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First published online June 15, 2007
Journal of Experimental Biology 210, 2267-2277 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003178
CoCl2 induces protective events via the p38-MAPK signalling pathway and ANP in the perfused amphibian heart
Department of Animal and Human Physiology, School of Biology, University of Athens, Panepistimioupolis, 157 84 Athens, Greece
* Author for correspondence (e-mail: ibeis{at}biol.uoa.gr)
Accepted 26 March 2007
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Summary |
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Key words: CoCl2, hypoxia, frog heart, p38-MAPK, ANP, cardioprotection, oxidative stress, signalling
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Holden and Storey, 1997).
Preservation of cardiac muscle performance under such stressful conditions is
crucial for survival. In particular, when exposed to hypoxic conditions,
amphibians face the challenge of maintaining intracellular acid–base
status, defending high energy phosphate homeostasis and metabolic substrates
in the ventricular muscle (Hermes-Lima and
Storey, 1996; Rocha and
Branco, 1998; Andersen et al.,
2003). The tolerance that these organisms exhibit has motivated a
plethora of studies in an effort to elucidate the mechanisms regulating their
respective responses.
Given their physiology, it was of great interest to evaluate amphibian
heart response to chemically induced hypoxia, i.e. to cobalt chloride.
CoCl2 is a well-known hypoxia mimetic agent
(Goldberg et al., 1988) that
has been demonstrated to act as a pro-oxidant, inducing an increase in the
levels of reactive oxygen species (ROS)
(Zou et al., 2001;
Xi et al., 2004; Kotake-Nora
and Saida, 2006) in diverse cell types
(Tomaro et al., 1991;
Chandel et al., 2000). Co (II)
has been identified as an oxidative stress-inducing factor producing ROS
via a Fenton-type reaction
(Moorhouse et al., 1985;
Wang et al., 1993).
ROS at low levels are known to act as signalling molecules
(Kamata and Hirata, 1999;
Li and Jackson, 2002;
Droge, 2002) while having
deleterious effects in high concentrations
(Droge, 2002). Cells possess
multiple antioxidant systems so as to counteract ROS, including specific
enzymes such as catalase (CAT), which is classified as an enzyme known to
convert H2O2 to H2O and molecular oxygen
(Fridovich, 1999), and
superoxide dismutase (SOD), whose main role is to convert the superoxide anion
(O2–) to H2O2
(McCord and Fridovich, 1969).
Nitric oxide (NO) is another member of the free radical family that diffuses
rapidly and does not readily react with most biomolecules
(Lancaster, 1994). However, NO
reaction with O2– generates peroxynitrite
(OONO–), which is highly cytotoxic
(Beckman and Koppenol, 1996).
Therefore, in the context of, for example, SOD competing with NO for binding
to the superoxide anions, it becomes evident that SOD exerts its
cardioprotective function by regulating formation of other ROS as well
(peroxynitrite in this case). Given the interaction between these reactions
one can deduce that, in this context, this is also the case for CAT.
Oxidative stress (exemplified by perfusion with H2O2)
has also been found to transcriptionally upregulate atrial natriuretic peptide
(ANP) levels in the amphibian heart
(Vassilopoulos et al., 2005).
There is also data implicating NO in the mechanism of this cardiac peptide
hormone regulation (Sanchez-Ferrer et al.,
1990; Carnio et al.,
2004). Therefore, ANP appears to constitute another factor
involved in the cardiac compensatory response to stimuli that could disturb
extracellular fluid volume and electrolyte balance
(Glass et al., 1996;
Silberbach and Roberts, 2001),
including redox perturbations.
Oxidative stress may trigger activation of a plethora of signalling
cascades (Kannan and Jain,
2000) including mitogen-activated protein kinase (MAPK) pathways
(Feuerstein and Young, 2000).
The three major subfamilies characterized in mammals are: the extracellular
signal-regulated kinases (ERKs), the c-jun N-terminal kinases (JNKs) and p38
reactivating kinase (p38-MAPK) (Kyriakis
and Avruch, 2001; Pearson et
al., 2001). The respective MAPKs have also been identified in the
amphibian heart (Aggeli et al.,
2001a). These kinases interact with their substrates in both the
cytoplasm and the nucleus, transducing a variety of molecular signals
(Bogoyevitch, 2000). Amphibian
heart p38-MAPK, in particular, has been demonstrated to be activated by
various forms of environmental stress, including mechanical and hyperosmotic
as well as thermal (Aggeli et al.,
2001b; Aggeli et al.,
2002). Additionally, certain antioxidants (SOD and CAT) have been
demonstrated to reverse H2O2-induced amphibian heart
p38-MAPK activation (Gaitanaki et al.,
2006a). H2O2-induced (oxidative stress)
p38-MAPK interacts with MAPK-activated-protein-kinase 2 (MAPKAPK2), a kinase
that subsequently phosphorylates the small heat shock protein Hsp27
(Gaitanaki et al., 2003).
Phosphorylation of Hsp27 contributes to stabilization of the actin
cytoskeleton, protecting cells against unfavourable stressful conditions
(Paul et al., 2002;
Concannon et al., 2003).
p38-MAPK is also involved in ANP gene expression, a cardiac hormone that has
been found to regulate adaptation to hypoxia
(Drexler et al., 1989;
Loennechen et al., 2001).
The overall aim of the present study was to assess and characterize the nature of the signal transduction mechanisms triggered by CoCl2 in the perfused amphibian heart, focusing on p38-MAPK cascade and ANP expression. Overall, our results indicate for the first time that CoCl2 simulates oxidative stress conditions in this particular experimental setting. Thus, we have detected p38-MAPK pathway activation along with an enhanced p38-MAPK-dependent presence of ANP, under the conditions examined, possibly implicating them in the mechanism regulating the compensatory response to this form of stress.
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Materials and methods |
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Rabbit polyclonal antibodies specific for dually phosphorylated p38-MAPK (#9211), MAPKAPK2 (#3041) as well as Hsp27 (#2401) were purchased from Cell Signalling (Beverly, MA, USA). The anti-actin (A2103) antibody was from Sigma-Aldrich (St Louis, MO, USA). Secondary antibodies were from DakoCytomation (Glostrup, Denmark). Pre-stained molecular mass markers were from New England Biolabs (Beverly, MA, USA). Super RX film was purchased from Fuji photo film GmbH (Dusseldorf, Germany). Most general laboratory reagents used were purchased from Sigma-Aldrich. The antioxidants used (Trolox and Lipoic acid) were synthesized and kindly provided by Dr Koufaki's group at the Institute of Organic and Pharmaceutical Chemistry at the National Hellenic Research Foundation. Catalase (C-30) and Superoxide Dismutase (SOD)-(S2515) were from Sigma-Aldrich.
Animals
Frogs (Rana ridibunda Pallas) weighing 120–150 g were caught
in the vicinity of Thessaloniki, Greece, and were supplied by a local dealer.
They were kept in containers in freshwater and received humane care in
accordance to the Guidelines for the Care and Use of Laboratory Animals
published by the Greek Government (160/1991) based on EC regulations (86/609).
They were used a week after arrival.
Heart perfusions
Frogs were anaesthetized by immersion in 0.01% (w/v) MS222 and sacrificed
by decapitation. The hearts were excised and mounted onto an aortic cannula.
Perfusions were performed in a non-recirculating Langendorff mode at a
pressure of 4.5 kPa (31.5 mmHg) with a bicarbonate-buffered saline (23 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 7.35 at 25°C) supplemented with 10 mmol
l-1 glucose and equilibrated with 95%:5%
O2:CO2. Heart and perfusate temperatures were maintained
at 25°C using a water-jacketed apparatus. All hearts were equilibrated for
15 min under these conditions. Following the equilibration period, hearts were
perfused with various concentrations of CoCl2 for 15 min or with
500 µmol l-1 CoCl2 for periods of time ranging from
0.5 to 60 min. After the 15 min equilibration hearts were also perfused with
500 µmol l-1 CoCl2 for 15 min in the presence of
Trolox (Tr) or Lipoic acid (LA) (both at 10 µmol l-1 final
concentration). Control hearts for the effect of these antioxidants were also
perfused with Tr or LA alone. Perfusions were also performed in the presence
of 1 µmol l-1 SB203580, both in the equilibration period and in
parallel with 500 µmol l-1 CoCl2, for 15 min. To
investigate ROS scavenging by CAT and SOD, perfusions were conducted in the
presence of 150 U ml-1 of CAT or 12.5 U ml-1 of SOD
after the equilibration period, along with 500 µmol l-1
CoCl2 for 15 min. Control hearts were also perfused with 150 U
ml-1 of CAT or 12.5 U ml-1 of SOD alone. At the end of
the perfusions, atria were removed and the ventricles, after immersion in
liquid N2, were pulverized under liquid N2. Powders were
stored at –80°C. During all the treatments the electromechanical
heart activity was monitored with no significant changes observed. No
viability loss was detected in any experimental intervention.
Protein extraction
Heart powders were homogenized with 3 ml g-1 of buffer G [20
mmol l-1 Tris-HCl pH 7.5, 20 mmol l-1
ß-glycerophosphate, 2 mmol l-1 EDTA, 10 mmol l-1
benzamidine, 20 mmol l-1 NaF, 0.2 mmol l-1
Na3VO4, 200 µmol l-1 leupeptin, 10 µmol
l-1 trans-epoxy-succinyl-L-leucylamido-(4-guanidino) butane (E-64),
5 mmol l-1 dithiothreitol (DTT), 300 µmol l-1
phenylmethylsulphonyl fluoride (PMSF) and 0.5% (v/v) Triton X-100]. After
extraction on ice for 15 min, samples were centrifuged (13 400
g, 5 min, 4°C), and the supernatants boiled with 0.33 vol.
of sodium dodecyl sulphate (SDS) sample buffer [SB4X: 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 reagent
Immunoblotting
Proteins (50 µg) were separated by SDS-PAGE on 10% (w/v) polyacrylamide
gels and transferred electrophoretically onto nitrocellulose membranes.
Nonspecific binding sites were blocked with 1% (w/v) bovine serum albumin
(BSA) in TBST [20 mmol l-1 Tris-Hcl, pH 7.5, 137 mmol
l-1 NaCl, 0.05% (v/v) Tween 20] for 30 min at room temperature.
Subsequently, the membranes were incubated overnight with the appropriate
primary antibody (1:1000) at 4°C. After washing in TBST (3x5 min)
blots were incubated with the respective horseradish peroxidase-conjugated
secondary antibody 1:5000 in TBST containing 1% (w/v) BSA (60 min, room
temperature, RT). After washing in TBST (3x5 min), bands were
detected using enhanced chemiluminescense (ECL), exposed to Super RX film
(Fuji photo film GmbH Dusseldorf, Germany) and quantified by laser scanning
densitometry (Gel Analyzer v. 1.0).
RNA preparation, cDNA synthesis and ratiometric reverse transcription PCR (RT-PCR)
The expression of endogenous ANP and ß-actin was determined by
ratiometric reverse transcription of total RNA followed by semi-quantitative
PCR analysis. Total RNA was extracted from heart ventricles using Trizol
(Invitrogen Life Technologies, CA, USA), according to the manufacturer's
instructions. For cDNA synthesis, 2 µg of total RNA was denatured in the
presence of 5 pmol oligo-dT primer in a reaction volume of 13.5 µl at
65°C for 5 min. Reverse transcription was performed with M-MLV Reverse
Transcriptase (Invitrogen Life Technologies), first strand buffer (Promega,
Madison, WI, USA), dithiothreitol (Promega) and deoxy-nucleotide triphosphates
(dNTPs) (Promega). The first strand reaction was incubated at 37°C for 1
h. Termination of the reaction was achieved by inactivation of the reverse
transcriptase at 70°C for 5 min. PCR for ANP was performed using 1.5 U Taq
(Bioron GmbH, Ludwigshafen, Germany) with sense 5'-CGA GGA GGT CCA GCA
TAC AAC TCC-3' and antisense 5'-CTA GAA CCT ACG GCC ACA ACC
CAT-3' primers (GenBank accession no. D01043). These primers amplify a
375-base pair (bp) PCR product. After a 5 s denaturation at 94°C, PCR was
carried out for 30 cycles (94°C for 30 s, 45°C for 30 s and 72°C
for 45 s), and then a final extension was done at 72°C for 5 min. PCR (30
cycles) for ß-actin was performed using the following primers: sense
5'-ATC TGG CAT CAC ACC TTC TAC-3' and antisense 5'-CTC CTG
CTT GCT GAT CCA C-3' (Takase et al.,
2002). cDNA samples derived from `control' and treated cells were
always amplified simultaneously. PCR products were separated on a 1.2% for ANP
and 1% for ß-actin (w/v) agarose gel supplemented with ethidium bromide
(EtBr) at a final concentration of 100 µg l-l. Band intensities
were determined and quantified by scanning densitometry using an appropriate
image analysis programme (Gel Analyzer v. 1.0). All values were normalized for
the amount of ß-actin mRNA and estimation of fragment band size (ANP 375
bp, ß-actin 900 bp) was performed by comparison with GeneRuler 100 bp DNA
ladder (Fermentas Life Sciences Inc., Hanover, USA).
Statistical evaluations
Western blots shown are representative of at least three independent
experiments. Each data point is presented as the mean ± s.e.m. of
samples from at least three separate hearts perfused under the respective
conditions. Comparisons between control and treatments were performed using
one-way analysis of variance (ANOVA) followed by Newman–Keuls post
hoc test. A value of at least P<0.05 was considered to be
statistically significant. p38-MAPK, MAPKAPK2 and Hsp27 activation in
`control' hearts was set at 1 and their stimulated activation in treated
hearts was expressed as `-fold' activation versus control hearts.
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)
or 30 µmol l-1 H2O2
(Gaitanaki et al., 2003).
MAPKAPK2 being an established p38-MAPK substrate
(Rouse et al., 1994), we
looked into its activation under the same experimental conditions. The kinase
phosphorylation pattern was found to be similar to that of p38-MAPK, maximized
at 500 µmol l-1 CoCl2 (4.2±0.61-fold relative
to control hearts, P<0.001)
(Fig. 1Aii,D). Hsp27, which is
phosphorylated by MAPKAPK2 (Stokoe et al.,
1992), was subsequently examined. In particular, Hsp27 exhibited a
response that did not differ from the aforementioned ones, with robust
phosphorylation (5.1±0.5-fold relative to control hearts,
P<0.001) also at 500 µmol l-1 CoCl2
(Fig. 1Aiii,E). Equal protein
loading was confirmed by probing with an anti-actin antibody
(Fig. 1Aiv).
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Next, so as to determine the time course of CoCl2 effect on p38-MAPK and Hsp27 activation, the kinase-stimulated phosphorylation was examined in samples from hearts perfused with 500 µmol l-1 CoCl2 for time periods ranging from 0.5 to 60 min (Fig. 1B). Under these conditions, p38-MAPK phosphorylation levels were rapidly elevated from as early as 0.5 min (8.1±0.77-fold relative to control, P<0.001) decreasing thereafter with a second maximal peak at 15 min (10.12±1.8-fold relative to control, P<0.001) that was sustained for at least 60 min (Fig. 1Bi,F). Hsp27 phosphorylation was observed to be equally prolonged, being statistically significant from 0.5 min, reaching maximal values at 15 min (5±0.67-fold relative to control, P<0.001) (Fig. 1Bii,G). Equal protein loading was once more confirmed using the anti-actin antibody (Fig. 1Biii).
CoCl2 activity as an oxidative stress stimulator is depicted in
Fig. 2. In the presence of two
different antioxidant agents, i.e. Trolox (Tr) and Lipoic acid (LA), the
phosphorylation of p38-MAPK and its substrates was considerably attenuated. In
particular, Tr inhibited CoCl2-induced p38-MAPK phosphorylation by
85±3.5% and LA was equally effective, with only a 20±1.5%
of the CoCl2-stimulated activation of the kinase remaining in its
presence (Fig. 2Ai,B). While Tr
almost abrogated MAPKAPK2 phosphorylation by CoCl2 (inhibition by
95±2.4%) the latter was markedly reduced in the presence of LA (by
60±3.1%) (Fig.
2Aii,C). Analogously, Tr was found to inhibit Hsp27
CoCl2-induced phosphorylation by 75±4% with LA
conferring a 50±3.7% inhibition
(Fig. 2Aiii,D). Note that in
order to determine the net effect of the antioxidants or inhibitors used in
parallel with CoCl2, any observed effect that these compounds have
on their own has to be deducted.
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; Scott et al.,
1994; Brookes et al.,
1998). In an attempt to specify the species of ROS inducing the
observed response, after perfusion of the amphibian heart with
CoCl2, we next examined the potential antioxidant effect of
catalase and superoxide dismutase. CAT is a known hydrogen peroxide
(H2O2) scavenger
(Fridovich, 1999;
Halliwell, 1999) while SOD is
known to mainly remove the superoxide anion (O –2)
(McCord and Fridovich, 1969).
Accordingly, both antioxidants were found to almost ablate p38-MAPK
(Fig. 3Ai,B), MAPKAPK2
(Fig. 3Aii,C) as well as Hsp27
(Fig. 3Aiii,D) phosphorylation,
thus activation. Blotting with the antibody raised against actin, we once more
confirmed equal protein loading (Fig.
3Aiv).
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Following a study by our group
(Vassilopoulos et al., 2005)
indicating p38-MAPK-mediated ANP transcript upregulation by direct oxidative
stress (perfusion with H2O2), we next tried to evaluate
mRNA levels of this natriuretic peptide, using semi-quantitative ratiometric
PCR, in samples from hearts perfused with CoCl2, in the presence or
absence of Tr (10 µmol l-1), LA (10 µmol l-1) and
1 µmol l-1 SB203580 (a known specific p38-MAPK inhibitor).
Perfusion for 15 min with 500 µmol l-1 CoCl2 was
found to significantly increase ANP mRNA levels (4.3±0.35-fold
relative to control, P<0.001)
(Fig. 4A, top panel;
Fig. 4B). We also assessed
ß-actin mRNA levels as an internal control
(Fig. 4A, bottom panel). ANP
response at the transcriptional level was almost abrogated by both
antioxidants tested, as well as SB203580
(Fig. 4).
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; Driedzic and
Gesser, 1994), e.g. as part of their winter hibernation in
northern latitudes (Hermes-Lima and
Storey, 1996). Hypoxia is defined as a state when oxygen tension
drops below normal limits, playing a pivotal role in pathological conditions
including cardiovascular diseases (Giaccia
et al., 2003; Jiang et al.,
2007; Muir et al.,
2007). In particular, myocardial hypoxia is associated with major
cellular ionic and metabolic changes leading to mechanical impairment
(Frist et al., 1978;
Kentish, 1986;
Allen and Orchard, 1987).
Nevertheless, it should be noted that frog cardiac muscle has been shown to
be resistant to hypoxia in terms of mechano-energetic properties
(Allen and Orchard, 1987).
Thus, it seemed exquisitely interesting to investigate the response of the
amphibian heart to perfusion with CoCl2, an agent that has been
widely reported to act as a chemical inducer of a hypoxic effect
(Goldberg et al., 1988). Data
addressing which signalling pathways are activated in hypoxia are
controversial. However, recent studies have indicated an emerging role for
MAPKs, especially p38-MAPK and JNKs
(Greenway and Storey, 2000;
Cowan and Storey, 2003).
Focusing on p38-MAPK, we first examined the dose- and time-dependent profile
of this kinase activation along with those of MAPKAPK2 and Hsp27
(Fig. 1). Both of the latter
proteins consist downstream components of the p38-MAPK signalling transduction
pathway (Kyriakis and Avruch,
2001) and have been shown to exert a cytoprotective effect. Hsp27,
in particular, is widely known to help maintain the cytoskeleton integrity and
hence contribute to cardioprotection under stressful conditions
(Huot et al., 1996).
Many reports show that CoCl2 can mimic the hypoxic response in
many aspects (Goldberg et al.,
1988) but also function as an oxidative stress-inducing factor
since Co (II) can react with H2O2 by a Fenton-type
reaction to produce ROS (Moorhouse et al.,
1985; Wang et al.,
1993). This water-soluble substance has been demonstrated to
increase ROS generation, and thus oxidative stress, by a non-enzymatic,
non-mitochondrial mechanism (Chandel et
al., 2000) in divergent cell types
(Tomaro et al., 1991;
Chandel et al., 2000). The
concentration used (500 µmol l-1) is applied routinely in
studies investigating this compound's effect
(Zou et al., 2002).
Among the various products of redox reactions one can distinguish ROS
(oxygen-derived) as well as RNS (nitrogen-derived) free radicals. ROS include
the superoxide anion, hydrogen peroxide and lipid peroxides, while nitric
oxide and peroxynitrite belong to the RNS
(Kyaw et al., 2004). In
excessive quantities, these reactive species formed by aerobic organisms
constitute a threat for the integrity of various biomolecules, but at
physiological concentrations they are actually required for normal cell
function (Nordberg and Arner,
2001), playing a significant physiological role in intracellular
signalling and redox regulation. NO has been identified as a signalling
molecule (Furchgott, 1995) and
a modulating factor of gene expression
(Bogdan, 2001). Furthermore,
myocardial protection in terms of ischemic preconditioning may be mediated by
ROS or NO (Baines et al., 1997;
Lochner et al., 2002)
via activation of downstream protein kinase cascades, i.e. p38-MAPK
(Rakhit et al., 2001).
Beneficial biological effects exerted by NO also include: vasodilatation,
inhibition of superoxide accumulation, attenuation of platelet adhesion and
protection of cardiac myocyte function
(Cotton et al., 2002).
Although NO can even ameliorate post-ischemic reperfusion myocardial injury it
may also confer deleterious effects via reaction with
O2– and formation of peroxynitrite, a strong
oxidizing agent (Oishi et al.,
2006). In particular, peroxynitrite (OONO–) has
been identified as a toxic metabolite of NO that causes myocardial
dysfunction, but there are other reports of its protective function
via conversion to S-nitrosothiols in blood and tissues
(Vinten-Johansen, 2000). Low
concentrations of peroxynitrite have been found to protect against
ischemia-reperfusion-induced arrhythmias
(Altug et al., 2001), and its
prolonged vasorelaxant action under physiological conditions
(Dowell and Martin, 1997)
shares many characteristics in common with NO
(Jothianandan and Furchgott,
1996). In particular, these may be attributed to its oxidant
properties or the induction of cGMP levels and activation of
K+-ATP-dependent (KATP) channels
(Moro at el., 1994). Beguin et
al. (Beguin et al., 2005) have
also demonstrated a protective role for the ATP-sensitive potassium channel in
delayed hypoxia preconditioning. What is more, KATP channels have
been identified in various tissues including the myocardium, where opening of
the mitochondrial KATP channels especially stimulates generation of
ROS that exert significant physiological roles as intracellular messengers
(Irani, 2000;
Pain et al., 2000;
Krenz et al., 2002),
conferring cardioprotection (Nagata et
al., 2003). Further supporting evidence for this scenario suggests
that mito KATP channel opening leads to the release of ROS that may
stimulate p38-MAPK activity, resulting in the observed salutary effect on the
myocardium (Yue et al.,
2002).
The sustained and prolonged timewise phosphorylation pattern of p38-MAPK
and Hsp27 observed in the present study, could probably underlie the pivotal
role of this intracellular pathway in maintaining cellular homeostasis under
the stressful conditions examined. Our group has previously shown that the
p38-MAPK
MAPKAPK2Hsp27 pathway transduces the oxidative stress
signal as exemplified by perfusion with H2O2
(Gaitanaki et al., 2003).
Hsp27 phosphorylation being the endpoint of this response, we may deduce that
it mediates cytoprotection, since it has been reported to act as a molecular
chaperone, to stabilize F-actin fibers and to inhibit the mitochondrial
apoptotic pathway by preventing cytochrome c release
(Guay et al., 1997;
Paul et al., 2002;
Concannon et al., 2003). The
biphasic phosphorylation profile of p38-MAPK
(Fig. 1Bii,D) could reflect the
key role played by the kinase in transuding the particular signal in a rapid
but at the same time, prolonged manner.
|
). Given the property of CoCl2 to produce ROS
(Moorhouse et al., 1985;
Wang et al., 1993), an effort
was made to elucidate the nature of this compound's effect on p38-MAPK
pathway. In order to decipher whether perfusion with CoCl2
represents an indirect oxidative stress stimulus, two different antioxidants
were used: both trolox (a vitamin E analogue) and lipoic acid (a disulphide
compound) inhibited CoCl2-induced p38-MAPK phosphorylation
(Fig. 2Ai,B), being equally
effective in reducing MAPKAPK2 (Fig.
2Aii,C) and Hsp27 (Fig.
2Aiii,D) CoCl2-stimulated phosphorylation levels. These
agents are known to exert their biological antioxidant activity directly by
free radical quenching as well as indirectly via promoting recycling
of other cellular antioxidants (Packer et
al., 1995).
Subsequently, in order to further identify and determine which reactive
oxygen species actually mediate the observed response of p38-MAPK pathway, we
investigated the effect of superoxide dismutase and catalase. These
antioxidant enzymes function to protect cells from the lethal effects of
excessive ROS formation (McCord and
Fridovich, 1969; Kirkman and
Gaetani, 1984). Accordingly, as shown in
Fig. 3, SOD as well as CAT
significantly attenuated the CoCl2 effect. SOD constitutes a
defense system against the superoxide anion (O –2)
(McCord and Fridovich, 1969)
while CAT removes hydrogen peroxide (H2O2)
(Fridovich, 1999;
Halliwell, 1999).
Nevertheless, given the complexity, crosstalk and multiplicity of the
metabolic pathways responsible for the generation as well as the
detoxification of free radicals, the likelihood of other species being
involved arises. Thus, although SOD and CAT are classified and extensively
used as scavengers of superoxide and hydrogen peroxide, respectively
(Li et al., 2005), one cannot
exclude their role as limiting factors of peroxynitrite formation. SOD in
particular, competes with NO over the fate of the superoxide anions, thus
minimizing OONO– formation, and the redox equilibrium
attained depends on reaction rates, targets concentration, rate constants etc.
(Beckman et al., 1992). In
addition to this, peroxynitrite is involved in the nitration of protein
tyrosine residues, a reaction where SOD is once more involved as a catalyst
(Ischiropoulos et al.,
1992).
Therefore, our results indicate that under the experimental conditions
studied, O2–, H2O2, NO and
possibly OONO– may influence, directly or indirectly,
triggering of p38-MAPK cascade activation. Our findings are further
substantiated by previous reports noting the rapid response of p38-MAPK signal
transduction pathway to oxidative stress, i.e. in the case of direct exposure
to H2O2 both in our experimental model
(Gaitanaki et al., 2003) and
in rat neonatal cardiac myocytes (Clerk et
al., 1998). The molecular mechanisms triggered during the
amphibian heart response to cobalt perfusion have been evaluated to be
specific to this metal, since our group has demonstrated that perfusion with
copper, another metal that can induce rapid generation of ROS via a
Haber–Weiss or Fenton-like reaction
(Gutteridge, 1985;
Shi and Dalal, 1992;
Gaetke and Chow, 2003), does
not stimulate p38-MAPK phosphorylation through O2–
or H2O2 (Gaitanaki
et al., 2006b).
The amphibian cardiac muscle is secreting an atrial natriuretic peptide
(ANP) very similar to the mammalian one
(Netchitailo et al., 1988) in
order to prevent heart overloading under stressful conditions
(Gisbert and Fischmeister,
1988; Netchitailo et al.,
1988; Cerra et al.,
2003). Given the fact that direct oxidative stress (i.e.
H2O2) has been found to elevate ANP mRNA levels
via the p38-MAPK signalling cascade in the frog heart
(Vassilopoulos et al., 2005),
an attempt was made to investigate ANP response to CoCl2 perfusion.
Accordingly, CoCl2 was found to stimulate ANP mRNA levels to a
significant degree, a response ablated by Trolox, Lipoic acid and SB203580
(Fig. 4). Thus, it is tenable
to postulate that ANP transcript levels appear to be modulated by reactive
species in a p38-MAPK-dependent manner. De Vito et al.
(De Vito et al., 2003) have
proposed in their study a role for ANP to aleviate oxidative stress-induced
damaging effects in rat aortic smooth muscle cells, while Kiemer et al.
(Kiemer et al., 2002) have
found ANP to be a defensive effector counteracting ischemia–reperfusion
injury, which is known to reproduce oxidative stress conditions. Furthermore,
several studies mark ANP regulation by free radicals, especially nitric oxide,
which is known to be released by cardiac myocytes
(Sanchez-Ferrer et al., 1990;
Carnio et al., 2004).
Supporting our results, Chun et al. (Chun
et al., 2003) have reported ANP transcriptional upregulation by
CoCl2 in H9c2 cardiac myoblasts. In agreement with the above
results, Thuerauf et al. (Thuerauf et al.,
1998) have also shown a role for p38-MAPK in ANP gene
transcriptional regulation in cardiac myocytes. The sustained p38-MAPK
activation by CoCl2 might act in concert with ANP transcriptional
upregulation to ensure a compensatory response to CoCl2 perfusion.
ANP could therefore function as a sensitive marker of cardiac stress, also
mediating cardioprotection.
Evidently, further studies are needed in order to fully delineate the exact
nature of reactive species mediating the observed CoCl2-induced
effect in the perfused amphibian heart. ROS have been regarded as toxic
byproducts of aerobic metabolism following reoxygenation after periods of
ischemia or hypoxia (McCord,
1985). However, accumulating data now suggest that ROS play a
vital physiological role in the context of signal transduction mechanisms
regulating redox cellular homeostasis
(Irani, 2000). Among the
factors contributing to cardioprotection under oxidative stress conditions,
recent studies have emphasized the role of nitric oxide, peroxynitrite and the
mito KATP channels, which have as end-effectors the ROS generated
or signalling molecules that are `activated' (i.e. p38-MAPK cascade members or
ANP). Hypoxia itself has been recently shown to confer a salutary effect
against prolonged ischemia in the rat via NO and KATP
channels (Beguin et al.,
2005), with p38-MAPK implicated in triggering this delayed PC
effect (Beguin et al.,
2007).
A hypothetical schematic model outlining the amphibian heart response to perfusion with CoCl2 can be seen in Fig. 5. Our results are indicative of p38-MAPK cascade and ANP playing a significant role in concert, contributing to protection of the amphibian heart against CoCl2 perfusion.
In conclusion, the hypoxia mimetic agent CoCl2 has been found to
exert an oxidative stimulus effect in our experimental model. The novel
findings of the present study implicate the p38-MAPK pathway to serve a
cytoprotective role in this context, by mediating both Hsp27 phosphorylation
and ANP mRNA upregulation. The precise identification and characterization of
the physiological role played by this network of kinases, in order to achieve
compensation for any adverse environmental conditions encountered by these
organisms in vivo remain to be determined. Overall, the amphibian
heart constitutes an excellent experimental model for the investigation of
mechanisms triggered in response to CoCl2-stimulated oxidative
stress. Amphibians have developed strategies to adapt to and overcome hypoxic
as well as oxidative stress conditions that they encounter, either due to low
oxygen availability or abrupt changes in environmental temperature or body
dehydration levels (Hermes-Lima and
Storey, 1996; Hermes-Lima and
Storey, 1998; Greenway and
Storey, 2000). Therefore, any data concerning their response to
such adverse conditions is of high physiological interest and gives insight to
the signal transduction mechanisms triggered at the whole organ level rather
than in a cell culture.
List of abbreviations
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Paul, C., Maner
P<0.05 vs control (untreated)
hearts.
