Evidence for a beta 2-Adrenergic/Arachidonic Acid Pathway in Ventricular Cardiomyocytes
REGULATION BY THE beta 1-ADRENERGIC/cAMP PATHWAY*

Catherine PavoineDagger , Sandrine Magne, Anne Sauvadet, and Françoise Pecker

From INSERM Unité 99, Hôpital Henri Mondor, 94010 Créteil, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The signaling pathway mediating the contractile effect of beta 2-adrenergic receptors (beta 2-AR) in the heart is still matter of debate. By using embryonic chick ventricular cardiomyocytes that express both functional beta 1-and beta 2-ARs, we show here that the specific beta 2-AR agonist, zinterol, increases the amplitude of Ca2+ transients and cell contraction of electrically stimulated cells. Zinterol, up to 10 µM, did not stimulate adenylyl cyclase activity, and its effect on Ca2+ transients was unmodified by the specific cAMP antagonist, (Rp)-cAMPS. In contrast, zinterol (10-100 nM) triggered arachidonic acid (AA) release from [3H]AA-loaded cells via the activation of the cytosolic phospholipase A2 (cPLA2). Stimulation of the Ca2+ transients by zinterol was abolished by the cPLA2 inhibitor, AACOCF3, and was mimicked by AA (0.3-3 µM). Both stimulations of [3H]AA release and of [Ca2+]i cycling by zinterol were abolished after treatment of the cardiomyocytes with pertussis toxin. Although cell responses to beta 2-AR stimulation were mediated by AA, they were under cAMP control as follows: (i) the beta 1-AR stimulation exerted a cAMP-mediated negative constraint on the beta 2-AR/cPLA2 pathway; (ii) cAMP potentiated AA action downstream beta -AR stimulation. We conclude that, in cardiomyocytes, beta 2-AR is coupled to cPLA2 activation via a pertussis toxin-sensitive G protein. These results demonstrate the involvement of the cPLA2/AA pathway in mediating positive inotropic effects, which could potentially compensate for a defective cAMP pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

beta 1- and beta 2-Adrenergic receptors (beta 1- and beta 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, beta 1-ARs represent 80% of the total beta -ARs but mediate about 60% only of beta -adrenergic-induced ventricular contractility (3). In the human failing heart, the beta 1/beta 2-AR ratio decreases, and the contribution of beta 2-AR to the contractile responses becomes predominant over that of beta 1-AR, in particular at low adrenaline concentrations (3, 4). For these reasons, the potential role of beta 2-AR for improving cardiac performance has received considerable attention. In fact, the myocardial-targeted overexpression of beta 2-ARs in transgenic mice significantly enhanced myocardial left ventricular contractility (5).

It is well documented that beta 1-AR and beta 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, beta 2-ARs are more efficiently coupled to adenylyl cyclase than beta 1-ARs (6-10). However, during cardiac failure, beta 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 beta 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 beta 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 beta -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 beta 1-AR (19, 20), also respond to beta 2-AR stimulation. We compared the beta 1- and beta 2-AR-mediated effects on adenylyl cyclase, [Ca2+]i transients, cell contraction, and AA release. Our results demonstrate that cAMP is the messenger of beta 1-AR responses. In contrast, cell responses to beta 2-AR stimulation were mediated by AA but under cAMP control.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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). [alpha -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 [alpha -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

beta 1- and beta 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 beta 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 beta 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 beta 1-AR agonist at concentrations below 100 nM, and as a mixed beta 1/beta 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 beta 1-AR antagonist CGP 20712A or 100 nM of the specific beta 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.

Zinterol, a specific, partial beta 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 beta 2-AR antagonist, ICI 118551, but was not affected by 300 nM of the selective beta 1-AR antagonist, CGP 20712A (Fig. 2B).


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Fig. 2.   Zinterol, the specific beta 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 beta 2-AR antagonist ICI 118551 or 300 nM of the specific beta 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.

Both beta 1- and beta 2-AR stimulatory effects on [Ca2+]i cycling were correlated with increases in the amplitude of cell contraction; 30 nM zinterol (beta 2-AR agonist) and 100 nM isoproterenol (at a concentration at which the agonist functioned as a beta 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.

beta 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 beta 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 beta 1-ARs but not to beta 2-ARs.


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Fig. 4.   Adenylyl cyclase activation and cyclic AMP mediate beta 1-AR response but do no support the increase in [Ca2+]i cycling triggered by beta 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 beta 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 (beta 2-AR agonists), 300 nM prenalterol (beta 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.

To analyze further cAMP dependence of [Ca2+]i cycling modulations in response to beta 2- and beta 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 beta 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 beta 1-AR-mediated effect of 100 nM isoproterenol (from 178 ± 9 to 118 ± 3%, Fig. 4B). In contrast, (Rp)-cAMPS failed to inhibit the beta 2-AR response to either zinterol (30 nM) or fenoterol (100 nM) (Fig. 4B). These findings demonstrate that adenylyl cyclase and cAMP govern beta 1-AR agonist-induced stimulation of [Ca2+]i cycling but are not involved in beta 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 beta 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 beta 1-AR agonist prenalterol, as well as isoproterenol <= 100 nM, when functioning in the beta 1-AR mode, had no effect on [3H]AA release (Fig. 5A). Only at concentrations above 1 µM did isoproterenol, functioning in the mixed beta 1/beta 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 beta 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 (beta 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 (beta 2-AR agonists) and 300 nM prenalterol or 100 nM isoproterenol (beta 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.

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.

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 beta 2-AR agonists zinterol and fenoterol (Fig. 5B). In contrast, AACOCF3 did not affect the beta 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 beta 2-AR stimulation elicited AA release by stimulating the cPLA2, sensitive to AACOCF3. It may be noted that AACOCF3 completely inhibited beta 2-AR-mediated effects on [Ca2+]i cycling, whereas it had only a partial effect on beta 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 beta 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 beta 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.

The beta 1-AR/cAMP Pathway Occludes Cell Responses to beta 2-AR Stimulation-- Next, we looked for a possible cross-talk between beta 1- and beta 2-AR responses. In a first series of experiments, cells were electrically stimulated and exposed to 300 nM of the beta 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 beta 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 beta 1-AR effect and blockade by beta 1-ARs of the beta 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 beta 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.

In a second series of experiments, we examined the response to beta 2-AR stimulation of cells under two extreme conditions: (i) after 3 min exposition to prenalterol, when beta 1-ARs are fully activated; (ii) after 45 min exposition to prenalterol, when beta 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 beta 1-AR-desensitized cells evoked a 2-fold increase in the amplitude of [Ca2+]i transients. Those results suggested a negative constraint exerted by beta 1-AR activation on the beta 2-AR-stimulated [Ca2+]i cycling.

8-Br-cAMP reproduced prenalterol effect and inhibited the beta 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 beta 1-AR agonist, prenalterol, on the cell response to beta 2-AR stimulation (Fig. 8, B and C). Taken together, those data suggest that cAMP, via PKA activation, exerts an inhibitory constraint on beta 2-AR stimulation.


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Fig. 8.   cAMP, via PKA activation, exerts an inhibitory constraint on beta 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.

cPLA2 Activation by beta 2-AR Agonists Is Sensitive to Pertussis Toxin Treatment-- beta 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 beta 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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, we show that beta 1- and beta 2-ARs are both expressed in embryonic chick ventricular cardiomyocytes, and this model allowed us to demonstrate the following: (i) beta 2-ARs are specifically coupled to cPLA2 via a Gi protein; (ii) cAMP exerts a dual tuning on cell responses to beta 2-AR stimulation.

In electrically stimulated embryonic chick ventricular cardiomyocytes, 30-100 nM zinterol, a specific partial beta 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 beta 2-AR agonists is undisputed. Nevertheless, in contrast to beta 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 beta 2-AR agonists, and the possible coupling of beta 2-AR to cAMP-independent pathways, are still a matter of debate. According to Bristow et al. (6), beta 2-ARs in the non-failing human heart are tightly coupled to adenylyl cyclase since a numerically small beta 2-AR fraction (19% of the total beta 1- and beta 2-ARs) accounts for the majority of adenylyl cyclase stimulation. Such an inherent efficacy for the human beta 2-AR in activating adenylyl cyclase, compared with that of its beta 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 beta 1- and beta 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 beta 2-AR stimulation was less efficiently used than cAMP produced upon beta 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, beta 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 beta 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 beta 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, beta 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, beta 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 beta 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 beta 2-AR agonist concentrations although it could contribute in the effects of high beta 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 beta 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 beta 2-AR activation of the cPLA2 via a pertussis toxin-sensitive G protein (Fig. 8). Such a coupling of beta 2-ARs to pertussis toxin-sensitive G protein(s) has been already reported in rat cardiomyocytes (30) and in cells sur-expressing beta 2-ARs (31).

cAMP exerts a dual tuning on cell responses to beta 2-AR stimulation. On the one hand, we show that cAMP, produced upon beta 1-AR stimulation, evokes a quenching of cell responses to beta 2-AR stimulation; thus, after 3 min exposure to prenalterol, cells did not respond further to beta 2-AR stimulation, whereas following complete desensitization of beta 1-ARs, beta 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 beta 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, beta 2-AR agonists elicit a positive inotropic effect via cPLA2 activation and AA release. Contrary to the beta 1-AR/cAMP pathway, beta 2-AR/cPLA2 pathway involves a pertussis toxin-sensitive G protein. cAMP exerts a dual regulation on the beta 2-AR/AA pathway; it inhibits the cell response to beta 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 beta 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 beta 2-AR/cPLA2/AA pathway could be determinant in failing hearts that have lost 50% of beta 1-ARs and show a parallel decrease in agonist-stimulated adenylyl cyclase activity (4, 6, 7).

    ACKNOWLEDGEMENTS

We thank S. Lotersztajn and G. Guellaën for helpful discussions and J. Hanoune for the constant support.

    FOOTNOTES

* This work was supported by INSERM, the French Ministère de la Recherche et de la Technologie, and the Unité de Formation et de Recherche de Médecine, Créteil, Paris-Val de Marne.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: INSERM Unité 99, Hôpital Henri Mondor, 94010 Créteil, France. Tel.: (33) 1 49 81 35 34; Fax: (33) 1 48 98 09 08; E-mail: pavoine{at}im3.inserm.fr.

The abbreviations used are: beta -AR, beta -adrenergic receptor; AA, arachidonic acid; [Ca2+]i, cytosolic free Ca2+ concentration; cPLA2, cytosolic phospholipase A2; PTX, pertussis toxin; (Rp)-cAMPS, (Rp)-adenosine-3',5'-cyclic monophosphorothioate; PKA, protein kinase A.
    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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