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J. Biol. Chem., Vol. 280, Issue 19, 18881-18890, May 13, 2005

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The Cytosolic Phospholipase A₂ Pathway, a Safeguard of ₂-Adrenergic Cardiac Effects in Rat^*

Bouziane Ait-Mamar, Michel Cailleret, Catherine Rucker-Martin¶, Anissa Bouabdallah, Gabriele Candiani, Christophe Adamy, Philippe Duvaldestin, Francoise Pecker, Nicole Defer, and Catherine Pavoine||

From the Inserm, U581, University of Paris, XII-Val de Marne, Créteil F-94010, France, the Service Anesthésie et Réanimation, Hôpital Henri Mondor, Créteil, F-94010, France, and ^¶CNRS, UMR-8078, Hôpital Marie-Lannelongue, Le Plessis-Robinson, F-92350, France

Received for publication, September 8, 2004 , and in revised form, January 31, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We have recently demonstrated that in human heart, ₂-adrenergic receptors (₂-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A₂ (cPLA₂) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117–1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of ₂-ARs triggered an increase in the amplitude of electrically stimulated [Ca²⁺]_i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA₂ inhibitor, AACOCF₃. The ₂-AR/cPLA₂ inhibitory pathway involved G_i and MSK1. Potentiation of ₂-AR/AC/PKA-induced Ca²⁺ responses by AACOCF₃ did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser¹¹⁷⁷) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser¹⁶). The constraint exerted by the ₂-AR/cPLA₂ pathway on the ₂-AR/AC/PKA-induced Ca²⁺responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca²⁺ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of ₂-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac ₂-AR signaling pathway that limits ₂-AR/AC/PKA-induced Ca²⁺ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cardiovascular diseases are becoming the leading cause of death worldwide, with heart failure as a major symptom. Most patients with heart failure show dysregulation of the sympathetic system, and the resulting chronic elevation of plasma catecholamine levels correlates with the prognosis (1). Among the identified cardiac catecholamine receptors, ₁-adrenergic receptors (₁-AR)¹ and ₂-AR received the most interest as mainstay regulators of cardiac performance. Noteworthy, ₁-AR and ₂-AR functionally diverge because of clear discrepancies in signaling transduction pathways (2). Chronic over-stimulation of cardiac ₁-AR, associated with exclusive activation of the G_s/adenylyl cyclase (AC) pathway, is detrimental and leads to hypertrophy, fibrosis, and heart failure (3). In sharp contrast, sustained ₂-AR stimulation protects cardiomyocytes against apoptosis. In addition, transgenic mice with cardiac moderate ₂-AR overexpression display not only long term improved cardiac function but also normal life expectancy (4–6). The beneficial effect of ₂-AR stimulation appears to rely on the ability of ₂-AR to couple with pertussis toxin (PTX)-sensitive G proteins (G_i2 and G_i3), leading to a ₂-AR/G_s/AC signaling spatially confined to the caveolar structures and functionally moderated, as compared with the ₁-AR/G_s/AC pathway (7–9). Identification of ₂-AR/G_i-downstream effectors is of major interest to elucidate, and to further exploit the cardiac protective ₂-AR potential.

Recently, on human cardiac ventricular and auricular biopsies, we identified a new ₂-AR-stimulated pathway, namely the G_i/cytosolic phospholipase A₂ (cPLA₂) pathway, in addition to the classical ₂-AR/G_s/AC pathway (10). cPLA₂ is a high molecular mass enzyme (85 kDa), activated by submicromolar concentrations of Ca²⁺, that displays a unique selectivity for arachidonyl in the sn-2 position of phospholipids (11). cPLA₂ is fully activated by both phosphorylation by mitogen-activated protein kinase and increases in [Ca²⁺]_i. cPLA₂ activation is associated with its translocation from the cytosol to intracellular membranes, such as endoplasmic/sarcoplasmic reticulum, Golgi apparatus, and nuclear envelope. The essential role of cPLA₂ in inflammation, asthma, neurodegenerative diseases, and bleomycin-induced pulmonary fibrosis is now well documented, and cPLA₂ has been identified as an attractive therapeutic target in the development of new inflammatory drugs (12–15). In contrast, a dearth of information exists on the possible functional impact of the cPLA₂ and its role in the development of cardiac diseases. Only one recent study has established the antihypertrophic potential of the cPLA₂ pathway in cardiac and skeletal muscles, using the model of the cPLA₂ knockout mice (16). In the present study, we examined the impact of ₂-AR-G_i/cPLA₂ and ₂-AR-G_s/Ac pathways on Ca²⁺ signaling and contraction in isolated adult rat cardiomyocytes. We show that ₂-AR agonists trigger an increase in the amplitude of electrically stimulated [Ca²⁺]_i transients that relies on the ₂-AR-G_s/AC/PKA pathway and is under the negative control of the ₂-AR-G_i/MSK1/cPLA₂ pathway. We provide evidence that upon ₂-AR stimulation cPLA₂ translocates to caveolae/sarcoplasmic reticulum (SR)-functional platforms and bridles phosphorylation of endothelial nitric-oxide synthase (eNOS) and phospholamban (PLB). Using fluorescence imaging, we show that the ₂-AR/cPLA₂ pathway neutralizes NO production. Our study highlights the essential physiological role of the ₂-AR/G_i/cPLA₂ pathway in limiting ₂-AR/AC/PKA-induced Ca²⁺ responses.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials
Zinterol was kindly supplied by Bristol-Myers Squibb Co (Stanford, CT). (±)isoproterenol, CGP20712A, ICI118551, epinephrine, forskolin, NaF, pertussis toxin, Prazosin, RO 31-8220, Filipin III, sucrose, L-NAME, L-arginine were from Sigma (Saint Quentin Fallavier, France). AACOCF₃ was purchased from Biomol Research laboratories (Plymouth Meeting, PA). Liberase Blendzyme III was from Roche Applied Sciences (Indianapolis, IN). DAF-FM diacetate and Fura 2-AM were purchased by Molecular Probes (Montluçon, France).

Rabbit polyclonal antibodies against human phospho-eNOS (Ser¹¹⁷⁷) and rabbit polyclonal antibodies against human phospho-MSK1 (Thr⁵⁸¹) were from Cell Signaling Technology (Beverly, MA). Sheep immunoaffinity-purified IgG antibodies against human MSK1 were from Upstate Biotechnology (Lake Placid, NY); mouse monoclonal IgG1 antibodies against rat caveolin-3 were from BD Biosciences Pharmingen (Le Pont de Claix, France), mouse monoclonal IgG antibodies against phospholamban (clone A1), rabbit polyclonal antibodies against phosphorylated phospholamban (Ser¹⁶), and phosphorylated phospholamban (Thr¹⁷) were from Cyclacel (Dundee, UK); mouse monoclonal antibodies against the N-terminal part of the human cPLA₂ were from Santa Cruz Biotechnology. Peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated donkey anti-sheep IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA), and peroxidase-conjugated rabbit anti-mouse IgG were from Biosys (Compiègne, France). Goat anti-mouse IgG (H+L) highly cross-absorbed coupled to Alexa Fluor® 594 and goat anti-rabbit IgG (H+L) highly cross-absorbed coupled to Alexa Fluor® 647 were from Molecular Probes. Fluorescein isothiocyanate (FITC)-conjugated phalloidin was from Sigma and Vectashield mounting medium containing DAPI was from Vector Laboratories (Peterborough, UK). Polyclonal antibodies against sarcoplasmic reticulum Ca²⁺ pump (SERCA) 2a were kindly provided by Dr. Wuytack (Leuven, Belgium). Chemiluminescent detection kit (SuperSignal West Dura) was from Pierce.

Methods
Cardiomyocyte Isolation—The care and the use of animals were in accordance with institutional guidelines. Adult male Wistar rats (180–250 g, Janvier, Le Genest St Isle, France) were used. Calcium-tolerant myocytes were isolated by cardiac retrograde aortic perfusion as previously described, but using liberase blendzyme III (30 µg/ml) instead of collagenase (17, 18). Freshly isolated cardiomyocytes were plated on laminin (3 µg/ml; Sigma) and cultured up to 3 days according to Sambrano et al. (19).

Measurement of [Ca²⁺]_i Transients and Cell Fractional Shortening— Plated cardiomyocytes were loaded with Fura 2-AM (1.5 µM) for 20 min and submitted to electrical stimulation (square waves, 0.5 Hz). [Ca²⁺]_i imaging experiments were performed, as previously described in a saline buffer containing 10 mM glucose, 130 mM NaCl, 5 mM KCl, 10 mM Hepes pH 7.4, 1 mM MgCl₂, 2 mM CaCl₂ (BSS buffer) (18, 20, 21). Results are shown as mean ± S.E. for 5 to 15 cells obtained from three different isolations or as a typical representation.

Cell fractional shortening was determined, as described previously (18, 20), from the fluorescence images recorded to measure F₃₆₀/F₃₈₀, with Scion Image Software (Scion, Frederick, MD). Results are shown as a typical representation.

Coordinated NO and Ca²⁺ Imaging in Isolated Cardiomyocytes—NO synthesis was visualized with the cell permeable fluorescent precursor, DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein) diacetate. Inside cells, this dye is hydrolyzed by cytosolic esterases to the nonpermeable DAF-FM. In the presence of nitric oxide and oxygen, the relatively non-fluorescent DAF-FM is converted into the highly fluorescent and photostable triazole form, DAF-FM T, whose fluorescence intensity is directly proportional to the NO concentration. This dye was chosen as the most sensitive fluorescent probe allowing NO imaging (detection from 3 nM [NO]_i), and displaying the important advantages of a fluorescent spectrum independent of pH variations above pH 5.5 and a resistance to bleaching (22). Experiments were performed at room temperature, and cells were exposed to 1 µM DAF-FM diacetate and 1.5 µM Fura 2-AM, for 20 min, to combine [NO]_i and [Ca²⁺]_i imaging. Cells were washed in BSS solution containing 100 µM L-arginine and submitted to electrical stimulation. Because of Fura-2 loading, cells were checked for appropriate [Ca²⁺]_i transient responses to electrical stimulation, as well as to agonists (zinterol or zinterol plus AACOCF₃), before and after the NO imaging experiment. DAF-FM diacetate fluorescence was excited at 480 nm, and emitted cellular fluorescence was recorded at 540 nm. Changes in cellular DAF-FM diacetate fluorescence intensities (F) in each experiment were normalized to the level of fluorescence recorded before agonist application (F₀), and changes in [NO]_i are expressed as F/F₀. L-Arginine was omitted when L-NAME was used to block NO synthase. Images were recorded every 6 s. Results are described as mean ± S.E. for 10–15 cells obtained from three different isolations or shown as a typical representation.

Adenylyl Cyclase Assay—A particulate fraction was obtained from freshly isolated cardiomyocytes suspended in lysis buffer containing 50 mM Hepes, pH 7.4, 2 mM EDTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride, disrupted by sonication, and centrifuged for 30 min at 20,000 x g. The pellet was resuspended in 50 mM Hepes, pH 7.4 and stored in liquid nitrogen. Adenylyl cyclase was measured as described previously (10, 23). Results were obtained from triplicate determinations.

Drug Treatment—Note that a maximal 1 µM zinterol concentration was chosen in order to focus on selective ₂-AR-mediated effects. AACOCF₃ (10 µM), RO 318220 (1 µM), L-NAME (1 mM), and H89 (3 µM) were added 10 min, or 30 min concerning H89, before addition of other stimuli, respectively. PTX treatment (500 ng/ml for 24 h) was carried out as described previously (23). Adult rat cardiomyocytes were incubated with Filipin III (0.2 µg/ml) at room temperature for 30 min before zinterol (1 µM) exposure. It is noteworthy that this dose of Filipin III allowed us to perform experiments on Fura-2-loaded cells that were devoid of any sign of Fura-2 leakage and were responsive to electrical stimulation for at least an additional 1 h experimental duration, thus arguing for peripheral cellular membrane integrity.

Preparation of Low Density Caveolin-3-enriched Membrane Fractions—Fractions enriched in the muscle-specific caveolin-3 isoform were prepared from isolated adult rat cardiomyocytes according to a detergent-free purification scheme as described previously (24, 25). Freshly isolated cardiomyocytes (1.5 x 10⁶ cells/condition) were submitted to a 10 min of pretreatment with or without 10 µM AACOCF₃ followed by a 10-min incubation with or without 1 µM zinterol in BSS buffer at 37 °C. All subsequent steps were carried out at 4 °C. Cells suspended in 1 ml of solution A (0.5 M Na₂CO₃, pH 11, containing phosphatase inhibitors (10 mM NaF, 100 µM Na₃VO₄, and 5 mM Na₄P₂O₇)) were sequentially disrupted by homogenization with a loose fitting Dounce homogenizer (10 strokes), a Polytron tissue grinder (three 10-s bursts), and a bath sonicator (one 5-min burst). 1 ml of homogenate was then adjusted to 45% sucrose (% Brix measured with a refractometer) by adding 3 ml of 60% sucrose prepared in 2 volumes of MES-buffered saline (MBS: 25 mM MES, pH 6.5, and 0.15 M NaCl) and 1 volume of solution A. The 45% sucrose fraction was placed on the bottom of an ultracentrifuge tube, overlaid with a 5–30% continuous sucrose gradient (sucrose solutions were prepared using equal volumes of MBS and solution A) and centrifuged at 39,000 rpm for 18 h at 4 °C in a SW41Ti rotor (Beckman Coulter). After centrifugation, eleven 1-ml fractions were collected from the top of the gradient, concentrated by precipitation with trichloroacetic acid (addition of 75 µl of a 100% solution). After centrifugation at 10,000 x g for 15 min at 4 °C, pellets were resuspended in 50 mM Tris, pH 8.8 added with phosphatase inhibitors (10 mM NaF, 100 µM Na₃VO₄, and 5 mM Na₄P₂O₇) and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 1 µg/ml pepstatin, and 2 µg/ml aprotinin). Following determination of the protein content, fractions were dissolved in Laemmli loading buffer.

Immunoblot Analysis of the Phosphorylation State of MSK1, eNOS, and PLB and Detection of Caveolin-3, PLB, MSK1, eNOS, SERCA 2A, and cPLA₂—Samples, 50 µg of each cellular extract (Fig. 5) or 5 µg of each gradient fraction (Fig. 7A), were subjected to SDS-PAGE (15% acrylamide gels) and proteins electrotransferred to polyvinylidene difluoride membranes (0.22 µm, Millipore). Membranes were first incubated with antibodies against caveolin-3 (1:5,000) (Fig. 7A), or P-(Ser¹⁶)-PLB (1:1,000) (Fig. 5), or P-(Thr¹⁷)-PLB (1:1,000) (not shown). Blots were then treated with secondary peroxidase-conjugated rabbit anti-mouse (1:20,000) or goat-anti rabbit (1:20,000) antibodies. The peroxidase activity was visualized with an enhanced chemiluminescent detection kit (Supersignal West Dura). 65 µg of each caveolin-3-enriched fraction (fraction 7) were loaded on preparative SDS-PAGE (8–16% acrylamide) and transferred to Hybond C Super (Amersham Biosciences) nitrocellulose. Immunoblots were cut in small pieces and analyzed with antibodies for cPLA₂ (1:500), MSK1 (1:1,000), P-(Thr⁵⁸¹)-MSK1 (1:500), PLB (1:5,000), P-(Ser¹⁶)-PLB (1:1,000), SERCA 2a (1: 1,000), eNOS (1:2,500), and P-(Ser¹¹⁷⁷)-eNOS (1:1,000). Blots were then treated with secondary peroxidase-conjugated rabbit anti-mouse (1: 20,000 dilution), goat-anti rabbit (1:20,000 dilution), or donkey anti-sheep (1:20,000 dilution) antibodies.

Immunocytochemistry and Confocal Laser Scanning Microscopy
Immunofluorescence Staining—Indirect immunofluorescence was performed on freshly isolated myocytes fixed with 4% formaldehyde at room temperature as described previously (26). Briefly, myocytes were incubated in phosphate-buffered saline containing 5% bovine serum albumin for 30 min to block nonspecific binding sites, followed by overnight incubation with a solution of mouse monoclonal antibodies against caveolin-3 (clone 26, 1:100) and rabbit polyclonal antibodies against SERCA 2a (1:100, see Ref. 27). After washes, cells were first incubated for 1 h with an excess of the secondary antibody goat anti-mouse IgG (H+L) highly cross-absorbed coupled to Alexa Fluor® 594 (1:100). Myocytes were then washed and treated with the secondary antibody goat anti-rabbit IgG (H+L) highly cross-absorbed coupled to Alexa Fluor® 647 (1:100) for 1 h. This was followed after washes by incubation with FITC-conjugated-phalloidin (1/30) for 90 min. After a final wash, coverslips were mounted in Vectashield mounting medium containing DAPI. Labeling of cells with secondary antibodies alone was carried out as a negative control.

Confocal Microscopy and Image Processing—Images were collected with a Zeiss LSM-510 multitracking laser scanning confocal microscope (Carl Zeiss SAS, Frankfurt, Germany). Fluorochromes were detected sequentially using laser lines 488 nm (FITC), 543 nm (Alexa Fluor® 594) and 633 nm (Alexa Fluor® 647). DAPI was visualized by UV excitation. Offset and gain for each channel was set in order to avoid any cross-talk between the three fluorochrome emission spectra. The images were coded white (FITC), red (Alexa Fluor® 594), and green (Alexa Fluor® 647) giving yellow co-localization in merge images (Alexa Fluor® 594 and Alexa Fluor® 647). The oil objective used was x63 (NA 1.4), giving a resolution of 100 nm in the x,y plane and 300 nm in the z axis (pinhole, 72 µm).

Quantification of Overlapping—We studied 10 individual myocytes and analyzed 55 ± 2 successive single sections acquired in each image stack. Overlapping was quantified with the LSM 510 3 software (Carl Zeiss). The overlapping of SERCA 2a and caveolin-3 stainings in the x,y plane was determined owing to analysis of fluorescence intensity profiles as previously described by Bolte et al. (28). Positive structures were evaluated using threshold values of 152 ± 17 (caveolin-3) and 144 ± 5 (SERCA 2a) pixel units on a grayscale of 0–255. A scatterplot of the individual pixels from each paired images was generated, and overlapped pixels were characterized by an overlap coefficient value of 1. The number of overlapped pixels was compared with the number of total positive pixels for each labeling (caveolin-3 or SERCA 2a).

Statistical Analysis—Results were analyzed by the unpaired two-tailed Mann-Whitney test. Differences were considered statistically significant at a value of p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The ₂-AR/cPLA₂ Pathway Restrains the ₂-AR/AC/PKA-induced Ca²⁺ and Contraction Responses in Isolated Cardiomyocytes—The amplitude of [Ca²⁺]_i transients was measured in electrically stimulated adult rat cardiomyocytes and loaded with Fura 2-AM, in response to the ₂-AR selective agonist, zinterol. Zinterol induced a dose-dependent stimulatory effect on the amplitude of [Ca²⁺]_i transients (maximal response of 182 ± 11% with zinterol (1 µM)) (Fig. 1, A and B). The cPLA₂ inhibitor, AACOCF₃ (10 µM), had no effect on basal [Ca²⁺]_i transient amplitude (Fig. 1B), but enhanced zinterol-induced Ca²⁺ responses (maximal response of 307 ± 28% with 1 µM zinterol) (Fig. 1, A and B). Typical traces of [Ca²⁺]_i transients and fractional shortening showed the correlation between the increase in the amplitude of [Ca²⁺]_i transients and that of contraction in cardiomyocytes in response to zinterol added alone, or after treatment with AACOCF₃ (Fig. 1A). Inhibition of PKA with H89 (3 µM) blunted the zinterol effect on the amplitude of [Ca²⁺]_i transients and contraction, in the absence (not shown) as well as in the presence of AACOCF₃ (Fig. 1A). These results identified PKA activation as an essential feature of zinterol-induced Ca²⁺ and contraction responses, and demonstrated that the cPLA₂ pathway exerted a constraint on PKA-dependent events.

View larger version (37K): [in this window] [in a new window]   FIG. 1. The 2-AR-induced increase in amplitude of [Ca2+]i transients and fractional shortening in electrically stimulated adult rat cardiomyocytes is enhanced by AACOCF3 and abrogated by H89. AC is not the target of AACOCF3 action. Cardiomyocytes, isolated from adult rats were loaded with Fura 2-AM, were electrically stimulated at 0.5 Hz, and exposed for 3 min to the 2-AR agonist, zinterol, in the absence (control) or in the presence of AACOCF3 (10 µM) added with or without H89 (3 µM), as described under "Experimental Procedures." A, typical traces of recorded [Ca2+]i transients and fractional shortenings are representative of at least six cells obtained from three different isolations. B, amplitude of [Ca2+]i transients was normalized to control values determined at time 0. Values are mean ± S.E. of effects observed on at least six cells obtained from three different isolations. C, adenylyl cyclase activity was measured in membranes prepared from isolated cardiomyocytes, as described under "Experimental Procedures." Results were obtained from triplicate determinations. *, p < 0.05 versus control.

The cPLA₂ Constraint Is Specific to ₂-AR and Does Not Affect ₁-AR-induced Ca²⁺ Response—The maximal increases in the amplitude of [Ca²⁺]_i transients elicited by two non-selective -AR stimuli, namely epinephrine (1 µM) added with the -AR antagonist, prazosin (1 µM) and isoproterenol (0.3 µM), were significantly enhanced in the presence of AACOCF₃ (Fig. 2). Maximal Ca²⁺ responses reached 302 ± 37 and 275 ± 18% in the presence of AACOCF₃, compared with 182 ± 12 and 204 ± 17%, in its absence, respectively. The ₂-AR stimulus, isoproterenol (0.3 µM) associated with the ₁-AR antagonist, CGP 20712A (0.3 µM), elicited a 119 ± 8% increase in the amplitude of [Ca²⁺]_i transients, that was markedly amplified (167 ± 19%) in the presence of the cPLA₂ inhibitor (Fig. 2). In contrast, selective ₁-AR stimulation triggered by isoproterenol (0.3 µM) associated with the ₂-AR antagonist, ICI 118551 (0.1 µM), induced similar increases in the amplitude of [Ca²⁺]_i transients in the absence or in the presence of AACOCF₃ (262 ± 21 versus 271 ± 47%, respectively) (Fig. 2). These results indicated that the constraint exerted by the cPLA₂ pathway selectively targeted the ₂-AR-induced Ca²⁺ response, without affecting the ₁-AR-induced Ca²⁺ response.

View larger version (25K): [in this window] [in a new window]   FIG. 2. AACOCF3 treatment enhances 2-AR- but not 1-AR-induced increase in amplitude of [Ca2+]i transients of electrically stimulated cardiomyocytes. Cardiomyocytes, isolated from adult rats, were loaded with Fura 2-AM, pretreated for 10 min, and maintained in cell medium with AACOCF3 (10 µM) or without AACOCF3 (control). Cardiomyocytes were electrically stimulated at 0.5 Hz and exposed for 3 min to non-selective -AR stimuli, i.e. isoproterenol (0.3 µM), or epinephrine (1 µM) combined with prazosin (1 µM); 1-AR stimulus, i.e. isoproterenol (0.3 µM) combined with the 2-antagonist ICI 118551 (0.1 µM); 2-AR stimulus, i.e. isoproterenol (0.3 µM) combined with the 1-antagonist CGP 20712A (0.3 µM). The amplitude of [Ca2+]i transients was normalized to control values determined at time 0. Values are mean ± S.E. of effects observed on at least six cells obtained from three different isolations. *, p < 0.05 versus control.

View larger version (20K): [in this window] [in a new window]   FIG. 3. PTX and RO 318220 treatments abrogate the enhancing effect of AACOCF3 on zinterol-induced Ca2+ response. Cardiomyocytes, isolated from adult rats treated (or not) for 24 h with PTX (500 ng/ml), were loaded with Fura 2-AM, pretreated for 10 min, and maintained in cell medium with or without AACOCF3 (10 µM) and/or RO 318220 (1 µM) as described under "Experimental Procedures." Cardiomyocytes were electrically stimulated and exposed for 3 min to the 2-AR agonist zinterol (1 µM). The amplitude of [Ca2+]i transients was normalized to control values determined at time 0. Values are mean ± S.E. of effects observed on at least five cells obtained from two different isolations. *, p < 0.05 versus control.

The ₂-AR/cPLA₂ Pathway Restrains NOS Activity and PLB Phosphorylation—In cardiomyocytes isolated from cat atria, ₂-AR stimulation has been reported to induce an increase in the L-type Ca²⁺ current (Ica,L) because of NO production (32, 33). In the cat atrial cardiomyocytes, NO acts via cGMP-mediated inhibition of the type III phosphodiesterase (PDE) activity, enhancing cAMP-dependent stimulation of Ica,L. Among NO-generating enzymes expressed in the cardiomyocytes, eNOS can be activated upon PKA- or PI3K-dependent phosphorylation of the Ser¹¹⁷⁷ residue (34). Because in adult rat cardiomyocytes, both PKA and PI3K activities are activated upon ₂-AR stimulation (35), we investigated whether zinterol did trigger NO production that could be amplified by AACOCF₃ treatment. Cells were loaded with the NO-sensitive fluorescent indicator, DAF-FM diacetate, and Fura 2-AM, to correlate NO and Ca²⁺ responses. In rat cardiomyocytes that were electrically stimulated, no change in DAF fluorescence was detected within the first 20 min of incubation time following addition of zinterol alone (not shown). In contrast, exposure to zinterol combined with AACOCF₃ produced a marked increase in DAF fluorescence (Fig. 4A), that reached a mean 2.1 ± 0.2-fold maximal increase in F/F₀ after 8 ± 1 min. The increase in DAF fluorescence was blocked in the presence of the NO synthase inhibitor, L-NAME (not shown), and could thus be attributed to NO production. An increase in the amplitude of [Ca²⁺]_i transients occurred together with NO release in cells stimulated with zinterol plus AACOCF₃. Interestingly, L-NAME treatment did not modify the increase in the amplitude of [Ca²⁺]_i transients in response to zinterol alone (Fig. 4B) but partially reversed the potentiation of zinterol-induced Ca²⁺ responses elicited by AACOCF₃ (Fig. 4B). These results suggested that, in adult rat cardiomyocytes, basal zinterol-induced Ca²⁺ responses were unrelated to NO production. In contrast, inhibition of the ₂-AR/cPLA₂ pathway by AACOCF₃, uncovered NO release in response to zinterol. The latter took part in the mechanism of amplification of PKA-dependent Ca²⁺ responses.

View larger version (16K): [in this window] [in a new window]   FIG. 4. The enhancing effect of AACOCF3 on zinterol-induced Ca2+ response is associated with NO production and abrogated by L-NAME treatment. Cardiomyocytes, isolated from adult rats, were loaded with Fura 2-AM and DAF-FM (A) or Fura2-AM alone (B), pretreated for 10 min, and maintained in cell medium with or without AACOCF3 (10 µM), and/or L-NAME (1 mM), as described under "Experimental Procedures." Cardiomyocytes were electrically stimulated at 0.5Hz and exposed to the 2-AR agonist zinterol (1 µM). Typical traces of [Ca2+]i transients were recorded before (t1) and after (t2) measurement of DAF fluorescence (A). The amplitude of [Ca2+]i transients was normalized to control values determined at time 0. Values are mean ± S.E. (B). Data are representative of effects observed on at least six cells obtained from three different isolations. *, p < 0.05 versus control.

Integrity of Caveolae Is Required to Observe the cPLA₂-dependent Constraint of ₂-AR-induced Ca²⁺ Responses—In adult rat cardiomyocytes, ₂-ARs are preferentially concentrated in caveolae, together with G proteins (i.e. G_s, G_i), effectors (i.e. AC), kinases (i.e. PKA, protein kinase C, mitogen-activated protein kinase) and adaptor proteins (i.e. AKAP, NHERF). Importantly, such a localization has been shown to dictate ₂-ARs downstream signaling pathways (25, 39, 40). Caveolae are characteristic flask-shaped invaginations of the plasma membrane that are distinctively enriched in cholesterol (41). Next, experiments were designed to evaluate the impact of caveolar structures on the constraint exerted by the cPLA₂ pathway on ₂-AR/AC/PKA-induced Ca²⁺ responses. We used Filipin III, a detergent that disrupts caveolar structures by binding to cholesterol. Cardiomyocytes loaded with Fura 2-AM were pretreated with Filipin III (0.2 µg/ml) for 30 min, including a pretreatment with or without AACOCF₃ (10 µM) during the last 10 min, and then exposed to zinterol (1 µM). Filipin III by itself produced a 149 ± 7% increase in the amplitude of basal [Ca²⁺]_i transients, that might reflect the accessibility of new targets to messengers (i.e. cAMP) or effectors (i.e. PKA) previously sequestrated inside caveolar structures (Fig. 6). Subsequent addition of zinterol (1 µM) led to a final 198 ± 10% increase. However, zinterol action was no more sensitive to activation by AACOCF₃ treatment (Fig. 6). These results suggested that, in adult rat cardiomyocytes, the integrity of caveolar structures was necessary for the cPLA₂ pathway to exert a constraint on zinterol-induced Ca²⁺ responses.

View larger version (22K): [in this window] [in a new window]   FIG. 5. AACOCF3 treatment uncovers phosphorylation of (Ser16)-PLB residue in response to zinterol. Cardiomyocytes, isolated from adult rats, were (or not) pretreated for 10 min, maintained with AACOCF3 (10 µM), and treated for 10 min with or without zinterol (1 µM). Cardiomyocytes obtained from each of four distinct conditions of incubation (control, zinterol, AACOCF3, AACOCF3 + zinterol) were homogenized, and protein samples (50 µg) were subjected to SDS-PAGE. Membranes were immunostained with antibodies against P-(Ser16)-PLB, as described under "Experimental Procedures." A representative immunoblot is shown in the upper panel. Average data (densitometric evaluation with arbitrary units) are presented in the bottom panel (mean ± S.E. of four experiments. *, p < 0.05 versus AACOCF3 treatment).

View larger version (21K): [in this window] [in a new window]   FIG. 6. Treatment with Filipin III abrogates the enhancing effect of AACOCF3 on zinterol-induced Ca2+ response. Cardiomyocytes, isolated from adult rats, were loaded with Fura 2-AM and electrically stimulated at 0.5 Hz. They were treated for 30 min with Filipin III (0.2 µg/ml), and with or without AACOCF3 (10 µM) during the last 10 min of incubation. Cardiomyocytes were then exposed for 3 min to the 2-AR agonist zinterol (1 µM). The amplitude of [Ca2+]i transients was normalized to control values determined at time 0, before the addition of Filipin III. Values are mean ± S.E. of effects observed on at least six cells obtained from two different isolations. *, p < 0.05 versus control

Next, experiments were performed using the most enriched caveolin-3 fraction, namely fraction 7, which was separated because of a 32 ± 1.5% sucrose density and corresponded to a mean 14 ± 3% of the total cellular proteins (Fig. 7A). Identical quantities of proteins from fraction 7, isolated from cells submitted to the four different treatments (described above), were separated on preparative gels. Following electroblotting, membranes were cut in small pieces and analyzed, in parallel, for the presence of caveolae markers (caveolin-3 and eNOS), ₂-AR signaling effectors (MSK1, P-(Thr⁵⁸¹)-MSK1 and cPLA₂), targets of the cPLA₂ constraint (P-(Ser¹¹⁷⁷)-eNOS and P-(Ser¹⁶)-PLB) and sarcoplasmic reticulum markers (PLB and SERCA 2a). As shown in Fig. 7, equal amounts of caveolin-3 were recovered in the four of the fraction 7 samples. cPLA₂ was exclusively detected in fraction 7 obtained from cardiomyocytes that have been treated with zinterol alone, illustrating the zinterol-induced translocation of the enzyme to the low density caveolin-3-enriched membrane fraction. Treatment with AACOCF₃ hindered cPLA₂ translocation to such a fraction. Similar levels of MSK1 were detected in each fraction 7, but only cells incubated with zinterol, in the absence or in the presence of AACOCF₃, displayed the MSK1-Thr⁵⁸¹-phosphorylated form, a prerequisite for MSK1 activation. Taken together, these results supported the role of MSK1 in the ₂-AR/cPLA₂ pathway, upstream cPLA₂ activation in view of the insensitivity of the ₂-AR induced MSK1 phosphorylation toward AACOCF₃ (see Fig. 3).

eNOS is a well known marker of caveolae, and the phosphorylation of eNOS at Ser¹¹⁷⁷ is usually correlated with eNOS activation. Similar levels of eNOS were detected in the four fraction 7 samples. Interestingly, a large increase in eNOS phosphorylation level was observed, in response to zinterol added together with AACOCF₃. Thus, zinterol-induced AACOCF₃-dependent production of NO (Fig. 4) could be related with phosphorylation and activation of eNOS, sedimented in the caveolin-3-enriched membrane fraction.

View larger version (24K): [in this window] [in a new window]   FIG. 7. Zinterol induces translocation of the cPLA2 to platforms integrating caveolin-3-enriched membranes, closely associated with sarcoplasmic reticulum membranes and allowing functional interaction between upstream activators (MSK1) and downstream targets (eNOS and PLB) of the cPLA2. Cardiomyocytes, isolated from adult rats, were pretreated (or not) for 10 min, maintained with AACOCF3 (10 µM), and treated for 10 min with or without zinterol (1 µM). A, cardiomyocytes obtained from each of four distinct conditions of incubation (control, zinterol, AACOCF3, AACOCF3 + zinterol) were homogenized and fractionated according to a detergent-free purification scheme, as described under "Experimental Procedures." Samples (5 µg of each gradient fraction) were subjected to SDS-PAGE. Membranes were immunostained with antibodies against caveolin-3, as described under "Experimental Procedures." The presence of caveolin-3, and the protein and sucrose distribution patterns were analyzed in parallel in the fractions corresponding to each gradient. Note that these patterns were equivalent under the four experimental conditions. A representative immunoblot is presented from experiments that have been repeated four times. B, caveolin-3-enriched fraction 7 of cardiomyocyte fractionations obtained from each of four distinct conditions of incubation was loaded on a preparative gel, as described under "Experimental Procedures." Each of the four membranes was cut in small pieces and analyzed, in parallel, for the presence of caveolae markers (caveolin-3, eNOS), upstream activators of the cPLA2 (MSK1, P-(Thr581)-MSK1), downstream effectors of the cPLA2 (P-(Ser1177)-eNOS, P-(Ser16)-PLB), and sarcoplasmic markers (SERCA 2a, PLB). Representative immunoblots are presented from experiments that have been repeated three times.

We performed confocal microscopy in isolated adult rat cardiomyocytes immunostained with SERCA 2a and caveolin-3 antibodies in order to compare intracellular distribution of the two proteins (Fig. 8). Immunostained cardiomyocytes displayed a striated and regular actin pattern as illustrated by phalloidin staining (Fig. 8A, panel b). As previously reported in adult mouse cardiomyocytes (27, 42), SERCA 2a exhibited a longitudinal and transverse distribution and was also concentrated around the nucleus (Fig. 8A, panel c). Caveolin-3 was present all along the peripheral cellular membrane, the intercalated disks and exhibited a punctuated distribution within the T-tubules (Fig. 8A, panel d). The merge image (Fig. 8A, panel a) showed partial overlapping of the two fluorescent stainings (yellow). Analysis of the overlapping of SERCA 2a and caveolin-3 stainings, using distribution of fluorescence intensities, as shown in the typical histogram in Fig. 8B, demonstrated a mean overlapping distance of 640 ± 36 nm largely above the limits of resolution of the confocal microscope (100 nm). Overlapping pixels represented 25 ± 5% of green pixels (SERCA 2a) and 43 ± 7% of red pixels (caveolin-3) (Fig. 8C, area 3). In addition to the well known major distribution of the SERCA 2a along the longitudinal SR and around the nucleus, these results strongly supported the close vicinity of a significant portion of SERCA 2a-enriched membranes (SR) with a portion of caveolin-3-enriched membranes (caveolae).

View larger version (49K): [in this window] [in a new window]   FIG. 8. Confocal microscopic analysis of SERCA 2a and caveolin-3 distribution in adult rat cardiomyocytes. A, double immunostaining (visualized by confocal microscopy) of isolated adult rat cardiomyocytes with antibodies directed against SERCA 2a (green) and caveolin-3 (red) and DAPI coloration of nuclei (panel a). Details of immunolabeling are shown in panel c (SERCA 2a) and panel d (caveolin-3). Phalloidin and DAPI labeling (panel b) were used to reveal actin distribution and nuclei, respectively. Bar, 20 µm. B, overlapping of SERCA 2a and caveolin-3 staining in the area previously indicated in A, panel c and A, panel d. The upper panel presents the region of interest, and the lower panel details distribution of green and red fluorescence intensities along the white line (about 3.5 µm). C, typical scatterplot of the individual pixels from paired images. The threshold levels of green (139) and red (169) signals determined the overlapping region (area 3); areas 2 and 1 corresponding to green and red pixels, respectively, with no color mixing.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The new findings of this study are 3-fold. First, we show that ₂-AR, through stimulation of a PTX-sensitive G protein and MSK1, evokes cPLA₂ activation. Second, we give evidence that ₂-AR-induced cPLA₂ activation is linked to the translocation of the enzyme to low density caveolin-3-enriched membrane fractions that are closely associated with SR membranes and constitute functional signaling platforms. Inside those platforms we identified two components of the ₂-AR/cPLA₂ pathway, namely eNOS and PLB, showing that the blockade of cPLA₂ unmasks zinterol-induced phosphorylation of eNOS and PLB. Finally, we demonstrate that the ₂-AR/cPLA₂ pathway, through inhibition of NOS phosphorylation and NO production, associated with the inhibition of PLB phosphorylation, selectively constrains the ₂-AR/AC/PKA-induced Ca²⁺ response.

In rat cardiomyocytes, cPLA₂ has been identified as a target of MSK1 in response to purinergic agonists stimulation (43, 44). We report for the first time that ₂-AR agonists trigger phosphorylation of MSK1 that we find associated within caveolin-3/SR platforms.

Caveolae constitute a unique endocytic and exocytic compartment of the cell membrane, capable of importing molecules, delivering them to specific locations within the cell, and compartmentalizing a variety of signaling activities (41). Caveolae are a site of Ca²⁺ storage and entry into the cell. Our study provides evidence for ₂-AR-induced translocation of the cPLA₂ to platforms constituted of caveolae tightly connected to SR membranes. PLA₂ activity has been implicated in constitutive membrane trafficking (45); however, only one recent study reported the presence of the cPLA₂ in caveolin 1-enriched membrane fractions isolated from hippocampal preparations (46). Most caveolin-interacting proteins identified so far contain a caveolin-binding motif located within their enzymatically active catalytic site (41). As a matter of fact, such a caveolin-binding motif is present within the catalytic domain of the cPLA₂. Nevertheless, up to date, activation of cPLA₂ has been essentially associated with its Ca²⁺-dependent translocation from the cytosol to intracellular membranes, including perinuclear, endoplasmic, or SR membranes, but not to plasma membranes (47). We observe that treatment with AACOCF₃ abrogates the association of the cPLA₂ to caveolin-3/SR-platforms. One possible explanation relies on recent data showing that the cPLA₂ catalytic domain modulates membrane association and membrane residence time of the enzyme (48). Because AACOCF₃ is expected to target the catalytic site of the enzyme, competing with endogenous substrates, it might also affect interaction between caveolin-3 or SR membranes and cPLA₂. This study describes the cosedimentation of caveolae markers (caveolin-3 and eNOS) with SR markers (PLB and SERCA 2a), suggesting the possible close interactions between the two types of membranes in cardiomyocytes. It is noteworthy that SR Ca²⁺ uptake is reported to take place mainly in the longitudinal SR (away from caveolae), which contains the highest SERCA 2a density (49). In contrast, most of the SR Ca²⁺ release would occur in proximity of the terminal cisternae (junctional and corbular SR), close to the T-tubules (49), where ryanodine receptors are concentrated (50). We provide confocal microscopic support for the major distribution of the SERCA 2a along the longitudinal SR and around the nucleus in adult rat cardiomyocytes. However, our experiments also demonstrate the close vicinity of a pool of SERCA 2a with caveolin-3-enriched membranes. These results are in agreement with the confocal microscopic study of Vangheluwe et al. (27), in adult mice cardiomyocytes, describing a close vicinity between SERCA and ryanodine receptor molecules, in an area close to the T-tubules. A similar localization of SERCA has been previously reported by Greene et al. (42). Interestingly, recent ultrastructural studies also describe the vicinity of the SR membranes with caveolae in airway smooth muscle cells (51), and caveolae are proposed to provide a platform of interaction between SR and plasmalemmal ion channels (52).

This study suggests that all components of the ₂-AR-cAMP pathway are located in caveolin-3/SR-interacting platforms where they can also be regulated by other signaling pathways, such as cPLA₂. The potentiating effect of AACOCF₃, which neutralizes cPLA₂ action, seems to rely on phosphorylating of target proteins already present inside such platforms rather than on a recruitment of effectors toward platforms. It is noteworthy that caveolae have been reported to retain eNOS in the inactive state, because eNOS activity is inhibited by binding to the caveolin scaffold domain. However, our results identify the phosphorylated form of eNOS in the caveolin-3-enriched membrane fraction, which argues for its activation inside caveolae. In fact, parallel imaging studies show that adult rat cardiomyocytes treated with AACOCF₃ plus zinterol produce NO.

Our findings that ₂-AR-induced cPLA₂ activation impairs ₂-AR-induced NO release agree with the previously reported inhibition of NO release by AA in PC12 cells (53). NO release, evoked by ₂-AR agonists in the presence of the cPLA₂ inhibitor, AACOCF₃, is associated with an augmented Ca²⁺ response. Data from the literature point out the complexity of the modulatory effects of NO on the contractile function. NO influences the positive -adrenergic inotropic effect in a bimodal fashion, depending not only on its concentration but also on that of catecholamines (54). And the group of Lipsius, in particular, reported an activatory role of NO on ₂-AR-mediated contractile effects (32, 33). Several mechanisms can underlie the potentiation of the Ca²⁺ response by NO, including inhibition of the cG_i-PDE that would favor cAMP pathways (55), and nitrosylation of the L-type Ca²⁺ channel (56) and the ryanodine receptor that would stimulate channel activities (57). Furthermore, through nitrosylation of enzymes of the respiratory complexes, NO enhances mechanoenergetic coupling (58). It should be noted that potentiation by NO of the ₂-AR/PKA-induced Ca²⁺ response, through the blockade of the cPLA₂ pathway, described herein in isolated adult rat cardiomyocytes, is not observed in cat atrial myocytes. In the latter, detectable NO production in response to zinterol does not require the blockade of cPLA₂ (32, 33).

Xiao and co-workers (35) previously identified PI3K as a downstream target of ₂-AR signaling, in adult rat cardiomyocyte. Our study highlights clear homology between ₂-AR/PI3K and ₂-AR/cPLA₂ pathways since they both confine and negate concurrent ₂-AR/G_s-mediated PKA signaling, without affecting ₁-AR-induced responses. PI3K inhibition, as well as cPLA₂ inhibition, enables the ₂-AR/PKA signaling to reach and to phosphorylate intracellular substrates such as phospholamban, and markedly enhance the ₂-AR-positive contractile response in cardiac myocytes. Likewise, both potentiating effects are mediated through a PTX-sensitive G protein and are not accompanied by an increase in cAMP formation. Because of their similar impact on ₂-AR/PKA signaling, a link between the two mechanisms cannot be ruled out. One hypothesis would be that both pathways share a common effector. One of the potential targets of arachidonic acid might be the protein phosphatase 5, a Ser/Thr phosphatase expressed in the heart, that is activated by lipids and calyculin A (59–61). Note that Xiao and co-workers have suggested that ₂-AR-induced PI3K activation resulted in the stimulation of a calyculin-sensitive protein phosphatase activity. However, a number of studies argue in favor of the divergence of cPLA₂ and PI3K signaling pathways. Thus, studies on transgenic mice have shown that ₂-AR signaling bifurcates at the level of two different PTX-sensitive G_i proteins, G_i2 and G_i3, leading to different effects depending of the G isoform. G_i2 takes an essential protective part in the chronic signaling of overexpressed ₂-AR, leading to prolonged survival and delayed cardiac pathology. In contrast, G₃ is supposed to mediate ₂-AR induced reduction of Ca²⁺ channel activity (8), potentially through the activation of the PI3K isoform, a downstream target of G_i3- signaling (32, 56). Knockout of the PI3K gene results in a net increase in cardiac contractility without impact on cardiomyocyte hypertrophy (35, 62). In contrast, the major role attributed to the cPLA₂ is a critical regulation of muscular cell size (16), and the knockout of the cPLA₂ gene produces cardiac hypertrophy without change in basal cardiac contractility. Studies performed in cardiomyocytes indicate that activation of PI3K stimulates rather than inhibits NO production (63, 64). In this report, we demonstrate that eNOS inhibition is an essential component of the cPLA₂ limiting action.

Our present study in adult rat ventricular cardiomyocytes further substantiate the major role in the heart of the ₂-AR/cPLA₂ pathway (65), beyond that of the ₂-AR/AC/PKA pathway, a role that we previously suspected from studies in human tissues (10) and embryonic chick cardiomyocytes (23, 66). It appears that the respective roles of each, ₂-AR/cPLA₂ and ₂-AR/AC/PKA pathways, depend on the species or the pathological state. Thus, in adult rat cardiomyocytes, the ₂-AR/AC/PKA pathway mediates the Ca²⁺ response triggered by ₂-AR agonists, and is constitutively depressed because of the concomitant activation of the ₂-AR/cPLA₂ pathway. In contrast, in embryonic chick heart cells, cPLA₂ activation exclusively mediates ₂-AR stimulation of [Ca²⁺] cycling and cell contraction, (23, 66). In human tissue, ₂-AR activates both AC and cPLA₂ activities, the ₂-AR/cPLA₂ pathway being favored under conditions of altered ₂-AR/AC/PKA signaling (10). Taken together, those data pose the question as to the contractile impact of the cPLA₂ in human heart, and its possible evolution in the course of heart failure or aging that both represent physiopathological situations associated with a degradation of the ₂-AR/AC coupling (1). Another remaining important question is whether activation of the cPLA₂ takes part in the cardiac protective effect of ₂-AR stimulation, with eNOS inhibition as an essential component of its limiting action on the AC/PKA pathway.

	FOOTNOTES

 
^* This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and The Fondation de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Unité INSERM 581, 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.

¹ The abbreviations used are: ₁-AR, ₁-adrenergic receptor; AC, adenylyl cyclase; FITC, fluorescein isothiocyanate; MES, 4-morpholineethanesulfonic acid; SERCA, sarcoplasmic reticulum Ca²⁺ pump; SR, sarcoplasmic reticulum; eNOS, endothelial nitric-oxide synthase; PLB, phospholamban; PKA, cAMP-dependent kinase; PLA₂, phospholipase A₂; PTX, pertussis toxin; PI3K, phosphatidylinositol 3-kinase; DAPI, 4',6-diamidino-2-phenylindole; DAF-FM (4-amino-5-methylamino-2',7'-difluorofluorescein.

	ACKNOWLEDGMENTS

 
We thank Dr. Wuytack for kindly providing SERCA 2a antibodies, Valérie Nicolas (Service d'imagerie confocale, IFR 75-ISIT, Chatenay-Malabry, France) for skillful assistance with confocal microscopy techniques, and G. Guellaen for permanent support.

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