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First published online January 17, 2007
Journal of Experimental Biology 210, 438-446 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02680

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Cu²⁺ and acute thermal stress induce protective events via the p38-MAPK signalling pathway in the perfused Rana ridibunda heart

Catherine Gaitanaki, Maria Pliatska, Konstantina Stathopoulou 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 (43K): [in this window] [in a new window]   Fig. 1. Perfusion protocols. For further details, see Materials and methods; for abbreviations, see List of abbreviations.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Phosphorylation of p38-MAPK and Hsp-27 by CuCl2. (A,C) Protein (A, 50 µg or C, 100 µg) from Rana ridibunda hearts perfused in the absence (Con) or presence of increasing concentrations of CuCl2 (50–500 µmol l–1) for 15 min was analysed by immunoblotting with anti-p38-MAPK (Ai) and anti-Hsp27 (C) phosphospecific antibodies. As a positive control, extracts from hearts perfused with 50 µmol l–1 H2O2 for 2 min were included. Identical samples were assayed with an anti-actin antibody as a control for protein loading (Aii). (B,D) Densitometric analysis of phospho-p38-MAPK (B) and phospho-Hsp27 (D) bands, by laser scanning. Results are means ± s.e.m. for three independent experiments. *P<0.05, **P<0.01, P<0.001 vs control value.

View larger version (6K): [in this window] [in a new window]   Fig. 3. Effect of the selective inhibitor SB203580 on the p38-MAPK and Hsp27 phosphorylation induced by CuCl2. Protein (50 µg, top and bottom, and 100 µg, middle) from hearts perfused without or with 500 µmol l–1 CuCl2 for 15 min in the absence or presence of 1 µmol l–1 SB203580 was analysed by immunoblotting with phosphospecific anti-p38-MAPK (top), phosphospecific anti-Hsp27 (middle) or anti-actin (bottom) antibodies. Western blots shown are representative of four independent experiments performed with similar results.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Time course of p38-MAPK phosphorylation in the amphibian heart, in response to reperfusion after CuCl2 treatment. (A) Phospho-p38-MAPK was detected in extracts (50 µg protein) from control hearts (Con), hearts perfused with 500 µmol l–1 CuCl2 for 15 min or hearts perfused for the indicated times with normal bicarbonate-buffered saline following the 15 min perfusion with 500 µmol l–1 CuCl2. As a positive control, extract from hearts perfused with 50 µmol l–1 H2O2 for 2 min was used (Ai). Equal loading was assessed, as previously, using an actin antibody (Aii). (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± s.e.m. for three independent experiments. Re, reperfusion. *P<0.05, **P<0.01, P<0.001 vs control value.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Phosphorylation of p38-MAPK and Hsp27 by the combined effects of CuCl2 and hyperthermia (42°C). (A,C) Phosphorylated p38-MAPK (A,top) and Hsp27 (C) levels were detected in extracts (50 and 100 µg of protein, respectively) from Rana ridibunda hearts perfused for 15 min with normal bicarbonate-buffered saline maintained at 25°C (Con) or at 42°C, either in the absence or presence of 500 µmol l–1 CuCl2. As a positive control, extracts from hearts perfused with 50 µmol l–1 H2O2 for 2 min were included. Identical samples were analysed using an actin antibody as a control for protein loading (A,bottom). (B,D) Densitometric analysis of phospho-p38-MAPK (B) and phospho-Hsp27 (D) bands by laser scanning. Results are means ± s.e.m. for three independent experiments. *P<0.05, **P<0.01, P<0.001 vs control value.

View larger version (10K): [in this window] [in a new window]   Fig. 6. Effect of L-ascorbic acid on the CuCl2–induced p38-MAPK phosphorylation. (A) Protein (50 µg) from Rana ridibunda hearts perfused without (Con) or with 100 µmol l–1 L-ascorbic acid (ASC) for 15 min, in the presence or absence of 500 µmol l–1 CuCl2, was analysed by immunoblotting with a phosphospecific anti-p38-MAPK antibody (top). Equal loading was verified by blotting identical samples with an anti-actin-specific antibody (bottom). (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± s.e.m. for three independent experiments performed with similar findings. *P<0.05, P<0.001 vs control value.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Effect of different antioxidants on the p38-MAPK phosphorylation induced by CuCl2 in the absence or presence of hyperthermia (42°C). Phospho-p38-MAPK was detected in extracts (50 µg protein) from control hearts (Con), hearts perfused for 15 min with 30 U ml–1 SOD alone or with 500 µmol l–1 CuCl2, either in the absence or the presence of 30 U ml–1 SOD, 150 U ml–1 CAT or the combination of SOD+CAT, at 25°C. In addition, hearts were perfused for 15 min with 500 µmol l–1 CuCl2 and identical combinations of antioxidant agents but at 42°C (Ai). Actin protein levels of identical samples were detected so as to confirm equal protein loading (Aii). Densitometric analysis of phospho-p38-MAPK bands by laser scanning was performed (B). Results are means ± s.e.m. for three independent experiments. *P<0.05, **P<0.01, P<0.001 vs control value.

View larger version (6K): [in this window] [in a new window]   Fig. 8. Absence of caspase-3 activation by CuCl2, at 25°C or at 42°C, in the absence or presence of CAT and SOD. Immunoblot analysis using a specific antibody that recognizes both the inactive full-length pro-caspase-3 and the fragmented active form of the protein led to the detection of only pro-caspase-3 in samples (100 µg protein) from hearts perfused without or with 500 µmol l–1 CuCl2 for 15 min, either in the absence or the presence of 30 U ml–1 SOD, 150 U ml–1 CAT or the combination of SOD+CAT, at 25°C or at 42°C. Western blot shown is representative of three independent experiments performed with similar results. The positions of marker proteins are shown on the left.