(cid:1) 2 -Adrenergic Receptor Agonists Increase Intracellular Free Ca 2 Concentration Cycling in Ventricular Cardiomyocytes through p38 and p42/44 MAPK-mediated Cytosolic Phospholipase A 2 Activation*

We have recently reported that arachidonic acid mediates (cid:1) 2 -adrenergic receptor (AR) stimulation of [Ca 2 (cid:2) ] i cycling and cell contraction in embryonic chick ventricular cardiomyocytes (Pavoine, C., Magne, S., Sauvadet, A., and Pecker, F. (1999) J. Biol. Chem. 274, 628–637). In the present work, we demonstrate that (cid:1) 2 -AR agonists trigger arachidonic acid release via translocation and activation of cytosolic phospholipase A 2 (cPLA 2 ) and in- crease caffeine-releasable Ca 2 (cid:2) pools from Fura-2-loaded cells. We also show that (cid:1) 2 -AR agonists trigger a rapid and dose-dependent phosphorylation of both p38 and p42/44 MAPKs. Translocation and activation of cPLA 2 , as well as Ca 2 (cid:2) accumulation in sarcoplasmic reticulum stores sensitive to caffeine and amplification of [Ca 2 (cid:2) ] i cycling in response to (cid:1) 2 -AR agonists, were blocked by inhibitors of the p38 or p42/44 MAPK pathway (SB203580 and PD98059, respectively), suggesting a role of both MAPK subtypes in (cid:1) 2 -AR determined using the blot detection shows that, heart membranes can occur at 37 °C in response to supraphysi- ological elevation of [Ca 2 (cid:1) ] i or at basal [Ca 2 (cid:1) ] i following MAPK activation (see “Results” and “Discussion”). In the experimental procedures, cell incubations were terminated by the addition of ice-cold Ca 2 (cid:1) -free lysis buffer containing phosphatase inhibitors. It should be noted that the inclusion of 2 m M Ca 2 (cid:1) (no EGTA) or its absence (2 m M EGTA added) in the ice-cold lysis did not affect cPLA 2 recovery in the 100,000 (cid:3) g fractions when analyzed by Western blotting. This suggested that (i) there is a stable binding of cPLA 2 to membranes that is not readily reversed by chelating Ca 2 (cid:1) at 4 °C in the presence of phosphatase inhibitors, and (ii) inclusion of Ca 2 (cid:1) in the ice-cold lysis buffer did not promote an artificial redistribution of the cytosolic enzyme toward the membranes. Immunofluorescence antibodies against

␤ 2 -Adrenergic receptor (AR) 1 stimulation improves cardiac contractile function (for a review, see Ref. 1). The ␤ 2 -AR-mediated signaling system plays a critical role in the regulation of cardiac performance, more particularly in the failing and aged heart, where there is selective down-regulation of ␤ 1 -ARs. ␤ 2 -AR agonists are mostly known as activators of adenylyl cyclase via coupling to G s (1)(2)(3)(4). However, the role of cAMP in the functional response to ␤ 2 -AR stimulation is controversial, and there is accumulating evidence that ␤ 2 -AR agonists could modulate cardiac excitation-contraction coupling via a cAMPindependent mechanism (1). We have recently identified an alternative pathway relaying the positive inotropic effect of ␤ 2 -AR agonists in embryonic chick heart cells (5). In these cells, stimulation of ␤ 2 -ARs triggers the production of arachidonic acid (AA) via a pertussis toxin-sensitive G protein pathway. We also demonstrated that AA induces 45 Ca 2ϩ accumulation in sarcoplasmic reticulum (SR) stores sensitive to caffeine, a mechanism likely to support positive inotropy (6). The ␤ 2 -ARelicited contractile effect is independent of adenylyl cyclase activation and cAMP production and totally relies on AA release. Although inhibition of the ␤ 2 -AR-induced responses by AACOCF 3 suggested the role of a Ca 2ϩ -dependent cytosolic phospholipase A 2 (cPLA 2 ) as the effector of ␤ 2 -AR, direct evidence for the coupling of ␤ 2 -AR to cPLA 2 activation remained to be established.
Phospholipase A 2 comprises a large family of enzymes that hydrolyze the sn-2-fatty acyl ester bonds of membranous phospholipids, leading to the liberation of free fatty acids including AA. PLA 2 enzymes are classified according to localization (extracellular versus intracellular), sequence homology, and biochemical characteristics (7). Extracellular PLA 2 enzymes, also referred as to secreted PLA 2 (sPLA 2 ) enzymes, represent a growing family of enzymes with five distinct mammalian sPLA 2 enzymes already identified (8). To date, four intracellular PLA 2 sequences have been reported: two Ca 2ϩ -dependent PLA 2 enzymes (cPLA 2 -␣ and the recently identified cPLA 2 -␤) (9,10) and two Ca 2ϩ -independent PLA 2 enzymes (iPLA 2 and cPLA 2 -␥) (11,12). Of particular interest is the Ca 2ϩ -dependent cPLA 2 -␣ (referred as to cPLA 2 below), which plays an essential role in hormone-induced AA release (13) with major implications in reproductive function and inflammation, as confirmed recently by studies using cPLA 2 -deficient mice (14,15). cPLA 2 is characterized by a molecular mass of 85 kDa, activation by low (micromolar) concentrations of calcium, selectivity for arachidonyl in the sn-2-position of phospholipids, and sensitivity to inhibitors such as AACOCF 3 and methyl arachidonyl fluorophosphate (MAFP) (16,17). cPLA 2 is fully activated by both phosphorylation and increases in intracellular calcium, which drive its translocation from the cytosol to membranes in a process utilizing a Ca 2ϩ -dependent phospholipid-binding domain (C2 domain) in the N-terminal region of the enzyme. In a variety of cell types, phosphorylation of cPLA 2 is achieved by p42/44 mitogen-activated protein kinases (MAPKs) and/or the MAPK homolog p38 (16,18).
MAPKs are a family of Ser/Thr kinases that comprises the extracellular signal-regulated kinases (ERKs or p42/44 MAPKs), the c-Jun N-terminal kinases (JNKs), and p38 MAPKs (for a review, see Ref. 19). In cardiac myocytes, the ERK cascade is activated principally by G protein-coupled receptor agonists and peptide growth factors. JNKs and p38 MAPKs are stimulated by hyperosmotic shock, hypoxia/reoxygenation, reactive oxygen species, and mechanical stress, but also by G protein-coupled receptors such as endothelin-1, phenylephrine (␣-AR agonist), and angiotensin II (for a review, see Ref. 20). There is considerable evidence that all MAPKs participate in the long-term myocyte hypertrophic response triggered by endothelin-1, phenylephrine, and phorbol 12myristate 13-acetate (PMA). Thus, the literature clearly demonstrates the immense potential significance of the regulation of MAPK pathways in the myocardium with respect to its reactions to pathological stresses (e.g. hypoxia, ischemia, reperfusion injury, hypertension, and inflammatory diseases). In contrast, the participation of MAPKs in cardiac physiological intracellular signaling is poorly documented.
In this study, we show that, in embryonic chick heart cells, ␤ 2 -AR stimulation triggers cPLA 2 translocation to the membranes, AA release, Ca 2ϩ accumulation in SR stores sensitive to caffeine, and amplification of [Ca 2ϩ ] i cycling. We also demonstrate that ␤ 2 -AR-induced responses rely on activation of both p42/44 and p38 MAPKs. This study highlights the participation of MAPKs in the positive inotropic effect elicited by ␤ 2 -AR agonists. This MAPK/cPLA 2 pathway is selective for ␤ 2 -AR stimulation and is not involved in ␤ 1 -AR-induced responses.
Fura-2 Loading and [Ca 2ϩ ] i Imaging-Cells were plated on plastic dishes, the bottoms of which were replaced with glass coverslips coated with laminin (1 g/ml), and were incubated at 37°C in humidified 5% CO 2 and 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 MgCl 2 , and 2 mM CaCl 2 ) and were incubated for 20 min at 25°C with 1.5 M Fura-2/AM in the presence of 1 mg/ml bovine serum albumin to improve Fura-2 dispersion and to facilitate cell loading. Cells were then washed two times with saline buffer B 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 (5,6,21,22) according to the procedure of Donnadieu et al. (23). Under usual loading conditions, the average intracellular concentration of Fura-2 was 15 M. Ca 2ϩ imaging was essentially performed as described by Sauvadet et al. (6,21). Field electrical stimulation (square waves, 10-ms duration, amplitude 20% above threshold, and 0.5 Hz) was supplied through a pair of platinum electrodes connected to the output of a HAMEG stimulator. MAPK inhibitors and/or selective ␤-AR antagonists were added 1 h or 10 min, respectively, before application of selective ␤-AR agonists.
To evaluate the caffeine-releasable Ca 2ϩ pool, electrical stimulation was omitted. MAPK or PLA 2 inhibitors were added 1 h or 10 min, respectively, before an additional 10-min preincubation with zinterol. Caffeine was applied a few seconds after initiation of image recordings.
Phospholipase A 2 Assay-Embryonic chick heart cells (5 ϫ 10 5 cells/ ml), suspended in buffer A supplemented with 5% fetal calf serum, were plated in 100-mm plates and incubated in humidified 5% CO 2 and 95% air at 37°C. After 24 h, the culture medium was changed to serum-free buffer A; and 24 h later, cells were exposed to various agonists and/or enzymatic inhibitors diluted in buffer A and incubated for the indicated periods of time at 37°C. Incubation was stopped by the removal of the medium, two successive washes with ice-cold phosphate-buffered saline (PBS), and the addition of ice-cold buffer C (40 mM Tris (pH 7.5), 250 mM sucrose, 1 mM EDTA, and protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 10 mM Na 4 P 2 O 7 , 200 M Na 3 VO 4 , and 50 mM NaF)). Cells were scraped and disrupted by three repeated freeze-thaw cycles, followed by sonication. Cell sonicates were transferred to microcentrifuge tubes and centrifuged at 1000 ϫ g for 10 min in a Sigma Model 1k15 centrifuge at 4°C. The 1000 ϫ g supernatant was submitted to a 100,000 ϫ g centrifugation for 40 min at 4°C.
Phospholipase A 2 activity in 100,000 ϫ g pellet and supernatant fractions was assessed by measuring the release of radiolabeled arachidonic acid from the sn-2-position of 1-stearoyl-2-[1-14 C]arachidonylphosphatidylcholine (PC). The reaction was carried out with 30 -50 g of proteins from the 100,000 ϫ g supernatant or pellet fraction and incubated for 30 min at 37°C in a 200-l final volume of buffer D (40 mM Tris (pH 7.5), 100 mM NaCl, 2 mM CaCl 2 , 50 mM NaF, 200 M Na 3 VO 4 , 10 mM Na 4 P 2 O 7 , and 1 mg/ml fatty acid-free bovine serum albumin) with or without inhibitors of iPLA 2 (10 M HELSS), sPLA 2 (2 mM DTT), and/or cPLA 2 (10 M AACOCF 3 ) and [ 14 C]PC (2 M, 1.5-2 ϫ 10 5 cpm/assay) substrate vesicles freshly prepared as follows. [ 14 C]PC (18 -24 nmol, 4.5-6 ϫ 10 6 cpm) was dried under liquid nitrogen, resuspended in 750 l of 100 mM Tris (pH 7.5) and 100 mM NaCl, and sonicated 3 ϫ 5 min at 4°C before addition to the assay medium at 25 l. The reaction was terminated by adding 800 l of Dole's reagent (32% isopropyl alcohol, 67% n-heptane, and 1% of 1 N H 2 SO 4 ) and 0.1 mg of unlabeled arachidonic acid. After vortexing, samples were centrifuged at 3000 ϫ g for 2 min at 4°C, and 400 l of the upper phase was mixed with silicic acid (50 mg) before centrifugation at 3000 ϫ g for 2 min at 4°C. The supernatant was collected and submitted to a second separation step on silicic acid. Following centrifugation at 3000 ϫ g for 2 min at 4°C, the supernatant was added to 10 ml of Ready-Solv solution, and radioactivity was counted in a liquid scintillation counter. Assays for cPLA 2 activity were routinely performed in the presence of both HELSS and DTT. Results are expressed in disintegrations/min since specific activity in the assays depended on the unknown endogenous content of phospholipids. It should be noted that, due to the preferential hydrolysis of endogenous phospholipids compared with exogenously added radiolabeled PC, cPLA 2 activity evaluated in the 100,000 ϫ g pellet fraction was probably underestimated compared with cPLA 2 activity in the 100,000 ϫ g supernatant fraction.
[ 3 H]Arachidonic Acid Release-Embryonic chick heart cells (5 ϫ 10 5 cells/ml), suspended in buffer A supplemented with 5% fetal calf serum, were plated in 24-well plates, left for 24 h in humidified 5% CO 2 and 95% air at 37°C, and then incubated with 1.5 Ci/ml [ 3 H]AA (6.75 nM). After 24 h, [ 3 H]AA release was determined by stimulating prelabeled cells in medium containing 0.2% fatty acid-free bovine serum albumin and counting total radioactivity in the cell medium as previously described (5). Bovine serum albumin served to trap the released AA (24).
Preparation of Cell Lysates for Study of cPLA 2 and MAPK Phosphorylation-Embryonic chick heart cells (5 ϫ 10 5 cells/ml), suspended in buffer A supplemented with 5% fetal calf serum, were plated in 60-mm plates and incubated in humidified 5% CO 2 and 95% air at 37°C. After 24 h, the culture medium was changed to serum-free buffer A. 24 h later, cells were exposed to various agonists and/or enzymatic inhibitors diluted in buffer A and incubated for various periods of time at 37°C. Incubation was terminated by the removal of the medium and the addition of ice-cold PBS. Cells were washed and resuspended in ice-cold lysis buffer (50 mM HEPES (pH 7.4), 0.5% (v/v) Nonidet-P40, 10% (v/v) glycerol, 135 mM NaCl, 1 mM EGTA, 10 mM NaF, 10 mM vanadate, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 2 g/ml aprotinin, 1 g/ml pepstatin, 40 mM ␤-glycerophosphate, and 100 M DTT). Cells were scraped and allowed to lyse for 1 h at 4°C. In the first series of experiments (cPLA 2 study; see Immunoblot Analysis of the Phosphorylation State of p38 and p42/44 MAPKs-Electrophoresis was performed on 10% SDS-polyacrylamide gel (25 mA/gel). Proteins were transferred to Protran nitrocellulose transfer membranes (Schleicher & Schü ll) by electroblotting using Tris/ glycine/SDS buffer containing 20% methanol (120 mA for 1 h at 4°C). Protein transfer was evaluated by staining the gel with Coomassie Blue. Equal loading of proteins in each lane was checked by Ponceau red staining of the membrane. The nitrocellulose blots were agitated for 30 min at room temperature in TBST (10 mM Tris-HCl (pH 8), 150 mM NaCl, and 0.05% Tween 20) and then for 1 h in TBST supplemented with 5% nonfat dry milk prior to overnight incubation with primary antibodies against phospho-MAPKs (1:2000 dilution) in TBST supplemented with 5% nonfat dry milk at 4°C. Membranes were washed three times with TBST and incubated with peroxidase-conjugated swine anti-rabbit IgG (1:1000 dilution) for 1 h at room temperature in TBST containing 5% nonfat dry milk. Membranes were washed three times with TBST, and the peroxidase activity was determined using the enhanced chemiluminescence Western blot detection system (ECL, Amersham Pharmacia Biotech).
Immunoblot Analysis of the Phosphorylation State of Both cPLA 2 and MAPKs-Electrophoresis was performed on 24-cm 7.5% SDS-polyacrylamide gel (45 mA/gel). Proteins were transferred to polyvinylidene difluoride (PVDF) microporous membrane (Millipore Corp.) by electroblotting using Tris/glycine/SDS buffer containing 20% methanol (40 V for 16 h at 4°C). Protein transfer was evaluated by staining the gel with Coomassie Blue. Equal loading of proteins in each lane was checked by Ponceau red staining of the membrane. The PVDF blots were agitated overnight at room temperature in TBST supplemented with 5% nonfat dry milk and then in TBST supplemented with 5% nonfat dry milk containing primary antibodies against cPLA 2 (1:1000 dilution) overnight at 4°C. Membranes were washed three times with TBST and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10,000 dilution) for 1.5 h at room temperature in TBST containing 5% nonfat dry milk. Membranes were washed three times with TBST, and the alkaline phosphatase activity was determined using the enhanced chemifluorescence Western blot detection system (ECF, Amersham Pharmacia Biotech). It is noteworthy that the amino acid sequence of chicken cPLA 2 differs from the sequences of the human and mouse enzymes by 20 -30% and that immunochemical detection of chicken cPLA 2 is rather difficult due to the lack of antibodies specifically raised against the chicken enzyme.
Following cPLA 2 detection, MAPK phosphorylation was evaluated on the same PVDF blots, which had been treated for 30 min in 100% methanol and washed three times with TBST to remove the ECF reaction precipitate. PVDF blots were incubated in stripping buffer (100 mM ␤-mercaptoethanol, 2% SDS, and 62.4 mM Tris (pH 6.7)) for 45 min at 50°C with agitation and washed twice with TBST at room temperature. After 24 h at room temperature in TBST supplemented with 5% nonfat dry milk, PVDF blots were incubated overnight with primary antibodies against phospho-MAPKs (1:2000 dilution) in TBST supplemented with 5% nonfat dry milk at 4°C. Membranes were washed three times with TBST and incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:10,000 dilution) for 1.5 h at room temperature in TBST containing 5% nonfat dry milk. Membranes were washed three times with TBST, and the alkaline phosphatase activity was determined using the ECF Western blot detection system.
This study shows that, in embryonic chick heart cells, cPLA 2 trans-location to membranes can occur at 37°C in response to supraphysiological elevation of [Ca 2ϩ ] i or at basal [Ca 2ϩ ] i following MAPK activation (see "Results" and "Discussion"). In the experimental procedures, cell incubations were terminated by the addition of ice-cold Ca 2ϩ -free lysis buffer containing phosphatase inhibitors. It should be noted that the inclusion of 2 mM Ca 2ϩ (no added EGTA) or its absence (2 mM EGTA added) in the ice-cold lysis buffer did not affect cPLA 2 recovery in the 100,000 ϫ g fractions when analyzed by Western blotting. This suggested that (i) there is a stable binding of cPLA 2 to membranes that is not readily reversed by chelating Ca 2ϩ at 4°C in the presence of phosphatase inhibitors, and (ii) inclusion of Ca 2ϩ in the ice-cold lysis buffer did not promote an artificial redistribution of the cytosolic enzyme toward the membranes. Immunofluorescence Microscopy-Embryonic chick heart cells (3 ϫ 10 5 cells/ml), suspended in buffer A supplemented with 5% fetal calf serum, were plated on plastic chamber slides (Lab-Tek, Nunc) coated with laminin (1 g/ml) and incubated in humidified 5% CO 2 and 95% air at 37°C. After 24 h, the culture medium was changed to serum-free buffer A. After a 1-h pretreatment with or without MAPK inhibitors and/or a 10-min preincubation with or without selective ␤-AR antagonists, cells were incubated for the indicated periods of time with or without selective ␤-AR agonists. Cells were washed twice briefly with PBS and allowed to dry. Cells were fixed for 10 min at room temperature in acetone, washed with PBS, and then permeabilized in 0.2% Triton X-100 and PBS for 15 min at room temperature. After three brief washes, blocking was performed in PBS containing 10% sheep serum for 30 min at 37°C. Cells were incubated with anti-cPLA 2 antibody (1:250 dilution) overnight at 4°C and washed three times for 5 min with PBS at room temperature. Incubation with Cy3-conjugated anti-rabbit antibody (1:500 dilution) was then performed for 1 h at room temperature in the dark. The cells were washed 3 ϫ 10 min with PBS and rinsed for 5 min with water. 10 l of Vectashield mounting medium was applied to the cell surface, and coverslips were mounted. Controls were carried out by replacing the primary antibody with PBS. Slides were viewed by fluorescence microscopy on a Zeiss Axoplan microscope (magnification ϫ 630).
Statistics-Results are expressed as means Ϯ S.E. of n experiments and were analyzed by unpaired Student's t test, one-way analysis of variance, or non-parametric analysis of variance, as appropriate, with p Ͻ 0.05 considered to be significant.

RESULTS
Distribution of PLA 2 Activities in the 100,000 ϫ g Fractions of Embryonic Chick Ventricular Cells-We measured PLA 2 activities in the 100,000 ϫ g fractions prepared from embryonic chick ventricular cells. We used the preferential substrate of cPLA 2 , 1-stearoyl-2-[1-14 C]arachidonylphosphatidylcholine, in the absence or presence of specific inhibitors of the different PLA 2 isoforms, viz. DTT, HELSS, and AACOCF 3 , to block the sPLA 2 , iPLA 2 , and cPLA 2 activities, respectively. In the absence of inhibitors, total PLA 2 activity in the 100,000 ϫ g supernatant fraction was 4 -7 times higher than in the 100,000 ϫ g pellet fraction. Thus, we used the 100,000 ϫ g supernatant fraction to determine optimal conditions for the measurement of the AACOCF 3 -sensitive PLA 2 activity (cPLA 2 ). As shown in Fig. 1A, in the absence of inhibitors, the AACOCF 3 -sensitive PLA 2 activity (cPLA 2 ) represented 73% of the total PLA 2 activity. The addition of the sPLA 2 inhibitor DTT strongly and selectively improved the AACOCF 3 -sensitive cPLA 2 activity by 200%, presumably through stabilization of this oxidation-sensitive enzyme (25). In contrast, the cPLA 2 activity was not significantly affected upon addition of HELSS.
In the presence of DTT and HELSS, cPLA 2 assay was linear with respect to time and protein (data not shown) and displayed Ca 2ϩ -dependent characteristics of cPLA 2 , with maximal activation obtained at 1 M Ca 2ϩ and half-maximal stimulation occurring at 0.3 M Ca 2ϩ (Fig. 1B). This specific assay for cPLA 2 was validated in both the 100,000 ϫ g supernatant and pellet fractions of embryonic chick heart cells by the total blockade of PLA 2 activity upon addition of 10 M AACOCF 3 (Fig. 1C).
␤ 2 -AR Agonists Activate cPLA 2 in Embryonic Chick Heart Cells-Embryonic chick heart cells were exposed to ␤ 2 -AR stim-ulation before preparation of 100,000 ϫ g subcellular fractions and subsequent measurement of cPLA 2 activity in the presence of DTT and HELSS. Zinterol, a specific partial ␤ 2 -AR agonist, induced a dose-dependent decrease in cPLA 2 activity in the 100,000 ϫ g supernatant fraction, with a maximal effect observed at 30 nM zinterol ( Fig. 2A). In parallel, 30 nM zinterol elicited an increase in cPLA 2 activity in the 100,000 ϫ g pellet fraction ( Fig.  2A). Zinterol effects on cPLA 2 activity were reproduced with a distinct ␤ 2 -AR stimulus, achieved with isoproterenol, a nonselective but complete ␤-agonist, added together with CGP 20712A, a selective ␤ 1 -AR antagonist ( Fig. 2A). AACOCF 3 abrogated the effects of ␤ 2 -AR agonists on cPLA 2 activity (data not shown). Experiments performed in embryonic chick heart cells prelabeled with [ 3 H]AA indicated that zinterol stimulation resulted in a rapid (detected within 30 s) and sustained AA release over the 15 min examined (Fig. 2B).
Zinterol-induced translocation of cPLA 2 was examined by immunofluorescent staining of myocytes using anti-cPLA 2 antibody (Fig. 3). Unstimulated cells showed diffuse fluorescence throughout the cell (Fig. 3B). This staining specifically represented cPLA 2 as demonstrated by the negative staining pattern obtained when the anti-cPLA 2 primary antibody was omitted (Fig. 3A). In contrast, after stimulation with 30 nM zinterol, the FIG. 1. PLA 2 activities in the 100,000 ؋ g subcellular fractions of embryonic chick ventricular cells. A, effect of DTT and HELSS on the AACOCF 3 -sensitive PLA 2 activity in the 100,000 ϫ g supernatant fraction; B, Ca 2ϩ -dependent stimulation of PLA 2 activity measured in the presence of DTT and HELSS in the 100,000 ϫ g supernatant fraction; C, AACOCF 3 -sensitive PLA 2 activity measured in the presence of DTT and HELSS. The 100,000 ϫ g supernatant and pellet fractions of embryonic chick ventricular cells were prepared, and PLA 2 activity was measured using 1-stearoyl-2-[1-14 C]arachidonylphosphatidylcholine as substrate as described under "Experimental Procedures." The reaction was carried out with 30 -50 g of proteins from the 100,000 ϫ g supernatant or pellet fraction. Assays were performed in the absence or presence of 10 M AACOCF 3 and/or 2 mM DTT and/or 10 M HELSS as indicated. In B, free Ca 2ϩ concentrations were prepared using Ca 2ϩ / EGTA buffers and determined by measurement of Fura-2 fluorescence. PLA 2 activity is expressed in total disintegrations/min (A and C) or as a percentage of maximal stimulation (B; with 100% maximal stimulation corresponding to 10,240 Ϯ 275 total dpm). Total disintegrations/ min corresponded to 450, 870, and 995 g of total proteins in supernatants (A-C, respectively) and 660 g of total proteins in the pellet (C). Total [ 14 C]PC radioactivity added to assays was 150,000 dpm (A) and 200,000 dpm (B and C). Values are the means Ϯ S.E. of triplicate determinations from a typical experiment that was repeated twice. *, p Ͻ 0.01. staining appeared to be concentrated at a perinuclear region of the cells (Fig. 3, C-F). These results are in agreement with a zinterol-induced redistribution of cPLA 2 from the cytoplasm to the nuclear and/or SR membranes, both networks being tightly associated in our cells. cPLA 2 translocation was almost maximal as soon as 30 s after stimulation with zinterol (Fig. 3C) and persisted during the first 30-min period examined (Fig. 3F). In keeping with these results, the linear kinetics of zinterol-induced AA release shown in Fig. 2 also argued for maximal activation of cPLA 2 as soon as 30 s after zinterol application. It should be noted that immunofluorescent staining of cPLA 2 in zinterol-treated cells appeared to be frequently and consistently more intense than in untreated cells. This was most likely due to a greater loss of soluble cPLA 2 than of membranebound cPLA 2 throughout the staining procedure. A similar interpretation has already been proposed in at least two other studies (see Ref. 26). Alternatively, as proposed by Schievella et al. (26), binding of cPLA 2 to membrane structures might result in a better exposure of the epitope for the primary antibody, allowing a more efficient antibody binding.
p38 and p42/44 MAPKs Are Phosphorylated upon ␤ 2 -AR Stimulation-Since phosphorylation of p38 and p42/44 MAPKs is a prerequisite for MAPK activation, we evaluated the phosphorylation state of either p38 or p42/44 MAPK in response to ␤ 2 -AR stimulation using specific anti-phospho-MAPK antibodies. Zinterol increased the phosphorylation of both p38 and p42/44 MAPKs in a dose-dependent manner, with maximal stimulation of p42/44 and p38 MAPKs occurring at 3 and 30 nM, respectively (Fig. 4A). The phosphorylation of both MAPK subtypes in response to 30 nM zinterol was totally prevented when cells were preincubated for 10 min with the selective ␤ 2 -AR antagonist ICI 118551 at 100 nM prior to zinterol application, suggesting that zinterol induction of MAPK phosphorylation results from a strict ␤ 2 -AR agonist effect (Fig. 4B).
Zinterol Does Not Induce Detectable Phosphorylation of cPLA 2 at Ser 505 -Immunoblot analysis of whole cell homogenates using anti-cPLA 2 antibody revealed a single 80-kDa band in control untreated cells (Fig. 5). An additional band with decreased electrophoretic mobility, characteristic of cPLA 2 phosphorylated at Ser 505 , was detected after treatment with 100 nM PMA for 2.5 min (Fig. 5). In contrast, zinterol stimulation did not impact on cPLA 2 gel mobility (Fig. 5). It should be noted that analysis of the same homogenates with anti-phospho-MAPK antibodies showed that 30 nM zinterol induced the phosphorylation of both p38 and p42/44 MAPKs. Phosphorylation of p38 and p42/44 MAPKs was detected as soon as 30 s after zinterol addition; was maximal after 2.5 and 5 min, respectively; and remained detectable over the 15-min period examined (Fig. 5). PMA also induced phosphorylation of both MAPK subtypes (Fig. 5).
␤ 2 -AR Stimulation Induces cPLA 2 Activation through a p38 and p42/44 MAPK Pathway-We next used SB203580, the p38 MAPK inhibitor acting at the catalytic site of the p38 MAPK enzyme (27), and PD98059, the MEK kinase inhibitor reported to impair p42/44 MAPK phosphorylation and activation (28), to investigate the impact of the p38 and p42/44 MAPK pathways on zinterol-induced AA release and cPLA 2 translocation. As shown in Fig. 6, the presence of either 1 M PD98059 or 1 M SB203580 totally blocked zinterol-induced AA release. Similarly, treatment of cardiomyocytes with either 1 M PD98059 or 1 M SB203580 did not affect the pattern of cPLA 2 in unstimulated cells (Fig. 7, C and E compared with A), but prevented its translocation in response to zinterol (D and F compared with B). Taken together, these results demonstrate that, upon ␤ 2 -AR challenging, both p38 and p42/44 MAPKs play a critical role upstream of cPLA 2 translocation and activation.
To examine the contribution of [Ca 2ϩ ] i in zinterol-induced cPLA 2 translocation, cells were pretreated for 10 min with 1 mM EGTA. Incubation of the cells with EGTA did not affect the . Cell lysates were prepared and separated (50 g of proteins) on 10% SDS-polyacrylamide gel. Proteins were transferred to a nitrocellulose sheet and probed with rabbit polyclonal antibodies raised against human phospho-p38 MAPK or human phospho-p42/44 MAPK as described under "Experimental Procedures." pattern of cPLA 2 in unstimulated cells (Fig. 7, G compared with  A). In contrast, it impaired zinterol-induced translocation of cPLA 2 (Fig. 7, H compared with B), suggesting that preservation of basal [Ca 2ϩ ] i is a crucial factor for redistribution of the enzyme in response to zinterol. ␤ 2 -AR Stimulation of [Ca 2ϩ ] i Cycling Requires p38 and p42/44 MAPK Activation and Relies on Zinterol-induced Calcium Loading of Caffeine-sensitive Stores-As previously demonstrated (5), zinterol stimulation triggered a rapid increase in the amplitude of [Ca 2ϩ ] transients of electrically stimulated embryonic chick ventricular cells (Fig. 8B), which was totally blunted upon addition of the cPLA 2 inhibitor AACOCF 3 (Fig.  8C). We show here that the zinterol effect on [Ca 2ϩ ] i cycling was unaffected in the presence of HELSS, a selective iPLA 2 blocker (Fig. 8F). Thus, our present data rule out the possible participation of iPLA 2 and confirm the selective role of cPLA 2 in ␤ 2 -AR-induced responses. The aim of the next experiments was to evaluate the role of p38 and p42/44 MAPKs in the effect of ␤ 2 -AR agonists on [Ca 2ϩ ] i cycling. Preincubation for 1 h with 1 M SB203580 (Fig. 8E), an inhibitor of p38 MAPK, or 1 M PD98059 (Fig. 8D), an inhibitor of the p42/44 MAPK pathway, totally blunted the zinterol effect. These results demonstrate that, in electrically stimulated cells, zinterol-induced stimulation of [Ca 2ϩ ] i cycling requires p38 and p42/44 MAPKs and selective cPLA 2 activation.
In the absence of electrical stimulation, it is noteworthy that zinterol was without effect on [Ca 2ϩ ] i (Fig. 8A). To evaluate the role of SR calcium stores as targets for zinterol action, we used caffeine, previously reported to increase the opening probability of the calcium release channels of the SR compartments (29). We examined the effect of zinterol on [Ca 2ϩ ] transients triggered by caffeine in Fura-2-loaded cells in the absence of electrical stimulation. The application of 10 mM caffeine produced a unique [Ca 2ϩ ] i transient (Fig. 9A). In contrast, the application of caffeine together with zinterol resulted in a train of [Ca 2ϩ ] i transients (Fig. 9B). These experiments demonstrate that, in the absence of electrical stimulation, zinterol action leads to Ca 2ϩ loading of caffeine-sensitive SR stores. Zinterol potentiation of caffeine-induced Ca 2ϩ mobilization from the SR was blunted in the presence of AACOCF 3 , PD98059, or SB203580 (Fig. 9, C-E, respectively), whereas it was insensitive to HELSS (Fig. 9F). Taken together, these results demonstrate that zinterol induces Ca 2ϩ loading of caffeine-sensitive SR stores through the activation of p38 and p42/44 MAPKs and  C, E, and G). After 10 min, cells were washed, fixed, permeabilized, and stained with rabbit anti-cPLA 2 antibody prior to incubation with Cy3-conjugated secondary antibody as described under "Experimental Procedures." Immunofluorescent staining was visualized by microscopy (magnification ϫ 630). cPLA 2 . It is noteworthy that Ca 2ϩ loading of SR stores induced by zinterol did not impact on basal cytosolic [Ca 2ϩ ] i in the absence of electrical stimulation (Fig. 8A) (Fig. 10C). In contrast, 100 nM isoproterenol, a ␤ 2 -AR stimulus, added together with 100 nM CGP 20712A, a selective ␤ 1 -AR antagonist, mimicked the effect of zinterol and induced translocation of the enzyme (Fig. 10B).
We previously demonstrated that, in embryonic chick ventricular cells, the ␤ 1 -AR-mediated increase in [Ca 2ϩ ] i cycling and contraction relies on a rise in intracellular cAMP via activation of adenylyl cyclase and is independent of AA production (5). In particular, ␤ 1 -AR stimulation does not trigger [ 3 H]AA release (5). However, as previously reported by others in adult rat cardiac myocytes, we observed that ␤ 1 -AR stimulation induced a rapid phosphorylation of p38 and p42/44 MAPKs in embryonic chick heart cells (data not shown). Next, imaging studies were performed to evaluate the role of p38 and p42/44 MAPKs in ␤ 1 -AR stimulation of [Ca 2ϩ ] i cycling compared with ␤ 2 -AR activation. We measured the amplitude of [Ca 2ϩ ] transients of cells incubated with 100 nM isoproterenol in the presence of either 100 nM ICI 118551, a selective ␤ 2 -AR antagonist, or 300 nM CGP 20712A, a selective ␤ 1 -AR antagonist. As expected, the addition of 1 M SB203580 and/or 1 M PD98059 reduced ␤ 2 -AR-mediated isoproterenol action (Fig. 11A). In contrast, blockade of the p38 and/or p42/44 MAPK pathway amplified ␤ 1 -AR-mediated isoproterenol action (Fig. 11B). These data indicate that ␤ 2 -AR-mediated effects on [Ca 2ϩ ] i cycling rely on p38 and p42/44 MAPK activation, in contrast to ␤ 1 -AR-mediated responses. DISCUSSION This study provides direct evidence for the role of cPLA 2 as a selective effector of ␤ 2 -AR in embryonic chick heart cells. We demonstrate a dose-dependent redistribution of cPLA 2 activity from the cytosol to membranes in response to low concentrations of zinterol, with the maximal effect being observed at 30 nM zinterol. This dose dependence correlates with our previous data on zinterol-induced AA release (5). At such doses, zinterol is unequivocally known to act through ␤ 2 -AR. Accordingly, zinterol-induced MAPK phosphorylation (this study) and [Ca 2ϩ ] i cycling stimulation (5) are totally blocked in the presence of 100 nM ICI 118551, a selective ␤ 2 -AR antagonist. Furthermore, zinterol effects are reproduced by a distinct ␤ 2 -AR stimulus, viz. the combination of isoproterenol and CGP 20712A, a selective ␤ 1 -AR antagonist.
Our results argue for the requirement of cPLA 2 activation in the ␤ 2 -AR stimulation of [Ca 2ϩ ] i cycling. We show that ␤ 2 -AR stimulation triggers a rapid translocation of cPLA 2 that results in an increase in cPLA 2 activity associated with the 100,000 ϫ g pellet subcellular fraction and subsequent release of AA, leading to SR Ca 2ϩ loading. The ␤ 2 -AR-induced responses (cPLA 2 translocation, AA release, and SR loading) are detected as soon as 30 s following the initiation of ␤ 2 -AR stimulation, in keeping with amplification of [Ca 2ϩ ] i cycling detected within 1 min.
Our results highlight the essential role of the p38 and p42/44 MAPK pathways in the regulation of cPLA 2 activity. Inhibitors of either the p38 or p42/44 MAPK pathway (SB203580 and PD98059, respectively) neutralize all zinterol-induced responses, viz. cPLA 2 translocation, induction of AA release, increase in SR Ca 2ϩ loading, and amplification of [Ca 2ϩ ] i cycling. Indeed, our results are the first to suggest that p38 and p42/44 MAPKs are key steps in the transduction of ␤ 2 -AR signaling and ␤ 2 -AR-mediated inotropic responses in embryonic chick heart cells. This study indicates that p38 and p42/44 MAPKs act independently and simultaneously to activate cPLA 2 and AA release. Similar dual MAPK activation has been reported in rat cardiomyocytes for cPLA 2 stimulation by ATP (30) and in human neutrophils for cPLA 2 activation by opsonized zymosan (31).
Our data indicate that the MAPK prerequisite concerns only ␤ 2 -AR-triggered pathways. ␤ 1 -AR-mediated effects on [Ca 2ϩ ] i cycling are rather amplified upon inhibition of the MAPK pathways. We have previously demonstrated that, in embryonic chick heart cells, as in myocytes of other species, ␤ 1 -AR-induced amplification of [Ca 2ϩ ] i cycling is mediated by cAMP and is neither associated with AA release nor sensitive to cPLA 2 blockers. However, we observed that ␤ 1 -AR stimulation elicits phosphorylation of p38 and p42/44 MAPKs in embryonic chick heart cells (data not shown). This suggests that MAPK activation by itself is not sufficient to induce cPLA 2 activation and that additional events are involved in ␤ 2 -AR-induced responses. An additional possibility is that the ␤ 1 -AR agonist second messenger, viz. cAMP, exerts a dominant-negative effect and mutes the activating effect of MAPK pathways on cPLA 2 . In fact, we have previously demonstrated that cAMP exerts a negative effect on the cPLA 2 /AA pathway in embryonic chick heart cells (5). Furthermore, inhibition of cPLA 2 activity upon protein kinase A-mediated phosphorylation has been reported in smooth muscle cells (32).
Several phosphorylation sites on cPLA 2 have been identified (33). Most studies have focused on MAPK-dependent phosphorylation of Ser 505 since it results in a characteristic decrease in electrophoretic mobility of the enzyme. The importance of Ser 505 phosphorylation in cPLA 2 activation appears to be cell type-and agonist-specific (for a review, see Ref. 34): although it is essential for Ca 2ϩ ionophore 4-bromo-A23187-induced AA release in Chinese hamster ovary cells overexpressing human cPLA 2 (26), it is not required for AA release from thrombinstimulated platelets (35). cPLA 2 activation by MAPKs, independent of Ser 505 phosphorylation, has been reported in mouse peritoneal macrophages and in human neutrophils (34). In fact, it has been shown that kinases downstream of MAPKs, MSK1