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First published online March 17, 2006
Journal of Experimental Biology 209, 1344-1354 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02134
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Extracellular pH changes activate the p38-MAPK signalling pathway in the amphibian heart

Konstantina Stathopoulou, 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


Figure 1
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  		Fig. 1. Phosphorylation of p38-MAPK by extracellular alkalosis (pH 8.5). (A) Protein (50 µg) from Rana ridibunda hearts perfused without (Co) or with Tris–Tyrode's buffer (pH 8.5) for the times indicated was assessed by immunoblot analysis using a phosphospecific anti-p38-MAPK antibody (top) or total p38-MAPK antibody as a control for equal loading (bottom). Extract from hearts perfused with 0.5 mol l–1 sorbitol (Sor) for 15 min was used as a positive control. (C) Time course of p38-MAPK phosphorylation induced by reperfusing hearts subjected to extracellular alkalosis (pH 8.5, 2 min; top). Equal loading was assessed by blotting identical samples with an anti-actin-specific antibody (bottom). (B,D) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Values are means ± s.e.m. for three independent experiments performed with similar findings. *P<0.05, **P<0.01, {dagger}P<0.001 vs control value.

 

Figure 2
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  		Fig. 2. Effect of the specific inhibitor SB203580 on the p38-MAPK phosphorylation induced by extracellular alkalosis (pH 8.5). (A) Protein (50 µg) from hearts perfused without (Co) or with an alkaline (pH 8.5) Tris–Tyrode's solution for 2 min in the absence (–) or presence (+) of 1 µmol l–1 SB203580 was assessed by immunoblot using phosphospecific anti-p38-MAPK (top) or anti-actin antibody (bottom). (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± s.e.m. for three independent experiments. *P<0.05 vs control value.

 

Figure 3
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  		Fig. 3. Phosphorylation of p38-MAPK by more intense extracellular alkalosis (pH 9.5) (A top, B) or acidosis (pH 6.5) (C top, D). (A) Phospho-p38-MAPK was detected in extracts (50 µg of protein) from control hearts (Co) and hearts perfused with a Tris–Tyrode's perfusion buffer of pH 9.5 for the indicated times (top). Total p38-MAPK levels were detected in identical samples as a control for loading (bottom). (C) The p38-MAPK phosphorylation was also measured by immunoblot analysis in samples from hearts subjected to extracellular acidosis (pH 6.5) for increasing periods of time using the MES–Tyrode's perfusion buffer (top), as described in Materials and methods. Equal loading was assessed, as previously, using a p38-MAPK antibody (bottom). As a positive control, extract from hearts perfused with 0.5 mol l–1 sorbitol (Sor) for 15 min was used. (B,D) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Results are means ± s.e.m. for three independent experiments. *P<0.05, **P<0.01, {dagger}P<0.001 vs control value.

 

Figure 4
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  		Fig. 4. Effect of inhibitors of the sodium exchangers, amiloride (Am), HOE642 and ouabain (Ou), on the alkalosis-induced phosphorylation of p38-MAPK. (A) 50 µg of protein from hearts perfused under alkaline extracellular conditions (pH 8.5, 2 min) in the presence or absence of 100 µmol l–1 amiloride, 5 µmol l–1 HOE642 and 100 µmol l–1 ouabain was immunoblotted against phospho-p38-MAPK (top) and total p38-MAPK (bottom) using the corresponding antibodies. (B) Densitometric analysis of phospho-p38-MAPK by laser scanning. Co, control. (C) Relation of the net p38-MAPK phosphorylation induced by each inhibitor in the presence of extracellular alkalosis to the alkalosis-induced p38-MAPK phosphorylation. Values are means ± s.e.m. for three independent experiments. *P<0.05, **P<0.01 vs control value.

 

Figure 5
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  		Fig. 5. (A) Extracts (100 µg of protein) from hearts perfused with an alkaline (pH 8.5) Tris–Tyrode's perfusion buffer for the indicated times were assayed for MAPKAPK2 phosphorylation through immunoblot analysis using an antibody specific for the phosphorylated form of MAPKAPK2 (top). Samples from hearts perfused with 0.5 mol l–1 sorbitol (Sor) for 15 min were used as positive control. Equal loading was assessed in identical samples using an antibody against total MAPKAPK2 (bottom). (B) Densitometric analysis of phospho-MAPKAPK2 bands by laser scanning. Results are means ± s.e.m. for three independent experiments. *P<0.05, **P<0.01 vs control value.

 

Figure 6
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  		Fig. 6. Phosphorylation of HSP27 induced by extracellular alkalosis (pH 8.5) and effect of the p38-MAPK specific inhibitor SB203580. (A) Protein (100 µg) from hearts perfused without (Co) or with a Tris–Tyrode's buffer of pH 8.5 for 2 min in the presence or absence of 1 µmol l–1 SB203580 was used to perform western blot analysis with antibodies specific for the phosphorylated HSP27 (top) or actin (bottom). (B) Densitometric analysis of phospho-HSP27 by laser scanning. Values are means ± s.e.m. for three independent experiments. *P<0.05 vs control value.

 

Figure 7
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  		Fig. 7. Immunohistochemical localisation of phospho-p38-MAPK (A–C) and phospho-HSP27 (D–F) in the ventricle of isolated amphibian heart perfused with Tris–Tyrode's perfusion buffers of various pHs: a normal (control heart) (A,D) or alkaline [(B,E), pH 8.5 or (C,F), pH 9.5] for 2 min. Cryosections were incubated with a phosphospecific anti-p38-MAPK (1:200 dilution) or a phosphospecific anti-HSP27 (1:200 dilution) antibody and processed as described in Materials and methods. Immunolocalisation deposits were visualized with Fast Red chromogen. Representative photographs of three independent experiments are shown. Green arrows indicate the perinuclear localisation. Scale bars, 20 µm.

 

Figure 8
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  		Fig. 8. Measurement of HSP70 protein levels by immunoblot analysis under extracellular alkaline (pH 8.5) conditions. (A) Protein (50 µg) from hearts perfused with a Tris–Tyrode's solution of pH 8.5 for the indicated times was immunoassayed with an anti-HSP70 antibody. Co, control. (B) Densitometric analysis of HSP70 bands by laser scanning. Results are means ± s.e.m. for three independent experiments. *P<0.05 vs control value. (C) Effect of the p38-MAPK specific inhibitor SB203580 (1 µmol l–1) on the protein levels of HSP70 in the presence of extracellular alkalosis.

 




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