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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

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CoCl₂ induces protective events via the p38-MAPK signalling pathway and ANP in the perfused amphibian heart

Catherine Gaitanaki, Theodora Kalpachidou, Ioanna-Katerina S. Aggeli, Panagiota Papazafiri and Isidoros Beis^*

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

View larger version (34K): [in this window] [in a new window]   Fig. 1. Western blots showing dose–response (A) and time course (B) of p38-MAPK, MAPKAPK2 and Hsp27 phosphorylation in the amphibian heart after CoCl2 treatment. (Ai) Phospho-p38-MAPK was detected in extracts (50 µg of protein) from control hearts (C) or hearts perfused for 15 min with the indicated concentration of CoCl2. (Aii) Phospho-MAPKAPK2 was detected in identical samples. (Aiii) Phospho-Hsp27 was detected in identical samples. An anti-actin antibody was used as a control for equal protein loading (Aiv). (Bi) Phospho-p38-MAPK was detected in extracts (50 µg of protein) from control hearts (C) or hearts perfused with 500 µmol l-1 CoCl2 for increasing periods of time. (Bii) Phospho-Hsp27 was detected in identical samples. An anti-actin antibody was used as a control for equal protein loading (Biii). (C–G) Densitometric analysis of phospho-p38 (C,F), phospho-MAPKAPK2 (D) and phospho-Hsp27 (E,G) bands was performed by laser scanning. Western blots shown are representative of at least three independent experiments; data are means ± s.e.m. for at least three independent experiments. *P<0.001 and P<0.05 vs control (untreated) hearts.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Western blots showing the effect of Trolox (Tr) and Lipoic acid (LA) on CoCl2-induced phosphorylation of p38-MAPK (Ai), MAPKAPK2 (Aii) and Hsp27 (Aiii) after CoCl2 treatment. The phosphorylated forms of the kinases were detected in extracts (50 µg of protein) from control hearts (control) or hearts perfused for 15 min with 500 µmol l-1 of CoCl2 in the presence or absence of TR (10 µmol l-1) and LA (10 µmol l-1). The effect of Tr and LA alone was also assessed. An anti-actin antibody was used as a control for equal protein loading (Aiv). Densitometric analysis of phospho-p38 (B), phospho-MAPKAPK2 (C) and phospho-Hsp27 (D) was performed by laser scanning. Western blots shown are representative of at least three independent experiments; data are means ± s.e.m. for at least three independent experiments. *P<0.001 vs control (untreated) hearts; P<0.05 vs CoCl2-treated hearts.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Western blots showing the effect of superoxide dismutase (SOD) and catalase (CAT) on CoCl2-induced phosphorylation of p38-MAPK (Ai), MAPKAPK2 (Aii) and Hsp27 (Aiii) after CoCl2 treatment. The phosphorylated forms of the kinases were detected in extracts (50 µg of protein) from control hearts (control) or hearts perfused for 15 min with 500 µmol l-1 of CoCl2 in the presence or absence of SOD (12.5 U ml-1) and CAT (150 U ml-1). The effect of SOD and CAT alone were also assessed. An anti-actin antibody was used as a control for equal protein loading (Aiv). Densitometric analysis of phospho-p38 (B), phospho-MAPKAPK2 (C) and phospho-Hsp27 (D) was performed by laser scanning. Western blots shown are representative of at least three independent experiments; data are means ± s.e.m. for at least three independent experiments. *P<0.001 vs control (untreated) hearts; P<0.001 vs CoCl2-treated hearts.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Effect of Trolox (Tr), Lipoic acid (LA) and SB203580 (SB) on atrial natriuretic peptide (ANP) mRNA levels in samples from CoCl2-perfused amphibian hearts. (A) RT-PCR analysis of RNA isolated from the ventricles of cardiac muscle perfused with 500 µmol l-1 CoCl2 in the presence or absence of Tr (10 µmol l-1), LA (10 µmol l-1) and SB203580 (1 µmol l-1). Amplification of ANP (top) and ß-actin (bottom) cDNA in identical samples was performed. (B) After densitometric analysis of the PCR products the results were normalized for ß-actin and presented as fold stimulation, expressed as ANP/ß-actin ratio. Results are means ± s.e.m. for at least three independent experiments. *P<0.001 vs control (untreated) hearts; P<0.001 vs CoCl2-treated hearts. The PCR products of ANP (375 bp) and ß-actin (900 bp) were analysed in 1.2% and 1% (w/v) agarose gel electrophoresis, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 5. A hypothetical schematic model for regulation of p38-MAPK, MAPKAPK2 and Hsp27 phosphorylation in CoCl2-perfused amphibian hearts. activation, –| inhibition.

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