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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.