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First published online November 17, 2005
Journal of Experimental Biology 208, 4427-4436 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01924
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Acute thermal stress and various heavy metals induce tissue-specific pro- or anti-apoptotic events via the p38-MAPK signal transduction pathway in Mytilus galloprovincialis (Lam.)

Erene Kefaloyianni, Eleni Gourgou, Vanessa Ferle, Efstathios Kotsakis, Catherine Gaitanaki and Isidoros Beis*

Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, Athens 157 84, Greece



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  		Fig. 1. Effect of various heavy metals on the p38-MAPK phosphorylation in M. galloprovincialis mantle tissue. (A) Phospho-p38-MAPK was detected in extracts (100 µg of protein) from control animals (C) or animals treated with either 1 µmol l–1 CuCl2 (top panel), 50 µmol l–1 ZnCl2 (middle panel) or 1 µmol l–1 CdCl2 (bottom panel) for the times indicated. Western blots shown are representative of four to six independent experiments. The molecular mass markers (kDa) are shown to the right of the panel. (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± S.E.M. for four to six independent experiments performed with similar findings. {dagger}P<0.001, *P<0.05, **P<0.01 versus control value.

 


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  		Fig. 2. Effect of SB203580 on the mantle tissue p38-MAPK activation by 1 µmol l–1 CuCl2. SB203580 (1 µmol l–1) was added to normal seawater and, after a 15 min equilibration period of the animals, it was present throughout the experiment. Phosphorylated (A, top panel) and total (A, bottom panel) p38-MAPK levels were assayed in mantle tissue extracts (100 µg of protein) from control animals, as well as from animals treated with 1 µmol l–1 CuCl2 in the absence or presence of the inhibitor. Western blots shown are representative of four to six independent experiments performed with similar findings. The molecular mass markers (kDa) are shown to the right of the panel. (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± S.E.M. for four independent experiments. {dagger}P<0.001 versus control value.

 


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  		Fig. 3. Effect of various heavy metals on the p38-MAPK phosphorylation in M. galloprovincialis gill tissue. (A) Phospho-p38-MAPK was detected in extracts (100 µg of protein) from control animals (C) or animals treated with either 1 µmol l–1 CuCl2 (top panel), 50 µmol l–1 ZnCl2 (middle panel) or 1 µmol l–1 CdCl2 (bottom panel) for the times indicated. Western blots shown are representative of four to six independent experiments. The molecular mass markers (kDa) are shown to the right of the panel. (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± S.E.M. for four to six independent experiments performed with similar findings. {dagger}P<0.001, **P<0.01 versus control value.

 


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  		Fig. 4. Time course of the effect of thermal stress and synergistic effect of Cu2+ combined with hyperthermia (30°C) upon p38-MAPK phosphorylation in M. galloprovincialis mantle tissue. Phosphorylated p38-MAPK was detected in extracts (100 µg of protein) from control animals maintained at 15°C (Con) or animals maintained at 4°C (A, top panel) or 30°C (A, bottom panel) for the indicated times. The molecular mass markers (kDa) are shown to the right of the panel. (C) Phosphorylated p38-MAPK (top panel) or total p38-MAPK (bottom panel) levels were detected in extracts (100 µg of protein) from control animals maintained at 15°C and animals maintained at 30°C for 30 min, either in the absence or presence of 1 µmol l–1 CuCl2. Western blots shown 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 performed with similar results. {dagger}P<0.001, *P<0.05, **P<0.01 versus control value.

 


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  		Fig. 5. DNA fragmentation and caspase-3 activation in the mantle and gill tissues from M. galloprovincialis specimens treated with various heavy metals. (A) (Left panel) DNA fragmentation induced by 1 µmol l–1 CuCl2 for 30 min, in the absence or presence of 1 µmol l–1 SB203580, or 50 µmol l–1 ZnCl2 for 30 min in the mantle tissue. (Right panel) DNA fragmentation induced by 1 µmol l–1 CuCl2 for 30 min, 50 µmol l–1 ZnCl2 for 30 min or 1 µmol l–1 CdCl2 for 60 min in the gill tissue. Gels shown are representative of three independent experiments performed with similar results. (B) Specimens (four animals per group) were incubated in normal seawater (controls) or treated with either 1 µmol l–1 CuCl2 in the absence or presence of 1 µmol l–1 SB203580, 50 µmol l–1 ZnCl2 or 1 µmol l–1 CdCl2 (for 30, 30 or 60 min, for each heavy metal, respectively). Endogenous full-length pro-caspase-3 and large active fragments of caspase-3 were detected using a specific rabbit monoclonal antibody in extracts (100 µg of protein) from mantle (top panel) and gill (bottom panel) tissues. Western blots shown are representative of four independent experiments performed with similar results. n.s., non specific.

 


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  		Fig. 6. Induction of Hsp70 expression by thermal stress and/or 1 µmol l–1 CuCl2 in M. galloprovincialis gill tissue. (A) Hsp70 was detected in extracts (100 µg of protein) from gill tissue of animals incubated at 30°C for 30 min either in the absence or presence of 1 µmol l–1 CuCl2 or at 15°C with 1 µmol l–1 CuCl2 in the absence or presence of 1 µmol l–1 SB203580 for 30 min. (B) Densitometric analysis of Hsp70 bands by laser scanning. Results are means ± S.E.M. for four independent experiments performed with similar results. The molecular mass markers (kDa) are shown to the right of the panel. {dagger}P<0.001, **P<0.01 versus control value.

 

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© The Company of Biologists Ltd 2005