Oxidative stress stimulates multiple MAPK signalling pathways and phosphorylation of the small HSP27 in the perfused amphibian heart
Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, Athens 157 84, Greece
* Author for correspondence (e-mail: ibeis{at}biol.uoa.gr)
Accepted 12 May 2003
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Summary |
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Key words: oxidative stress, amphibian heart, MAPK, p38-MAPK, HSP27, signal transduction, Rana ridibunda
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Introduction |
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Although the above-described mechanisms leading to oxidative stress are
common in all air-breathing organisms, there are some distinguishable
differences between higher and lower vertebrates and/or invertebrates. Mammals
are designed to function under high oxygen pressure and therefore show a
limited capacity for maintaining cellular homeostasis without oxygen and a
limited ability to deal with oxidative stress. On the other hand, many lower
vertebrate and invertebrate species deal naturally with wide variations and
rapid changes in oxygen availability and include some species that have a
well-developed capacity to sustain prolonged periods of complete anoxia
(Driedzic and Gesser, 1994).
Previous studies conducted in organs of anoxia-tolerant species have shown
that particular adaptations, including the induction or overexpression of
various antioxidant systems, exist in these organisms to allow them to deal
effectively with a rapid transition from anoxia back to normoxia, minimising
stress or injury resulting from a burst of ROS generation
(Hermes-Lima and Storey, 1996
;
Venditti et al., 1999
;
Hermes-Lima et al., 2001
;
Lushchak et al., 2001
;
Pritchard, 2002
).
Various reports have documented the involvement of the mitogen-activated
protein kinase (MAPK) signalling pathways in redox-stressed cells and tissues,
including mammalian cardiac myocytes and intact myocardium (reviewed in
Sugden and Clerk, 1998;
Franklin and McCubrey, 2000
).
However, the factors that modulate these signalling pathways have not been
described fully in any system studied to date.
MAPKs are members of a major intracellular signal transduction pathway that
has been demonstrated to play an important role in various physiological
processes (Widmann et al.,
1999; Kyriakis and Avruch,
2001
; Pearson et al.,
2001
). Three subfamilies of these serine/threonine kinases have
been clearly identified in mammals: the extracellularly responsive kinases
(ERKs), the c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. The third
subfamily, p38-MAPK, is activated by various forms of environmental stress,
including hyperosmolarity and heat shock (for a review, see
Bogoyevitch, 2000
;
Kyriakis and Avruch, 2001
).
The respective MAPK subfamilies in the amphibian heart have been recently
characterised in our laboratory (Aggeli et al.,
2001a
,b
,
2002a
,b
).
In the isolated perfused Rana ridibunda heart, the one isoform of ERK
(p43) detected was activated by phorbol esters [1 µmol l-1
4ß-phorbol 12-myristate 13-acetate (PMA)] and mechanical overload. The
two isoforms of JNKs identified (p46-JNK1 and p52-JNK2) were found to be
phosphorylated in response to 0.5 mol l-1 sorbitol, mechanical
overload and re-oxygenation following anoxia. p38-MAPK was also stimulated by
mechanical overload but was most potently activated by hyperosmotic and
thermal stresses.
Activated MAPKs are characterised by their localisation in both the
cytoplasm and the nucleus, where they interact with their substrates
(Bogoyevitch, 2000;
Aggeli et al., 2001b
). A
variety of substrates for the MAPKs has been identified, including several
transcription factors and other protein kinases, and these phosphorylations
are probably responsible for the ultimate cellular effects of MAPK activation.
One of the p38-MAPK substrates is MAPK-activated protein kinase 2 (MAPKAPK2),
which phosphorylates the small heat shock protein HSP27
(Stokoe et al., 1992
;
Rouse et al., 1994
). In
several cell types, phosphorylation of HSP27 is associated with stabilisation
of the actin cytoskeleton, protecting cells against damage
(Lavoie et al., 1995
;
Guay et al., 1997
;
Concannon et al., 2003
).
Here, we have investigated the effect of oxidative stress (as exemplified by H2O2) on the phosphorylation levels of the three MAPK subfamilies as well as MAPKAPK2 and HSP27 in the isolated perfused amphibian heart. We have also studied the immunolocalisation pattern of phosphorylated p38-MAPK and HSP27 induced by oxidative stress in the absence or presence of the selective p38-MAPK inhibitor SB203580. Overall, our results provide evidence that, despite the fundamental structural and functional differences between the mammalian and amphibian heart, common MAPK signal transduction pathways are involved in responses such as oxidative stress.
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Materials and methods |
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Rabbit polyclonal antibodies specific for the total and dually phosphorylated ERKs (#9102 and #9101, respectively), JNKs (#9252 and #9251, respectively) and p38-MAPK (#9212 and #9211, respectively), as well as for the total and phosphorylated (Thr334) MAPKAPK2 (#3042 and #3041, respectively) and phosphorylated (Ser82) HSP27 (#2401) were purchased from Cell Signalling (Beverly, MA, USA). A mouse monoclonal antibody specific for the total HSP27 (#2402) was also obtained from Cell Signalling. Pre-stained molecular mass markers were from New England Biolabs. Biotinylated anti-rabbit and anti-mouse antibodies were from DAKO A/S (Glostrup, Denmark). X-OMAT AR 13 cmx18 cm and Elite chrome 100 films were purchased from Eastman Kodak Company (New York, NY, USA).
Animals
Frogs (Rana ridibunda Pallas) weighing 120150 g were caught
in the vicinity of Thessaloniki, Greece and supplied by a local dealer. They
were kept in containers in freshwater and their care met the standards of Good
Laboratory Practice.
Heart perfusions
Frogs were anaesthetised by immersion in 0.01% (w/v) MS222 and sacrificed
by decapitation. The hearts were excised and mounted onto the aortic cannula
of a conventional Langendorff perfusion system. Perfusions were performed in a
non-recirculating Langendorff mode at a pressure of 4.5 kPa (31.5 mmHg) with
bicarbonate-buffered saline (23.8 mmol l-1 NaHCO3, 103
mmol l-1 NaCl, 1.8 mmol l-1 CaCl2, 2.5 mmol
l-1 KCl, 1.8 mmol l-1 MgCl2, 0.6 mmol
l-1 NaH2PO4, pH 7.4 at 25°C) supplemented
with 10 mmol l-1 glucose and equilibrated with
95%O2:5%CO2. The temperature of the hearts and
perfusates was maintained at 25°C using a water-jacketed apparatus. All
hearts were equilibrated for 15 min under these conditions. At the end of the
equilibration period, hearts were perfused with 100 µmol l-1
H2O2 for periods of time ranging from 30 s to 60 min. In
another set of experiments, hearts were perfused with different concentrations
of H2O2 (31000 µmol l-1) for 2 min
or 5 min (the time point of the respective maximal MAPK activation) after the
equilibration period. In addition, hearts perfused with either 1 µmol
l-1 PMA for 10 min or 0.5 mol l-1 sorbitol for 15 min
after the equilibration period were used as positive controls. Perfusions were
also conducted in the presence of 1 µmol l-1 SB203580 or 25
µmol l-1 PD98059 during both the equilibration period and the
perfusion with either 30 µmol l-1 or 100 µmol l-1
H2O2 for 2 min or 5 min.
At the end of the perfusions, atria were removed, and the ventricles, after being immersed in liquid N2, were pulverised under liquid N2. Powders were stored at -80°C.
Tissue extractions
Heart powders were homogenised with 3 ml g-1 of buffer [20 mmol
l-1 Tris-HCl, pH 7.5, 20 mmol l-1
ß-glycerophosphate, 20 mmol l-1 NaF, 2 mmol l-1
EDTA, 0.2 mmol l-1 Na3VO4, 5 mmol
l-1 dithiothreitol (DTT), 10 mmol l-1 benzamidine, 200
µmol l-1 leupeptin, 120 µmol l-1 pepstatin A, 10
µmol l-1
trans-epoxy-succinyl-L-leucylamido(4-guanidino)butane, 300 µmol
l-1 phenyl methyl sulphonyl fluoride (PMSF), 0.5% (v/v) Triton
X-100] and extracted on ice for 30 min. The samples were centrifuged (10 000
g, 5 min, 4°C) and the supernatants boiled with 0.33
volumes of sodium dodecyl sulphatepolyacrylamide gel electrophoresis
(SDSPAGE) sample buffer [0.33 mol l-1 Tris-HCl, pH 6.8, 10%
(w/v) SDS, 13% (v/v) glycerol, 20% (v/v) 2-mercaptoethanol, 0.2% (w/v)
Bromophenol Blue]. Protein concentrations were determined using the BioRad
Bradford assay.
SDSPAGE and immunoblot analysis
Proteins were separated by SDSPAGE on 10% (w/v) acrylamide, 0.275%
(w/v) bis-acrylamide slab gels and transferred electrophoretically onto
nitrocellulose membranes (0.45 µm). Membranes were then incubated in TBS-T
[20 mmol l-1 Tris-HCl, pH 7.5, 137 mmol l-1 NaCl, 0.05%
(v/v) Tween 20] containing 1% (w/v) bovine serum albumin (BSA) for 30 min at
room temperature. Subsequently, the membranes were incubated with the
appropriate antibody according to the manufacturer's instructions. After
washing in TBS-T (4x5 min), the blots were incubated with horseradish
peroxidase-conjugated anti-rabbit IgG antibodies [1:5000 dilution in TBS-T
containing 1% (w/v) BSA; 1 h at room temperature]. The blots were washed again
in TBS-T (4x5 min), and the bands were detected using the enhanced
chemiluminescence (ECL) reaction with exposure to X-OMAT AR film. Blots were
quantified by laser scanning densitometry.
Immunolocalisation of phospho-p38-MAPK and phospho-HSP27
At the end of the perfusions, atria were removed and ventricles were
immersed in isopentane pre-cooled in liquid N2 and stored at
-80°C. Tissues were sectioned with a cryostat at a thickness of 56
µm, fixed with ice-cold acetone (10 min at room temperature) and stored at
-30°C until use. Tissue sections were washed in TBS-T [containing 0.1%
(v/v) Tween 20], and non-specific binding sites were blocked with 3% (w/v) BSA
in TBS-T (1 h at room temperature). Specimens were incubated with primary
antibodies specific for phospho-p38-MAPK and phospho-HSP27, diluted in 3%
(w/v) BSA in TBST (overnight at 4°C), according to the method previously
described (Aggeli et al.,
2002a). All sections were immunostained by the alkaline
phosphatase method using a Kwik kit according to the manufacturer's
instructions. The alkaline phosphatase label was visualised by exposing the
sections to Fast Red chromogen, and nuclei were counterstained with
haematoxylin. Slides were mounted, examined with a Zeiss Axioplan microscope
and photographed with a Kodak Elite chrome 100 film.
Statistical evaluations
All data are presented as means ± S.E.M. Comparisons
between control and treatments were performed using the unpaired Student's
t-test. A value of P<0.05 was considered to be
statistically significant. MAPK activation in `control' hearts was set at 1,
and the stimulated MAPK activation in treated hearts was expressed as `-fold'
activation over control hearts.
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Results |
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Activation of JNKs by H2O2 was studied using specific antibodies for the dually phosphorylated forms of these kinases. The 46 kDa (p46-JNK1) and 52 kDa (p52-JNK2) JNKs were rapidly activated by 100 µmol l-1 H2O2 (Fig. 3A). Activation was apparent within 1 min and was maximal at approximately 2 min. p46-JNK1 was activated by approximately 2.5±0.01-fold (P<0.001, N=3) and p52-JNK2 by approximately 2.2±0.01-fold (P<0.001, N=3) relative to control values. Phosphorylation levels showed a progressive decline that reached control values after a period of 30 min. The blot presented in Fig. 3B shows that there were no changes in the total cellular pool of JNKs and therefore provides a control for protein loading under these conditions.
|
p38-MAPK is activated by dual phosphorylation of Thr and Tyr residues
within a Thr-Gly-Tyr motif. Activation of the kinase by
H2O2 was therefore studied by immunoblot using a
specific antibody that recognises the dually phosphorylated kinase in extracts
from amphibian hearts perfused with 100 µmol l-1
H2O2 for increasing time periods varying from 30 s to 60
min. The results of this study showed that the phosphorylation of p38-MAPK was
apparent within 30 s,maximal at 2 min (approximately 9.75±0.75-fold
relative to control values; P<0.001, N=4) and
progressively declined thereafter, reaching control values within 45 min of
treatment (Fig. 4A,B).
Interestingly, a second band corresponding to 39 kDa was also detected at 2
min of treatment with H2O2, a result quite similar to
that induced by either 0.5 mol l-1 sorbitol
(Aggeli et al., 2001a) or
mechanical overload (Aggeli et al.,
2001b
) in the perfused amphibian heart. Equivalent protein loading
was confirmed by probing identical samples with an antibody recognising the
total p38-MAPK levels (Fig.
4A,bottom panel). We also examined the dose-dependent activation
of p38-MAPK by H2O2. As can be seen in
Fig. 4C,D, 3 µmol
l-1 of this agent induced strong activation of the kinase
(3.40±0.77-fold relative to control value; P<0.01,
N=4) whereas 30 µmol l-1 H2O2
induced maximal activation of the kinase (9.94±0.76-fold relative to
control values; P<0.001, N=4). Lower phosphorylation
levels were elicited by concentrations of H2O2 of 300
µmol l-1 (approximately 5.42±1.05-fold relative to
control values; P<0.01, N=4) or 1 mmol l-1
(4.33±1.34-fold relative to control values; P<0.01,
N=4) (Fig. 4C,D). The
maximal phosphorylation of p38-MAPK induced by oxidative stress was comparable
to that induced by 0.5 mol l-1 sorbitol (9.18±1.00-fold
relative to control values; P<0.001, N=4;
Fig. 4C,D). SB203580 (1 µmol
l-1) abolished the phosphorylation of p38-MAPK induced by 100
µmol l-1 H2O2
(Fig. 5A,B). As a positive
control, extract from heart perfused with 0.5 mol l-1 sorbitol was
used (Fig. 5A,B). The bottom
panel in Fig. 5A shows that
there were no changes in the total cellular pool of p38-MAPK and therefore
provides a control for protein loading under these conditions.
|
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It is well established that MAPKAPK2 is phosphorylated and activated by p38-MAPK, and we therefore studied its phosphorylation state in the amphibian heart perfused with 30 µmol l-1 H2O2. H2O2 induced a strong phosphorylation of the kinase (Fig. 6A,B). In particular, the time course of the MAPKAPK2 phosphorylation by 30 µmol l-1 H2O2 showed that the kinase phosphorylation was apparent within 1 min, maximised within 5 min (2.57±0.10-fold relative to control values; P<0.01, N=3) and reached control values within 30 min of treatment. Furthermore, the p38-MAPK selective inhibitor SB203580 (1 µmol l-1) completely inhibited the MAPKAPK2 activation induced by 30 µmol l-1 H2O2. As a positive control, extract from heart perfused with 0.5 mol l-1 sorbitol for 15 min was used. The bottom panel in Fig. 6A shows that equivalent protein was loaded.
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HSP27 is phosphorylated at up to three sites (Ser15, Ser78 and Ser82) by
MAPKAPK2 and the related kinase MAPKAPK3. The phosphorylation state of HSP27
was assessed by immunoblot using a rabbit polyclonal antibody that detects the
phosphorylated HSP27 at Ser82 (#2401). As can be seen in
Fig. 7A,B, 30 µmol
l-1 H2O2 induced a strong phosphorylation of
this protein (5.34±0.25-fold relative to control value;
P<0.001, N=3). Moreover, the p38-MAPK selective inhibitor
SB203580 (1 µmol l-1) abolished the HSP27 phosphorylation
induced by oxidative stress, whereas the ERK pathway selective inhibitor
PD98059 at a concentration of 25 µmol l-1 had no significant
inhibitory effect. The bottom panel in Fig.
7A shows that there were no changes in the total cellular pool of
HSP27 and therefore provides a control for protein loading under these
conditions. All the above results support the conclusion that oxidative stress
induced by H2O2 leads to the activation of a
p38-MAPK-dependent signalling pathway and are consistent with a
p38-MAPKMAKAPK2/3
HSP27 phosphorylation cascade.
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In cryosections from hearts perfused with 30 µmol l-1 H2O2 for 2 min in the absence or presence of 1 µmol l-1 SB203580, we tried to immunolocalise the phosphorylated forms of p38-MAPK and HSP27. Neither in control hearts (Figs 8A, 9A) nor in specimens incubated either with the secondary antibody or the chromogen alone was any immunoreactivity detected (data not shown). In specimens from hearts perfused with 30 µmol l-1 H2O2 for 2 min, strong immunoreactivity staining for phosphorylated p38-MAPK was observed within the cytoplasm as well as in the perinuclear region (Fig. 8B). In particular, perinuclear clustering of deposits indicating phospho-p38-MAPK immunoproducts was more intense in specimens from hearts subjected to either oxidative stress (Fig. 8B) or 0.5 mol l-1 sorbitol treatment (Fig. 8C). This immunoreactivity pattern disappeared when the hearts were perfused with H2O2 in the presence of the selective p38-MAPK inhibitor SB203580 (Fig. 8D). In respective sections, the anti-phospho-HSP27 antibody produced a discrete pattern of phospho-HSP27 immunoreactivity staining. Observed phospho-HSP27-immunoproducts were localised in the perinuclear region but were also widely dispersed in the cytoplasm (Fig. 9B,C). Overall, our results reveal that the selective p38-MAPK inhibitor SB203580 (1 µmol l-1) abolished this HSP27 phosphorylation by oxidative stress (Figs 8D and 9D for phospho-p38-MAPK and phospho-HSP27, respectively), which is consistent with our results obtained using biochemical approaches.
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Discussion |
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We previously reported that neither anoxia nor anoxia/reoxygenation induces
p38-MAPK phosphorylation, whereas anoxia/reoxygenation leads to a strong
phosphorylation of JNKs (p46-JNK1 and p52-JNK2, respectively) in the perfused
amphibian heart (Aggeli et al.,
2001a). The present study therefore sought to investigate whether
oxidative stress induced by exogenous H2O2 causes
phosphorylation (hence activation) of the three well-established MAPK
superfamily members in the perfused amphibian heart as well as the possible
mechanisms involved in these responses.
Using antibodies that can distinguish between phosphorylated and
unphosphorylated forms of these kinases, we detected the activation of the
p43-ERK and JNKs with a different time-dependent profile. In particular,
p43-ERK was found to be activated by H2O2 from as early
as 30 s and this phosphorylation was sustained for as long as 30 min of
treatment (Fig. 1). Similar
time-dependent phosphorylation of the kinase was previously detected by
mechanical overload (Aggeli et al.,
2001b) and by the
1-adrenergic agonist
phenylephrine (Aggeli et al.,
2002b
) in the amphibian heart. Furthermore, p43-ERK was activated
by H2O2 in a dose-independent pattern, a result that is
different from those previously reported for either neonatal cardiac myocytes
(Clerk et al., 1998a
) or
perfused rat heart (Clerk et al.,
1998b
). However, our results are consistent with reports that
describe a common, widespread response of ERKs to various stimuli
(Steinberg, 2000
).
On the other hand, phosphorylation of JNKs by H2O2
was detected from as early as 1 min of treatment, with its maximal value
attained within 2 min and showing a progressive decline thereafter, reaching
control values within 30 min of treatment
(Fig. 3). A similar
time-dependent profile of JNK activation by oxidative stress has also been
reported for rat neonatal cardiac myocytes
(Clerk et al., 1998a;
Dougherty et al., 2002
) and
adult rat heart (Clerk et al.,
1998b
). However, the activation of JNKs detected in the present
study was low compared with their response to various other stressful stimuli
such as hyperosmotic stress, anoxia/reoxygenation or mechanical overload
previously examined in this experimental model (Aggeli et al.,
2001a
,b
).
Our studies on the stimulation of the principal stress-activated p38-MAPK
by H2O2 showed that this kinase is activated in a time-
and dose-dependent manner. The immediate phosphorylation of the kinase by
H2O2 (within the first 30 s of treatment) indicates the
importance of an early response of the amphibian ventricular cells to
oxidative stress. The immediate response of the p38-MAPK cascade to
H2O2 has been also reported for rat neonatal myocytes
(Clerk et al., 1998a). The
maximal phosphorylation of the kinase was detected within 2 min of
H2O2 treatment, while a progressive decline of the
kinase activation levels was observed thereafter
(Fig. 4). Stimulation of the
p38-MAPK phosphorylation was completely inhibited by its selective inhibitor
SB203580 at a concentration of 1 µmol l-1
(Fig. 5). This transient
activation of JNKs and p38-MAPK by oxidative stress may be regulated by either
dephosphorylation via the respective phosphatases, scaffolding
proteins or feedback mechanisms (Kyriakis
and Avruch, 2001
; Pearson et
al., 2001
).
To confirm that phosphorylation of p38-MAPK represents its activation, we also tried to examine the phosphorylation state of its specific substrate MAPKAPK2, utilising antibodies that have recently become available and that specifically detect either the phosphorylated (activated) or the total levels of the kinase. The results of these studies clearly showed that 30 µmol l-1 H2O2 induced a strong phosphorylation of MAPKAPK2 similar to the p38-MAPK activation time-dependent profile and that 1 µmol l-1 SB203580 abolished this response.
Using antibodies specific to the phosphorylated form of the small heat
shock protein HSP27, we also showed that oxidative stress induced a strong
phosphorylation of this protein. Overall, using these specific antibodies, we
showed a direct pathway from p38-MAPKMAPKAPK2
HSP27, since the
selective p38-MAPK inhibitor SB203580 abolished the oxidative stress-induced
phosphorylation of both MAPKAPK2 and HSP27 (Figs
6 and
7 for MAPKAPK2 and HSP27,
respectively). Our results are consistent with previous reports describing the
possible mechanisms involved in the protective responses of the mammalian
heart against oxidative stress (Clerk et al.,
1998a
,1998b
;
Wang et al., 1998b
).
Furthermore, the immunohistochemical studies we performed revealed that
oxidative stress induced a strong phosphorylation of both p38-MAPK and HSP27
in the ventricular cells of the amphibian heart (Figs
8 and
9 for p38-MAPK and HSP27,
respectively). The localisation pattern observed indicates that this
signalling pathway was activated by H2O2, which was
administered exogenously. Previous studies have established that the
expression of the cytoplasmic small HSPs is regulated at either the
transcriptional level (Frohli et al.,
1993) or by post-transcriptional modifications such as
phosphorylation, deamination and acylation
(Lavoie et al., 1995
). The
phosphorylation of HSP27 is catalysed by MAPKAPK2 in a stress-dependent manner
(Landry et al., 1992
;
Knauf et al., 1994
). Although
the influence of HSP27 phosphorylation on cellular stress responses is still
controversial and not clearly defined, it is well established that this
protein has a wide variety of different and seemingly unrelated functions
ranging from a molecular chaperone to a mediator of thermoresistance and
chemoresistance. It also inhibits actin polymerisation, regulates apoptosis
and protects ribonucleic acids (Sakamoto
et al., 2000
; Geum et al.,
2002
; Concannon et al.,
2003
).
The roles of the different MAPK subfamilies in vertebrate heart function
are still under investigation. In many differentiated cells, ERK activation is
associated with cell survival (Xia et al.,
1995), whereas the stress-activated JNKs and p38-MAPK may promote
apoptosis (Xia et al., 1995
;
Park et al., 1997
;
Turner et al., 1998
). In the
amphibian heart, however, p38-MAPK stimulation of MAPKAPK2 and HSP27
phosphorylation is potentially cytoprotective. Both we and others have
previously reported that anoxia or anoxia/reoxygenation does not stimulate the
p38-MAPK signalling pathway in the perfused R. ridibunda heart or in
tissues from the freeze-tolerant wood frog Rana sylvatica
(Greenway and Storey, 2000
;
Aggeli et al., 2001a
). Anoxia
or hypoxia tolerance is a common feature of animals that can often face and
sustain these environmental conditions in vivo. It has also been
reported that in various tissues of such tolerant animals the levels of
antioxidants are higher than in respective mammalian tissues, a feature
representative of the different adaptive mechanisms existing between mammals
and lower vertebrates (Hermes-Lima et al.,
2001
). In particular, amphibians, as ectotherms, can face
tremendous variations in vivo in both body temperature and body water
content, while their life style is consistent with a wide annual range of
activity that obviously modulates their metabolic demands according to the
status of their environment. These changes in metabolic demands require
compensatory changes in cardiac output. Therefore, environmental stresses
leading to cardiac mechanical overload, either directly or indirectly (i.e.
thermal or hyperosmotic stresses), could be strong modulators of metabolic ROS
overproduction, thus inducing oxidative stress in the amphibian heart.
In summary, we have shown that H2O2, a ROS that can be produced in the amphibian heart under various conditions of environmental stress, activates all three MAPK subfamilies (ERK, JNKs and p38-MAPK). Furthermore, activation of p38-MAPK stimulates phosphorylation of MAPKAPK2, which in turn phosphorylates the HSP27, possibly leading to cell survival. In addition, the immunohistochemical studies we performed provide evidence that under oxidative stress the presence of phosphorylated forms of p38-MAPK and HSP27 is enhanced. All these findings taken together could indicate a possible involvement of p38-MAPK and HSP27 in preservation of amphibian heart homeostasis under similar situations in vivo.
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Acknowledgments |
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References |
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