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INTRODUCTION |
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 Gs (1-4).
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 cAMP-independent
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 45Ca2+ accumulation in
sarcoplasmic reticulum (SR) stores sensitive to caffeine, a mechanism
likely to support positive inotropy (6). The
2-AR-elicited 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 AACOCF3 suggested the role of a
Ca2+-dependent cytosolic phospholipase
A2 (cPLA2) as the effector of
2-AR, direct evidence for the coupling of
2-AR to cPLA2 activation remained to be established.
Phospholipase A2 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. PLA2 enzymes are classified according to localization
(extracellular versus intracellular), sequence homology, and
biochemical characteristics (7). Extracellular PLA2
enzymes, also referred as to secreted PLA2
(sPLA2) enzymes, represent a growing family of enzymes with five distinct mammalian sPLA2 enzymes already identified
(8). To date, four intracellular PLA2 sequences have been
reported: two Ca2+-dependent PLA2
enzymes (cPLA2-
and the recently identified
cPLA2-) (9, 10) and two Ca2+-independent
PLA2 enzymes (iPLA2 and cPLA2-
)
(11, 12). Of particular interest is the
Ca2+-dependent cPLA2-
(referred as to cPLA2 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 cPLA2-deficient mice (14, 15).
cPLA2 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
AACOCF3 and methyl arachidonyl fluorophosphate
(MAFP) (16, 17). cPLA2 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
Ca2+-dependent phospholipid-binding domain (C2
domain) in the N-terminal region of the enzyme. In a variety of cell
types, phosphorylation of cPLA2 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 12-myristate 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 cPLA2
translocation to the membranes, AA release, Ca2+
accumulation in SR stores sensitive to caffeine, and amplification of
[Ca2+]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/cPLA2 pathway is selective for
2-AR stimulation and is not involved in
1-AR-induced responses.
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EXPERIMENTAL PROCEDURES |
Materials
Zinterol and CGP 20712A were kindly provided by Bristol-Myers
Squibb Co. and Ciba-Geigy (Basel, Switzerland), respectively. HELSS,
AACOCF3, PD98059, PMA, 4-bromo-A23187, and
arachidonic acid were purchased from BIOMOL Research Labs Inc.
(Plymouth Meeting, PA). Penicillin/streptomycin, trypsin, leupeptin,
aprotinin, nucleotides, (±)-isoproterenol, bovine serum albumin, sheep
serum, and ICI 118551 were obtained from Sigma (Saint-Quentin
Fallavier, France). SB203580 was purchased from France Biochem (Meudon,
France). Silicic acid and n-heptane were from Merck
(Chelles, France). Isopropyl alcohol was purchased by Carlo-Erba
(Val-de-Reuil, France). Fura-2/AM was from Molecular Probes, Inc.
(Interchim, Montluçon, France). Fetal calf serum, M199 medium,
and phosphate-buffered saline 2040 medium were from Life Technologies,
Inc. (Cergy Pontoise, France). 1-Stearoyl-2-[1-14C]arachidonyl-L-3-phosphatidylcholine
(53 mCi/mmol) was from Amersham Pharmacia Biotech (Les Ulis, France).
[5,6,8,9,11,12,14,15-3H]Arachidonic acid (180-240
Ci/mmol) was from PerkinElmer Life Sciences (Les Ulis). Vectashield
mounting medium was from Vector Labs, Inc. (Biovalley, Conches,
France). Rabbit antibodies against human phospho-p44/42
MAPK(Thr202/Tyr204) and human phospho-p38
MAPK(Thr180/Tyr182) were from New England
Biolabs Inc. (Ozyme, Saint-Quentin). Antibodies from
peroxidase-conjugated swine anti-rabbit IgG were purchased from Dako
(Trappes, France). Antibodies from alkaline phosphatase-conjugated goat
anti-mouse IgG + IgM were from Amersham Pharmacia Biotech. Antibodies
from Cy3-conjugated sheep anti-rabbit IgG were from Sigma. Rabbit
antibodies against human cPLA2 were kindly provided by Dr.
Tamayo (Genetics Institute, Cambridge, MA).
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 embryonic chick ventricles as previously
described (5, 6, 21, 22). Briefly, cells were dissociated by repeated cycles of trypsinization. The resulting cell suspension (5 × 105 cells/ml) was bubbled with 5% CO2 and 95%
air and kept at 4 °C in buffer A (M199 medium containing 0.1% (w/v)
NaHCO3, 0.01% (w/v) L-glutamine, and 0.1%
penicillin/streptomycin antibiotic solution) until used, for up to 5 days.
Fura-2 Loading and [Ca2+]i
Imaging--
Cells were plated on plastic dishes, the bottoms of which
were replaced with glass coverslips coated with laminin (1 µg/ml), and were incubated at 37 °C in humidified 5% CO2 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 MgCl2, and 2 mM
CaCl2) and were incubated for 20 min at 25 °C with 1.5 µM 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 µM. Ca2+ 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 Ca2+ pool, electrical
stimulation was omitted. MAPK or PLA2 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 A2 Assay--
Embryonic chick heart
cells (5 × 105 cells/ml), suspended in buffer A
supplemented with 5% fetal calf serum, were plated in 100-mm plates
and incubated in humidified 5% CO2 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 µg/ml leupeptin, 10 mM Na4P2O7, 200 µM Na3VO4, 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 × g for
10 min in a Sigma Model 1k15 centrifuge at 4 °C. The 1000 × g supernatant was submitted to a 100,000 × g centrifugation for 40 min at 4 °C.
Phospholipase A2 activity in 100,000 × g
pellet and supernatant fractions was assessed by measuring the release
of radiolabeled arachidonic acid from the sn-2-position of
1-stearoyl-2-[1-14C]arachidonylphosphatidylcholine (PC).
The reaction was carried out with 30-50 µg of proteins from the
100,000 × g supernatant or pellet fraction and
incubated for 30 min at 37 °C in a 200-µl final volume of buffer D
(40 mM Tris (pH 7.5), 100 mM NaCl, 2 mM CaCl2, 50 mM NaF, 200 µM Na3VO4, 10 mM
Na4P2O7, and 1 mg/ml fatty
acid-free bovine serum albumin) with or without inhibitors of
iPLA2 (10 µM HELSS), sPLA2 (2 mM DTT), and/or cPLA2 (10 µM AACOCF3) and [14C]PC (2 µM,
1.5-2 × 105 cpm/assay) substrate vesicles freshly
prepared as follows. [14C]PC (18-24 nmol, 4.5-6 × 106 cpm) was dried under liquid nitrogen, resuspended in
750 µl of 100 mM Tris (pH 7.5) and 100 mM
NaCl, and sonicated 3 × 5 min at 4 °C before addition to the
assay medium at 25 µl. The reaction was terminated by adding 800 µl
of Dole's reagent (32% isopropyl alcohol, 67% n-heptane,
and 1% of 1 N H2SO4) and 0.1 mg of
unlabeled arachidonic acid. After vortexing, samples were centrifuged
at 3000 × g for 2 min at 4 °C, and 400 µl of the
upper phase was mixed with silicic acid (50 mg) before centrifugation
at 3000 × 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 × 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
cPLA2 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, cPLA2 activity evaluated
in the 100,000 × g pellet fraction was probably
underestimated compared with cPLA2 activity in the
100,000 × g supernatant fraction.
[3H]Arachidonic Acid Release--
Embryonic chick
heart cells (5 × 105 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% CO2 and 95% air at
37 °C, and then incubated with 1.5 µCi/ml [3H]AA
(6.75 nM). After 24 h, [3H]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 cPLA2 and
MAPK Phosphorylation--
Embryonic chick heart cells (5 × 105 cells/ml), suspended in buffer A supplemented with 5%
fetal calf serum, were plated in 60-mm plates and incubated in
humidified 5% CO2 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 µg/ml
leupeptin, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 40 mM -glycerophosphate, and 100 µM DTT). Cells were scraped
and allowed to lyse for 1 h at 4 °C. In the first series of
experiments (cPLA2 study; see Fig. 5), whole cell
homogenates were used. In the second series of experiments (MAPK study;
see Fig. 4), cell homogenates were transferred to microcentrifuge tubes
and centrifuged at 20,000 × g for 15 min at 4 °C.
Samples of the 20,000 × g supernatant containing 50 µg of proteins (MAPK study; see Fig. 4) or whole cell homogenates
containing 100 µg of proteins (cPLA2 study; see Fig. 5)
were taken, lyophilized, resuspended in Laemmli loading buffer, frozen
in liquid nitrogen, and stored at 
80 °C. Samples were boiled for 5 min prior to electrophoresis.
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 & Schüll)
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
cPLA2 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 cPLA2 (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 cPLA2 differs from the sequences of the human and
mouse enzymes by 20-30% and that immunochemical detection of chicken
cPLA2 is rather difficult due to the lack of antibodies specifically raised against the chicken enzyme.
Following cPLA2 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,
cPLA2 translocation to membranes can occur at 37 °C in
response to supraphysiological elevation of
[Ca2+]i or at basal [Ca2+]i
following MAPK activation (see "Results" and "Discussion"). In
the experimental procedures, cell incubations were terminated by the
addition of ice-cold Ca2+-free lysis buffer containing
phosphatase inhibitors. It should be noted that the inclusion of 2 mM Ca2+ (no added EGTA) or its absence (2 mM EGTA added) in the ice-cold lysis buffer did not affect
cPLA2 recovery in the 100,000 × g fractions when analyzed by Western blotting. This suggested that (i)
there is a stable binding of cPLA2 to membranes that is not readily reversed by chelating Ca2+ at 4 °C in the
presence of phosphatase inhibitors, and (ii) inclusion of
Ca2+ 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 × 105 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 µg/ml) and incubated in
humidified 5% CO2 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-cPLA2 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 × 10 min with
PBS and rinsed for 5 min with water. 10 µl 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 × 630).
Statistics--
Results are expressed as means ± 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 < 0.05 considered to be significant.
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RESULTS |
Distribution of PLA2 Activities in the 100,000 × g Fractions of Embryonic Chick Ventricular Cells--
We measured
PLA2 activities in the 100,000 × g
fractions prepared from embryonic chick ventricular cells. We used the
preferential substrate of cPLA2,
1-stearoyl-2-[1-14C]arachidonylphosphatidylcholine, in
the absence or presence of specific inhibitors of the different
PLA2 isoforms, viz. DTT, HELSS, and
AACOCF3, to block the sPLA2, iPLA2,
and cPLA2 activities, respectively. In the absence of
inhibitors, total PLA2 activity in the 100,000 × g supernatant fraction was 4-7 times higher than in the
100,000 × g pellet fraction. Thus, we used the
100,000 × g supernatant fraction to determine optimal
conditions for the measurement of the AACOCF3-sensitive
PLA2 activity (cPLA2). As shown in Fig.
1A, in the absence of
inhibitors, the AACOCF3-sensitive PLA2 activity
(cPLA2) represented 73% of the total PLA2
activity. The addition of the sPLA2 inhibitor DTT strongly
and selectively improved the AACOCF3-sensitive
cPLA2 activity by 200%, presumably through stabilization
of this oxidation-sensitive enzyme (25). In contrast, the
cPLA2 activity was not significantly affected upon addition
of HELSS. In the presence of DTT and HELSS, cPLA2 assay was
linear with respect to time and protein (data not shown) and displayed
Ca2+-dependent characteristics of
cPLA2, with maximal activation obtained at 1 µM Ca2+ and half-maximal stimulation
occurring at 0.3 µM Ca2+ (Fig.
1B). This specific assay for cPLA2 was validated
in both the 100,000 × g supernatant and pellet
fractions of embryonic chick heart cells by the total blockade of
PLA2 activity upon addition of 10 µM
AACOCF3 (Fig. 1C).
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Fig. 1.
PLA2 activities in the
100,000 × g subcellular fractions of embryonic
chick ventricular cells. A, effect of DTT and HELSS on the
AACOCF3-sensitive PLA2 activity in the
100,000 × g supernatant fraction; B,
Ca2+-dependent stimulation of PLA2
activity measured in the presence of DTT and HELSS in the 100,000 × g supernatant fraction; C,
AACOCF3-sensitive PLA2 activity measured in the
presence of DTT and HELSS. The 100,000 × g supernatant
and pellet fractions of embryonic chick ventricular cells were
prepared, and PLA2 activity was measured using
1-stearoyl-2-[1-14C]arachidonylphosphatidylcholine as
substrate as described under "Experimental Procedures." The
reaction was carried out with 30-50 µg of proteins from the
100,000 × g supernatant or pellet fraction. Assays
were performed in the absence or presence of 10 µM
AACOCF3 and/or 2 mM DTT and/or 10 µM HELSS as indicated. In B, free
Ca2+ concentrations were prepared using
Ca2+/EGTA buffers and determined by measurement of Fura-2
fluorescence. PLA2 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 ± 275 total dpm). Total
disintegrations/min corresponded to 450, 870, and 995 µg of total
proteins in supernatants (A-C, respectively) and 660 µg
of total proteins in the pellet (C). Total
[14C]PC radioactivity added to assays was 150,000 dpm
(A) and 200,000 dpm (B and C). Values
are the means ± S.E. of triplicate determinations from a typical
experiment that was repeated twice. *, p < 0.01.
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2-AR Agonists Activate cPLA2
in Embryonic Chick Heart Cells--
Embryonic chick heart cells were
exposed to 2-AR stimulation before preparation of
100,000 × g subcellular fractions and subsequent
measurement of cPLA2 activity in the presence of DTT and
HELSS. Zinterol, a specific partial 2-AR agonist,
induced a dose-dependent decrease in cPLA2
activity in the 100,000 × g supernatant fraction, with
a maximal effect observed at 30 nM zinterol (Fig.
2A). In parallel, 30 nM zinterol elicited an increase in cPLA2
activity in the 100,000 × g pellet fraction (Fig.
2A). Zinterol effects on cPLA2 activity were
reproduced with a distinct 2-AR stimulus, achieved with
isoproterenol, a non-selective but complete -agonist, added together
with CGP 20712A, a selective 1-AR antagonist (Fig.
2A). AACOCF3 abrogated the effects of
2-AR agonists on cPLA2 activity (data not
shown). Experiments performed in embryonic chick heart cells prelabeled
with [3H]AA indicated that zinterol stimulation resulted
in a rapid (detected within 30 s) and sustained AA release over
the 15 min examined (Fig. 2B).
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Fig. 2.
2-AR stimulation
elicits redistribution of cPLA2 activity from the
100,000 × g supernatant fraction to the
100,000 × g pellet fraction (A)
and induces [3H]AA release (B).
A, cPLA2 activity was measured in 100,000 × g fractions (30-50 µg of proteins/assay) in the
presence of 2 mM DTT and 10 µM HELSS after
hormonal stimulation had been performed for 20 min as described under
"Experimental Procedures." PLA2 activity is expressed
as a percentage of the control (100% values were 2200 ± 400 and
12,200 ± 2000 total dpm in the pellet and supernatant fractions,
respectively, and corresponded to 528 ± 40 and 970 ± 96 µg of total proteins, respectively). Values are the means ± S.E. of nine experiments, performed in triplicate. *, p < 0.01. zint, zinterol; iso, isoproterenol;
CGP, 20712A. B, embryonic chick heart cells were
labeled for 24 h with 1.5 µCi/ml [3H]AA, washed
twice with saline buffer containing 0.2% fatty acid-free bovine serum
albumin, and incubated for the indicated periods of time with 30 nM zinterol as described under "Experimental
Procedures." [3H]AA release in control cells
corresponded to 1337 ± 187 dpm. Results expressed in
disintegrations/min were corrected for the release in control cells and
are the means ± S.E. of three different experiments.
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Zinterol-induced translocation of cPLA2 was examined by
immunofluorescent staining of myocytes using anti-cPLA2
antibody (Fig. 3). Unstimulated cells
showed diffuse fluorescence throughout the cell (Fig. 3B).
This staining specifically represented cPLA2 as
demonstrated by the negative staining pattern obtained when the
anti-cPLA2 primary antibody was omitted (Fig.
3A). In contrast, after stimulation with 30 nM
zinterol, the 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 cPLA2
from the cytoplasm to the nuclear and/or SR membranes, both networks
being tightly associated in our cells. cPLA2 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 cPLA2 as soon as 30 s
after zinterol application. It should be noted that immunofluorescent
staining of cPLA2 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 cPLA2 than
of membrane-bound cPLA2 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 cPLA2 to membrane
structures might result in a better exposure of the epitope for the
primary antibody, allowing a more efficient antibody binding.
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Fig. 3.
Time-dependent induction of
cPLA2 redistribution by zinterol in embryonic chick
ventricular cells. Embryonic chick ventricular cells were cultured
on plastic coverslips and incubated for 0 s (A and
B), 30 s (C), 2.5 min (D), 10 min
(E), and 30 min (F) in the presence of 30 nM zinterol. Cells were washed, fixed, permeabilized, and
stained with (B-F) or without (A) rabbit
anti-cPLA2 antibody prior to incubation with Cy3-conjugated
secondary antibody as described under "Experimental Procedures."
Immunofluorescent staining was visualized by microscopy
(magnification × 630).
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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).
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Fig. 4.
Zinterol enhances phosphorylation of p38 and
p42/44 MAPKs: dose dependence (A) and
sensitivity to ICI 118551 (2-AR
antagonist) (B) of zinterol action. Embryonic
chick ventricular cells were stimulated for 8 min in the presence of
increasing concentrations of zinterol (A) or for 8 min with
or without 30 nM zinterol in the absence or presence of 100 nM ICI 118551 (B). Cell lysates were prepared
and separated (50 µg 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."
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Zinterol Does Not Induce Detectable Phosphorylation of
cPLA2 at Ser505--
Immunoblot analysis of
whole cell homogenates using anti-cPLA2 antibody revealed a
single 80-kDa band in control untreated cells (Fig.
5). An additional band with decreased
electrophoretic mobility, characteristic of cPLA2
phosphorylated at Ser505, was detected after treatment with
100 nM PMA for 2.5 min (Fig. 5). In contrast, zinterol
stimulation did not impact on cPLA2 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).
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Fig. 5.
Zinterol evokes a time-dependent
phosphorylation of p38 and p42/44 MAPKs, but does not affect
cPLA2 electrophoretic mobility, in contrast to PMA.
Embryonic chick ventricular cells were stimulated for the indicated
periods of time with 30 nM zinterol or for 2.5 min in the
presence of 100 nM PMA. Whole cell lysates were separated
(100 µg of proteins) on 7.5% SDS-polyacrylamide gel. Proteins were
electroblotted onto PVDF membrane, and the same membrane was
successively probed with antibodies raised against human
cPLA2, human phospho-p38 MAPK, and human phospho-p42/44
MAPK as described under "Experimental Procedures." Proteins were
revealed using an alkaline phosphatase-linked immunoglobulin upon
addition of ECF substrate. cPLA2-S505-P indicates the slowly
migrating form of cPLA2 characteristic of cPLA2
phosphorylated at Ser505.
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2-AR Stimulation Induces cPLA2
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 cPLA2 translocation. As
shown in Fig. 6, the presence of either 1 µM PD98059 or 1 µM SB203580 totally blocked
zinterol-induced AA release. Similarly, treatment of cardiomyocytes
with either 1 µM PD98059 or 1 µM SB203580
did not affect the pattern of cPLA2 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 cPLA2 translocation and
activation.
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Fig. 6.
Inhibition of the p38 MAPK (SB203580) or
p42/44 MAPK (PD98059) pathway impairs zinterol-induced
[3H]AA release. Embryonic chick heart cells were
labeled for 24 h with 1.5 µCi/ml [3H]AA as
described under "Experimental Procedures." Radiolabeled cells were
incubated for 15 min with 30 nM zinterol in the absence or
presence of either 1 µM SB203580 or 1 µM
PD98059. Results expressed in disintegrations/min were corrected for
the release in control cells and are from a typical experiment that was
reproduced twice. N.D., non-detectable above background
levels.
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Fig. 7.
Zinterol-induced cPLA2
translocation is impaired by MAPK pathway inhibitors (SB203580 and
PD98059) and EGTA treatment. Embryonic chick ventricular cells
were cultured on plastic coverslips and preincubated in the absence
(A and B) or presence of either 1 µM SB203580 (C and D) or 1 µM PD98059 (E and F) for 1 h
or with 1 mM EGTA (G and H) for 10 min. 30 nM zinterol was then added (B,
D, F, and H) or not (A,
C, E, and G). After 10 min, cells were
washed, fixed, permeabilized, and stained with rabbit
anti-cPLA2 antibody prior to incubation with Cy3-conjugated
secondary antibody as described under "Experimental Procedures."
Immunofluorescent staining was visualized by microscopy
(magnification × 630).
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To examine the contribution of [Ca2+]i in
zinterol-induced cPLA2 translocation, cells were pretreated
for 10 min with 1 mM EGTA. Incubation of the cells with
EGTA did not affect the pattern of cPLA2 in unstimulated
cells (Fig. 7, G compared with A). In contrast,
it impaired zinterol-induced translocation of cPLA2 (Fig.
7, H compared with B), suggesting that
preservation of basal [Ca2+]i is a crucial factor
for redistribution of the enzyme in response to zinterol.
2-AR Stimulation of
[Ca2+]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
[Ca2+] transients of electrically stimulated embryonic
chick ventricular cells (Fig.
8B), which was totally blunted
upon addition of the cPLA2 inhibitor AACOCF3
(Fig. 8C). We show here that the zinterol effect on
[Ca2+]i cycling was unaffected in the presence of
HELSS, a selective iPLA2 blocker (Fig. 8F).
Thus, our present data rule out the possible participation of
iPLA2 and confirm the selective role of cPLA2
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 [Ca2+]i
cycling. Preincubation for 1 h with 1 µM SB203580 (Fig. 8E), an inhibitor of p38 MAPK, or 1 µM
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
[Ca2+]i cycling requires p38 and p42/44 MAPKs and
selective cPLA2 activation.
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Fig. 8.
Zinterol stimulation of
[Ca2+]i cycling is insensitive to
iPLA2 inhibition, but is impaired by cPLA2 and
p38 and p42/44 MAPK pathway inhibitors. Embryonic chick
ventricular cells were loaded with Fura-2 as described under
"Experimental Procedures." Cells were electrically stimulated at
0.5 Hz (B-F) or not (A) and perfused with
30 nM zinterol alone (A and B) or
with 10 µM AACOCF3 (C), 1 µM PD98059 (D), 1 µM SB203580
(E), or 10 µM HELSS (F). The
traces are representative of at least 15 cells obtained from
two different isolations.
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In the absence of electrical stimulation, it is noteworthy that
zinterol was without effect on [Ca2+]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
[Ca2+] transients triggered by caffeine in Fura-2-loaded
cells in the absence of electrical stimulation. The application of 10 mM caffeine produced a unique [Ca2+]i
transient (Fig. 9A). In
contrast, the application of caffeine together with zinterol resulted
in a train of [Ca2+]i transients (Fig.
9B). These experiments demonstrate that, in the absence of
electrical stimulation, zinterol action leads to Ca2+
loading of caffeine-sensitive SR stores. Zinterol potentiation of
caffeine-induced Ca2+ mobilization from the SR was blunted
in the presence of AACOCF3, PD98059, or SB203580 (Fig. 9,
C-E, respectively), whereas it was insensitive to HELSS
(Fig. 9F). Taken together, these results demonstrate that
zinterol induces Ca2+ loading of caffeine-sensitive SR
stores through the activation of p38 and p42/44 MAPKs and
cPLA2. It is noteworthy that Ca2+ loading of SR
stores induced by zinterol did not impact on basal cytosolic
[Ca2+]i in the absence of electrical stimulation
(Fig. 8A). In contrast, the release of Ca2+
accumulated in SR stores is likely to support zinterol-induced amplification of electrically stimulated [Ca2+]
cycling.
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Fig. 9.
Zinterol potentiates caffeine-induced
Ca2+ mobilization from the SR. Embryonic chick
ventricular cells were loaded with Fura-2 as described under
"Experimental Procedures." In the absence of electrical
stimulation, cells were perfused in the absence (A) or
presence of 30 nM zinterol (zint) alone
(B) or with 10 µM AACOCF3
(C), 1 µM PD98059 (D), 1 µM SB203580 (E), or 10 µM HELSS
(F) before the addition of 10 mM caffeine
(arrows). The traces are representative of at
least 15 cells obtained from two different isolations.
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1-AR Stimulation of
[Ca2+]i Cycling Does Not Rely on Either
cPLA2 Translocation or p38 and p42/44 MAPK Activation, in
Contrast to 2-AR-mediated
Responses--
1-AR stimulation achieved with 100 nM isoproterenol added with 100 nM ICI 118551, a selective 2-AR antagonist, was ineffective in
cPLA2 redistribution (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).
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Fig. 10.
Selective
2-AR induction of cPLA2
redistribution in embryonic chick ventricular cells. Embryonic
chick ventricular cells were cultured on plastic coverslips and
incubated for 30 min in the absence (A) or presence of
either 100 nM isoproterenol and 300 nM CGP
20712A (B) or 100 nM isoproterenol and 100 nM ICI 118551 (C). Cells were washed, fixed,
permeabilized, and stained with rabbit anti-cPLA2 antibody
prior to incubation with Cy3-conjugated secondary antibody as described
under "Experimental Procedures." Immunofluorescent staining was
visualized by microscopy (magnification × 630).
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We previously demonstrated that, in embryonic chick ventricular cells,
the 1-AR-mediated increase in
[Ca2+]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 [3H]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
[Ca2+]i cycling compared with 2-AR
activation. We measured the amplitude of [Ca2+]
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 µM SB203580 and/or 1 µM 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 [Ca2+]i cycling rely on p38 and p42/44
MAPK activation, in contrast to 1-AR-mediated
responses.
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Fig. 11.
Improvement of
1-AR-mediated versus
impairment of 2-AR-mediated
stimulation of [Ca2+]i cycling in response to
inhibition of the p38 and/or p42/44 MAPK pathway. Embryonic chick
ventricular cells were loaded with Fura-2 as described under
"Experimental Procedures." Cells were electrically stimulated at
0.5 Hz and perfused in the presence of 100 nM isoproterenol
with 300 nM CGP 20712A (A) or 100 nM
isoproterenol with 100 nM ICI 118551 (B) in the
absence or presence of 1 µM SB203580 or 1 µM PD98059, added separately or together. Values obtained
are the means ± S.E. of the effects observed in at least 15 cells
obtained from two different isolations. *, p < 0.01.
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DISCUSSION |
This study provides direct evidence for the role of
cPLA2 as a selective effector of 2-AR in
embryonic chick heart cells. We demonstrate a
dose-dependent redistribution of cPLA2 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
[Ca2+]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 cPLA2 activation
in the 2-AR stimulation of [Ca2+]i
cycling. We show that 2-AR stimulation triggers a rapid
translocation of cPLA2 that results in an increase in
cPLA2 activity associated with the 100,000 × g pellet subcellular fraction and subsequent release of AA,
leading to SR Ca2+ loading. The 2-AR-induced
responses (cPLA2 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
[Ca2+]i cycling detected within 1 min.
Our results highlight the essential role of the p38 and p42/44 MAPK
pathways in the regulation of cPLA2 activity. Inhibitors of
either the p38 or p42/44 MAPK pathway (SB203580 and PD98059, respectively) neutralize all zinterol-induced responses,
viz. cPLA2 translocation, induction of AA
release, increase in SR Ca2+ loading, and amplification of
[Ca2+]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 cPLA2 and AA release. Similar dual MAPK activation has been reported in rat cardiomyocytes for cPLA2 stimulation by ATP (30) and in
human neutrophils for cPLA2 activation by opsonized zymosan
(31).
Our data indicate that the MAPK prerequisite concerns only
2-AR-triggered pathways. 1-AR-mediated
effects on [Ca2+]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
[Ca2+]i cycling is mediated by cAMP and is
neither associated with AA release nor sensitive to cPLA2
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 cPLA2 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 cPLA2. In fact, we have previously demonstrated
that cAMP exerts a negative effect on the cPLA2/AA pathway
in embryonic chick heart cells (5). Furthermore, inhibition of
cPLA2 activity upon protein kinase A-mediated
phosphorylation has been reported in smooth muscle cells (32).
Several phosphorylation sites on cPLA2 have been identified
(33). Most studies have focused on MAPK-dependent
phosphorylation of Ser505 since it results in a
characteristic decrease in electrophoretic mobility of the enzyme. The
importance of Ser505 phosphorylation in cPLA2
activation appears to be cell type- and agonist-specific (for a review,
see Ref. 34): although it is essential for Ca2+ ionophore
4-bromo-A23187-induced AA release in Chinese hamster ovary cells
overexpressing human cPLA2 (26), it is not required for AA
release from thrombin-stimulated platelets (35). cPLA2 activation by MAPKs, independent of Ser505 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 and MNK1 (30, 36), can activate cPLA2. These
kinases are likely to phosphorylate cPLA2 at sites other
than Ser505 (in particular, Ser727) without
impact on cPLA2 gel mobility (18). Our results suggest that, in embryonic chick heart cells, cPLA2 can be
phosphorylated at Ser505 in response to the MAPK activator
PMA. In contrast, activation of cPLA2 by zinterol, which
also relies on MAPK activation, seems unrelated to the phosphorylation
of Ser505 since it occurs in the absence of detectable
impact on cPLA2 gel mobility. However, a limited
Ser505 phosphorylation of cPLA2 in response to
zinterol cannot be ruled out due to technical problems associated with
detection of the chicken cPLA2 sequence (see
"Methods").
It is well documented that calcium induces binding of cPLA2
to membranes through the C2 domain. Basal cPLA2 activity in
embryonic chick heart cells assayed in vitro exhibited a
typical Ca2+ requirement (Fig. 1). In addition, a
supraphysiological increase in [Ca2+]i promoted
by the addition of the Ca2+ ionophore 4-bromo-A23187 (2 µM for 10 min) led to the translocation of the enzyme to
membranes (data not shown). In many cell models, agonist-induced
cPLA2 activation and AA release require Ca2+
elevation (34). However, in Sf9 cells, okadaic acid-induced AA
release requires basal Ca2+ levels, but is observed without
an increase in [Ca2+]i (37). Similarly, in
embryonic chick heart cells that were not submitted to electrical
stimulation, i.e. in the absence of detectable
[Ca2+]i elevation, zinterol induced
cPLA2 translocation and AA release. Disruption of cellular
Ca2+ homeostasis by incubating cells with EGTA prevented
zinterol-induced cPLA2 translocation. This suggests that,
in response to zinterol stimulation, basal
[Ca2+]i is sufficient to support
cPLA2 translocation. Thus, our results indicate that, at
normal basal [Ca2+]i, MAPK activation plays an
essential role in regulating zinterol-induced translocation of
cPLA2. Whether additional mechanisms are involved remains
to be elucidated.
Zinterol-induced Ca2+ accumulation in SR stores could
result from the blockade of SR Ca2+ efflux through
ryanodine receptors or the potentiation of SR Ca2+ influx
via the SR Ca2+ pump. However, another attractive
hypothesis concerns the possible involvement of sarcolemmal
AA-activated Ca2+ channels. In fact, a non-capacitative
Ca2+ entry pathway, gated by AA and independent of store
depletion, has been identified in non-excitable cells and in A7r5
smooth muscle cells (38, 39). Whether AA-activated Ca2+
channels exist in cardiac cells and are responsible for
zinterol-induced SR loading remains an open question.
The coupling of -AR to MAPKs has already been documented. However,
studies focused on the long-term role of MAPKs, in particular in the
hypertrophic and/or mitogenic responses or in receptor desensitization
mechanisms. In heart, p42/44 MAPK activation is involved in the
induction of cardiomyocyte hypertrophy in response to isoproterenol, a
nonspecific -AR agonist (40). Studies performed in
2-AR-transfected HEK293 cells have demonstrated an
activation of p42/44 MAPKs by those receptors. In HEK293 cells, p42/44
MAPK activation follows 2-AR desensitization and relies
on a cAMP-mediated event: protein kinase A-mediated phosphorylation of
2-AR uncouples the phosphorylated receptor from the
adenylyl cyclase stimulatory G protein (Gs), a
process termed heterologous desensitization, and switches the
2-AR coupling to Gi, with subsequent
stimulation of p42/44 MAPK (41). Daaka and co-workers (42-44) have
proposed that this provides one mechanism by which 2-ARs
not only regulate their own internalization, but also initiate an
additional signal transduction pathway, in which the desensitized
receptor might function as a structural component of a mitogenic
signaling complex. Thus, mechanistically, 2-AR-mediated
MAPK activation in fibroblasts requires sequential coupling to
Gs and Gi. In the absence of any coupling of
2-AR to the Gs/adenylyl cyclase pathway, the
situation in embryonic chick heart cells is clearly different. Our
results argue for a primary coupling of 2-AR to a
PTX-sensitive G protein (hypothetically Gi) and MAPKs. In
addition, our data suggest that 2-AR coupling to MAPK
will represent an alternative to adenylyl cyclase in supporting cardiac
contractility rather than a termination signal.
Alternatively, other studies performed in rat, dog, and mouse hearts
have suggested a dual coupling of 2-AR to concurrent Gs and PTX-sensitive Gi signaling pathways
(45-47). In those species, the 2-AR/Gi
pathway seems to exert a negative control on the 2-AR/Gs pathway. Indeed, stimulant effects
of 2-AR agonists on murine myocyte contractions can be
detected only after PTX treatment (46). In rat and dog, PTX enhances
the positive inotropic response of 2-AR agonists.
Xiao and Lakatta (45) proposed that Gi signaling
limits the Gs pathway, inducing the compartmentalization of
2-AR-induced cAMP signaling.
Our data clearly demonstrate that, in chicken, an exclusive coupling to
a PTX-sensitive pathway mediates the positive inotropic response of
2-AR agonists. The relative coupling efficiency of 2-AR to either Gs/cyclase or
Gi/cPLA2 would determine the nature of the
messenger, cAMP or AA. This could vary depending on the cardiac tissue,
the animal species, and/or the pathophysiological state of the heart.
In healthy human hearts, 2-AR is essentially coupled to
Gs, and the positive inotropic response is mediated by
cAMP, with a limited negative influence of 2-AR coupled
to Gi. An attractive hypothesis is that
2-AR/cPLA2 coupling would be effective in
mediating inotropic responses in the case of defective coupling to
adenylyl cyclase. Thus, congestive human heart failure is associated
with alterations in the activation of the 2-AR/cAMP pathway. Part of the remaining contractile effect of
2-AR agonists could rely on the alternative production
of AA. In keeping with this hypothesis, preliminary results from our
laboratory clearly indicate the relevance of 2-AR
coupling to cPLA2 in human under certain
pathophysiological circumstances.
In conclusion, our results confirm that the 2-AR pathway
diverges from the 1-AR pathway in embryonic chick
ventricular cells. They establish the selective role of the
cPLA2/AA pathway and p38 and p42/44 MAPKs in
2-AR-mediated effects on
[Ca2+]i cycling. This study emphasizes that,
apart from their involvement in long-term -AR-induced hypertrophy,
MAPKs may play a major role as physiological regulators of the
2-AR-mediated contractile responses in heart. A complete
understanding of the cellular mechanisms involved in the coupling of
2-AR to cPLA2 should identify novel targets
for therapeutic intervention in failing hearts, known to present an
uncoupling of both 1-AR and 2-AR from the
adenylyl cyclase system.
2-Adrenergic Receptor Agonists
Increase Intracellular Free Ca2+ Concentration
Cycling in Ventricular Cardiomyocytes through p38 and p42/44
MAPK-mediated Cytosolic Phospholipase A2 Activation*
TOP
ABSTRACT
INTRODUCTION
