inhibitory constraint on
2-AR
stimulation.

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Fig. 8.
cAMP, via PKA activation, exerts an
inhibitory constraint on 2AR-stimulated
[Ca2+]i cycling. Embryonic chick ventricular
cardiomyocytes, loaded with Fura-2, were electrically stimulated at 0.5 Hz, as described under "Experimental Procedures." A, 30 nM zinterol was added to the perfusion medium after 3 min
incubation with 75 µM 8-Br-cAMP. B, cells were
preincubated for 1 h with 10 µM
(Rp)-cAMPS; 30 nM zinterol was added to the
perfusion medium after 3 min incubation with 300 nM
prenalterol. C, cells were preincubated for 30 min with 3 µM H89; 30 nM zinterol was added to the
perfusion medium after 3 min incubation with 300 nM
prenalterol. The traces are representative of at least 15 cells obtained from two different isolations.
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cPLA2 Activation by
2-AR Agonists Is
Sensitive to Pertussis Toxin Treatment--
2-AR can
couple to both Gs and Gi proteins (30, 31).
Thus, we investigated the possible role of Gi in the
specific coupling of
2-AR to cPLA2.
Treatment of the cells with PTX totally abolished the stimulatory
effects of zinterol on both [3H]AA release and
[Ca2+]i cycling (Fig.
9, A and B). The
efficiency of PTX treatment was checked by the blockade of
Gi-mediated acetylcholine inhibition of isoproterenol
effect on Ca2+ cycling (Fig. 9B). It should be
noted that treatment with pertussis toxin was without detectable impact
on basal or isoproterenol-stimulated [Ca2+]i
transients suggesting the absence of a tonic control by Gi,
in particular over Gs.

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Fig. 9.
Treatment with pertussis toxin abolishes the
effects of zinterol on [3H]AA release and
[Ca2+]i cycling. A, embryonic
chick ventricular cells were labeled for 24 h with 1.5 µCi/ml
[3H]AA in the presence or in the absence of 500 ng/ml
PTX, as described under "Experimental Procedures." Radiolabeled
cells were washed twice in saline buffer containing 0.2% fatty
acid-free bovine serum albumin and incubated for 30 min with varying
concentrations of zinterol. The amount of [3H]AA released
was expressed as percentage of control values (58 ± 6 dpm/µg).
Values are the means ± S.E. of two different experiments, done in
triplicate. Control curve was similar to that shown in Fig.
5A and is thus represented in dashed lines.
B, cells were treated for 24 h with or without 500 ng/ml pertussis toxin. The effects on the amplitude of
[Ca2+]i transients of zinterol, isoproterenol,
and isoproterenol plus acetylcholine were then examined in cells loaded
with Fura-2 and electrically stimulated at 0.5 Hz as described under
"Experimental Procedures." Values obtained are means ± S.E.
of the effects observed on at least 15 cells obtained from three
different isolations.
|
|
 |
DISCUSSION |
In the present study, we show that
1- and
2-ARs are both expressed in embryonic chick ventricular
cardiomyocytes, and this model allowed us to demonstrate the following:
(i)
2-ARs are specifically coupled to cPLA2
via a Gi protein; (ii) cAMP exerts a dual tuning on cell
responses to
2-AR stimulation.
In electrically stimulated embryonic chick ventricular cardiomyocytes,
30-100 nM zinterol, a specific partial
2-AR
agonist, elicited a 40-50% increase over basal in the amplitude of
[Ca2+]i transients (Fig. 2), which correlated
with increases in twitch amplitude and twitch velocity (Fig. 3). Such a
positive inotropic effect of
2-AR agonists is
undisputed. Nevertheless, in contrast to
1-AR-mediated
positive inotropic effect, which definitely relies on a rise in
intracellular cAMP, the contribution of cAMP to the positive inotropic
effect of
2-AR agonists, and the possible coupling of
2-AR to cAMP-independent pathways, are still a matter of
debate. According to Bristow et al. (6),
2-ARs in the non-failing human heart are tightly coupled
to adenylyl cyclase since a numerically small
2-AR
fraction (19% of the total
1- and
2-ARs)
accounts for the majority of adenylyl cyclase stimulation. Such an
inherent efficacy for the human
2-AR in activating
adenylyl cyclase, compared with that of its
1
counterpart, has been confirmed by expression of those receptors in
fibroblast cell lines (9, 10). However, Kaumann and Lemoine (7) have compared the relative contribution of
1- and
2-ARs to adenylyl cyclase stimulation and positive
inotropic effects of adrenaline and noradrenaline in pathological human
heart. They concluded that the positive inotropic response was not
straightforwardly correlated to adenylate cyclase stimulation. These
authors were also the first to suggest compartmentation of cAMP since
cAMP produced upon
2-AR stimulation was less efficiently
used than cAMP produced upon
1-AR stimulation by
cellular effectors involved in contractility. More recently, the group
of Lakatta (12) suggested that, in addition to coupling to adenylyl
cyclase,
2-AR stimulation activates other signal
transduction pathways to produce changes in
[Ca2+]i and contraction. This proposal relies on
two observations. First, in rat ventricular cells
2-AR
stimulation elicits a positive inotropic response that is dissociated
from cAMP increase (12). Evidence for the involvement of cAMP is given
only for high
2-agonist concentrations; indeed,
activation by 10 µM zinterol of both contraction (32) and
L-type Ca2+ current (2) is blocked by
(Rp)-cAMPS, the specific inhibitory cAMP analog. Second, in
electrically stimulated dog myocytes,
2-AR activation is
ineffective in stimulating adenylyl cyclase, whereas it produces
increases in [Ca2+]i transient and twitch
amplitudes (13). In this regard, we show here that, in embryonic chick
heart cells,
2-AR stimulation by zinterol triggers a
positive inotropic effect, independent of adenylyl cyclase activation
(Figs. 2, 3, and 4A). The absence of participation of cAMP
in this effect of zinterol is further confirmed by the fact that, in
contrast to the actions of
1-AR agonists, it is not
blocked by either (Rp)-cAMPS (Figs. 4B and 8B) or the PKA inhibitor, H89 (Fig. 8C). Thus, we
conclude that cAMP does not support the inotropic effect of low
2-AR agonist concentrations although it could contribute
in the effects of high
2-AR agonist concentrations.
Glucagon action in heart relies on the synergistic actions of glucagon
itself and its metabolite (19-29), mini-glucagon (15, 22). We have
demonstrated that cAMP mediates glucagon action and that AA is the
second messenger of mini-glucagon (15). In the present study, several
lines of evidence support the proposal that AA is also the second
messenger in response to stimulations by
2-AR agonists:
1) zinterol increases AA release from [3H]AA-prelabeled
myocytes in a dose-dependent manner from 3 to 100 nM (Fig. 5A); 2) AA, added to the cell medium at
concentrations as low as 1-3 µM, reproduces the effect
of zinterol on [Ca2+]i cycling in electrically
stimulated cardiomyocytes (Fig. 6). AA release results from
2-AR activation of the cPLA2 via a pertussis
toxin-sensitive G protein (Fig. 8). Such a coupling of
2-ARs to pertussis toxin-sensitive G protein(s) has been
already reported in rat cardiomyocytes (30) and in cells sur-expressing
2-ARs (31).
cAMP exerts a dual tuning on cell responses to
2-AR
stimulation. On the one hand, we show that cAMP, produced upon
1-AR stimulation, evokes a quenching of cell responses
to
2-AR stimulation; thus, after 3 min exposure to
prenalterol, cells did not respond further to
2-AR
stimulation, whereas following complete desensitization of
1-ARs,
2-AR stimulation was restored
(Fig. 7). The negative constraint exerted by cAMP is likely to rely on
PKA stimulation since H89, the PKA inhibitor, hampers it. It could be
due to phosphorylation, and inhibition, by protein kinase A of the
PTX-sensitive G protein coupling cPLA2 to
2-AR (33). On the other hand, downstream cPLA2 activation, cAMP potentiates AA-mediated stimulation
of [Ca2+]i cycling (Fig. 6). Those synergistic
actions of cAMP and AA would rely on the ability of AA to accumulate
Ca2+ into the sarcoplasmic reticulum stores and that of
cAMP to induce "Ca2+-induced Ca2+ release"
from these stores (15).
In conclusion, we show that, at low concentrations,
2-AR
agonists elicit a positive inotropic effect via cPLA2
activation and AA release. Contrary to the
1-AR/cAMP
pathway,
2-AR/cPLA2 pathway involves a
pertussis toxin-sensitive G protein. cAMP exerts a dual regulation on
the
2-AR/AA pathway; it inhibits the cell response to
2-AR stimulation but potentiates AA-mediated stimulation of [Ca2+]i cycling.
There is now accumulating evidence that hydrolytic products derived
from membrane phospholipids play important roles in cardiovascular signaling (34). Originally, attention mainly focused on diacylglycerol and eicosanoids (prostaglandins, prostacyclins, thromboxanes, leukotrienes, etc.) (35, 36). However, the list of bioactive lipidic
molecules now includes AA, the precursor of eicosanoids. Studies on
mice deficient in cPLA2 have demonstrated the major role of
this enzyme in allergic responses, reproductive physiology, and
pathophysiology of neuronal death (37, 38). The participation of
cPLA2 and/or AA in mediating positive inotropic response to various agents was suspected (14-18). Our results unequivocally establish that, at low concentrations of agonist, the
2-AR-mediated inotropic effect relies on the selective
activation of cPLA2 and AA release. Although this remains
to be demonstrated, it is tempting to speculate that the
2-AR/cPLA2/AA pathway could be determinant in failing hearts that have lost 50% of
1-ARs and
show a parallel decrease in agonist-stimulated adenylyl cyclase
activity (4, 6, 7).