Oxidative stress stimulates multiple MAPK signalling pathways and phosphorylation of the small HSP27 in the perfused amphibian heart
Catherine Gaitanaki,
Stathopoulou Konstantina,
Stavridou Chrysa and
Isidoros Beis*
Department of Animal and Human Physiology, School of Biology, Faculty
of Sciences, University of Athens, Panepistimioupolis, Athens 157 84,
Greece
View larger version (28K):
[in a new window]
|
Fig. 1. Activation of p43-ERK by H2O2. (A) Top panel: R.
ridibunda hearts were perfused in the absence (C) or presence of 100
µmol l-1 H2O2 for the times indicated, and
phosphorylation of p43-ERK (50 µg of protein) was assessed by immunoblot.
Bottom panel: an immunoblot of identical samples for total p43-ERK levels was
included as a control for protein loading. (C) H2O2
dose-dependent activation of p43-ERK. Hearts were perfused with 3–1000
µmol l-1 H2O2 for 5 min. As positive
controls, extracts from hearts perfused with 1 µmol l-1 PMA for
10 min were included. Blots are representative of three independent
experiments. (B,D) Densitometric analysis of phospho-p43-ERK bands by laser
scanning. Results are means ± S.E.M. for three independent
experiments performed with similar findings. *P<0.05,
**P<0.01 vs control value.
|
|
View larger version (17K):
[in a new window]
|
Fig. 2. Effect of selective inhibitors on the p43-ERK phosphorylation induced by
oxidative stress. Protein (50 µg) from hearts perfused with 30 µmol
l-1 H2O2 for 5 min in the absence or presence
of 25 µmol l-1 PD98059 or 1 µmol l-1 SB203580 was
assessed by immunoblot using phosphospecific anti-ERK (A, top panel) or total
(phosphorylation state independent) anti-ERK antibody (A, bottom panel). Blots
are representative of three independent experiments. (B) Densitometric
analysis of phospho-p43-ERK bands by laser scanning. Results are means
± S.E.M. for three independent experiments.
*P<0.05 vs control value.
|
|
View larger version (31K):
[in a new window]
|
Fig. 3. Time course of JNKs activation by 100 µmol l-1
H2O2. Protein (100 µg) from hearts perfused in the
absence (C) or presence of 100 µmol l-1
H2O2 for the times indicated was assessed by immunoblot
using phosphospecific anti-JNKs antibody (A, top panel) or total anti-JNKs
antibody (B). Blots are representative of three independent experiments. The
bottom panel in A shows quantification of JNK1 and JNK2 bands by laser
scanning densitometry. Results are means ± S.E.M. for three
independent experiments. *P<0.05,
 P<0.001 vs control value.
|
|
View larger version (31K):
[in a new window]
|
Fig. 4. Activation of p38-MAPK by H2O2. (A) Protein (100
µg) from R. ridibunda hearts perfused in the absence (C) or
presence of 100 µmol l-1 H2O2 for the
times indicated was assessed by immunoblot using phosphospecific anti-p38-MAPK
antibody (top panel) or total anti-p38-MAPK antibody (bottom panel). (C)
Hearts were perfused with increasing concentrations of
H2O2 (3–1000 µmol l-1) for 2 min. As
positive controls, extracts from hearts perfused with 0.5 mol l-1
sorbitol (S) for 15 min were included. Blots are representative of four
independent experiments. (B,D) Densitometric analysis of phospho-p38-MAPK
bands by laser scanning. Results are means ± S.E.M. for four
independent experiments. *P<0.05,
**P<0.01, P<0.001
vs control value.
|
|
View larger version (25K):
[in a new window]
|
Fig. 5. Effect of the selective inhibitor SB203580 on the p38-MAPK phosphorylation
induced by oxidative stress. Protein (100 µg) from hearts perfused with 100
µmol l-1 H2O2 for 2 min in the absence or
presence of 1 µmol l-1 SB203580 was assessed by immunoblot using
phosphospecific anti-p38-MAPK (A, top panel) or total anti-p38-MAPK antibody
(A, bottom panel). As a positive control, extract from hearts perfused with
0.5 mol l-1 sorbitol was included. Blots are representative of
three independent experiments. (B) Densitometric analysis of phospho-p38-MAPK
bands by laser scanning. Results are means ± S.E.M. for
three independent experiments. P<0.001
vs control value.
|
|
View larger version (30K):
[in a new window]
|
Fig. 6. Phosphorylation of MAPKAPK2 by 30 µmol l-1
H2O2. (A) Protein (100 µg) from control hearts (C),
hearts perfused with 0.5 mol l-1 sorbitol for 15 min (S), hearts
perfused with 30 µmol l-1 H2O2 in the
absence of inhibitor for the times indicated or the presence of 1 µmol
l-1 SB203580 for 2 min (I) was assessed by immunoblot using
phosphospecific (top panel) or total (bottom panel) anti-MAPKAPK2 antibodies.
Blots shown are representative of three independent experiments. (B)
Densitometric analysis of phospho-MAPKAPK2 bands by laser scanning. Results
are means ± S.E.M. for three independent experiments.
**P<0.01 vs control value.
|
|
View larger version (20K):
[in a new window]
|
Fig. 7. Phosphorylation of HSP27 by H2O2. Protein (100 µg)
from hearts perfused with 30 µmol l-1 H2O2
for 2 min in the absence or presence of 25 µmol l-1 PD98059 or 1
µmol l-1 SB203580 was assessed by immunoblot for phosphospecific
anti-HSP27 antibody (A, top panel) or total anti-HSP antibody (A, bottom
panel). As a positive control, extract from hearts perfused with 0.5 mol
l-1 sorbitol was included. Blots are representative of three
independent experiments. (B) Densitometric analysis of phospho-HSP27 bands by
laser scanning. Results are means ± S.E.M. for three
independent experiments. P<0.001 vs
control value. C, control; S, sorbitol.
|
|
View larger version (129K):
[in a new window]
|
Fig. 8. Immunohistochemical localisation of phosphorylated p38-MAPK in the
ventricle of isolated amphibian heart perfused with
H2O2. Hearts were perfused (A) under normal conditions,
with 30 µmol l-1 H2O2 for 2 min in (B) the
absence or (D) the presence of 1 µmol l-1 SB203580 or (C) with
0.5 mol l-1 sorbitol for 15 min. Cryosections were incubated with
phosphospecific anti-p38-MAPK antibody (1:200 dilution) and processed as
described in Materials and methods. Representative photographs from three
independent experiments are shown. Immunolocalisation deposits are visualised
with Fast Red chromogen. Scale bar, 20 µm.
|
|
View larger version (117K):
[in a new window]
|
Fig. 9. Immunohistochemical localisation of phosphorylated HSP27 in the ventricle
of isolated amphibian heart perfused with H2O2. Hearts
were perfused (A) under normal conditions, with 30 µmol l-1
H2O2 for 2 min in (B) the absence or (D) the presence of
1 µmol l-1 SB203580 or (C) with 0.5 mol l-1 sorbitol
for 15 min. Cryosections were incubated with phosphospecific anti-HSP27
antibody (1:200 dilution) and processed as described in Materials and methods.
Representative photographs from three independent experiments are shown. Scale
bar, 20 µm.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2003
