Hyperosmotic and thermal stresses activate p38-MAPK in the perfused amphibian heart
Ioanna-Katerina S. Aggeli1,
Catherine Gaitanaki1,
Antigone Lazou2 and
Isidoros Beis1,*
1 Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, Athens 157 84, Greece and
2 Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece
View larger version (40K):
[in a new window]
|
Fig. 1. Time course of p38-MAPK phosphorylation in the amphibian heart, in response to reperfusion after sorbitol treatment. (Ai) Phospho-p38-MAPK was detected in extracts (100 µg of protein) from control hearts (C), hearts perfused with 0.5 mol l–1 sorbitol for 15 min (S), or hearts reperfused for the indicated times with normal bicarbonate-buffered saline following a 15 min perfusion with 0.5 mol l–1 sorbitol. Molecular mass markers (kDa) are shown to the right. (Aii) Identical samples immunoblotted for total p38-MAPK levels, as a control for loading. (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. Re, reperfusion. The western blots are representative of three independent experiments. *Significantly different from control value (P<0.001).
|
|
View larger version (1K):
[in a new window]
|
Fig. 2. Time course of the effect of excessive NaCl concentration upon p38-MAPK phosphorylation in the amphibian heart. (Ai) Phospho-p38-MAPK was detected in extracts (100 µg of protein) from control hearts (C), hearts perfused with 0.5 mol l–1 sorbitol for 15 min (S), or hearts perfused with excess of NaCl (206 mmol l–1 final concentration) in bicarbonate-buffered saline for various time periods. (Aii) Total p38-MAPK levels in identical samples, as a control for loading. (Aiii) Phospho-p38-MAPK was also detected in extracts (100 µg of protein) from hearts reperfused with normal buffer after a 2 min perfusion with excess of NaCl. The western blots shown are representative of three independent experiments. Molecular mass markers (kDa) are shown to the right. (B,C) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. The results are means ± S.E.M. for three independent experiments. Asterisks indicate values significantly different from control values; *P<0.001, **P<0.01.
|
|
View larger version (44K):
[in a new window]
|
Fig. 3. Time course of the effect of excessive KCl concentration upon p38-MAPK phosphorylation in the amphibian heart. (Ai) Phospho-p38-MAPK was detected in extracts (100 µg of protein) from control hearts (C), hearts perfused with 0.5 mol l–1 sorbitol for 15 min (S), or hearts perfused with 16 mmol l–1 KCl in bicarbonate-buffered saline. (Aii) Phospho-p38-MAPK was also detected in extracts (100 µg) from hearts perfused for 30 s with excess of KCl and reperfused with normal bicarbonate-buffered saline. (Aiii) Total p38-MAPK levels were detected in identical samples as a control for loading. The western blots shown are representative of three independent experiments. Molecular mass markers (kDa) are shown to the right. (B,C) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Values are means ± S.E.M. for three independent experiments performed with similar results. Asterisks indicate values significantly different from control values; *P<0.001, **P<0.01,  P<0.05.
|
|
View larger version (21K):
[in a new window]
|
Fig. 4. Effect of SB 203580 on p38-MAPK activation by hyperosmotic stress. SB 203580 (1 µmol l–1) was added after a 15 min equilibration period and was present throughout the experiment. Phosphorylated (Ai) and total (Aii) p38-MAPK levels were assayed in extracts (100 µg of protein) from control hearts, as well as from hearts perfused under the various hyperosmotic conditions indicated, in the absence (–) or presence (+) of the inhibitor. The western blots shown are representative of three independent experiments performed with similar findings. Molecular mass markers (kDa) are shown to the right. (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Values are means ± S.E.M. for three independent experiments. *Value significantly different from control value (P<0.001).
|
|
View larger version (50K):
[in a new window]
|
Fig. 5. Time course of the effect of thermal stress upon p38-MAPK phosphorylation in the amphibian heart. Phosphorylated p38-MAPK was detected in extracts (100 µg of protein) from control hearts perfused at 25°C (C), hearts perfused with 0.5 mol l–1 sorbitol for 15 min at 25°C (S), or hearts perfused at 15°C (Ai) or 42°C (Aii) for the indicated times. (Aiii) Total p38-MAPK levels detected in samples from control hearts perfused at 25°C (C) or hearts perfused at 42°C for the indicated times, as a µcontrol for loading. The western blots shown are representative of three independent experiments. Molecular mass markers (kDa) are shown to the right. (B,C) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Values are means ± S.E.M. for three independent experiments performed with similar results. Values significantly different from controls are indicated; *P<0.05, **P<0.01, P<0.001.
|
|
View larger version (28K):
[in a new window]
|
Fig. 6. p38-MAPK activity in response to hyperosmolarity (NaCl 206 mmol l–1 for 2 min) and hyperthermia (42°C for 30 s) in the perfused amphibian heart. (A) The kinase activity was assayed by the in-gel kinase method in extracts (200 µg of protein) from control hearts (C), hearts perfused with hypertonic medium (H) or perfused at increased temperature (T), as described in the Materials and Methods. A typical autoradiogram is shown, representative of at least three independent experiments. The molecular mass marker (kDa) is shown to the right. (B) Densitometric analysis of phospho-p38-MAPK bands by laser scanning. Values are means ± S.E.M. for three independent experiments performed with similar results. Asterisks indicate values significantly different from control values; *P<0.001, **P<0.01.
|
|
View larger version (157K):
[in a new window]
|
Fig. 7. Immunohistochemical localisation of phosphorylated p38-MAPK in the ventricle of isolated amphibian heart perfused under various hyperosmotic conditions. Hearts were perfused under normal conditions (C), with 0.5 mol l–1 sorbitol in the presence of 1 µmol l–1 SB203580 (S+SB), or subjected to diverse forms of osmotic stress such as 0.5 mol l–1 sorbitol for 15 min (S), 0.5 mol l–1 sorbitol (15 min) followed by reperfusion (5 min) (S/Rep), 206 mmol l–1 NaCl for 2 min (NaCl), 206 mmol l–1 NaCl (2 min) followed by reperfusion (30 s) (NaCl/Rep), 16 mmol l–1 KCl for 30 s (KCl), 16 mmol l–1 KCl (30 s) followed by reperfusion (30 s) (KCl/Rep). After the removal of atria, ventricles were cryosectioned longitudinally and fixed with ice-cold acetone. Specimens were incubated with phospho-p38-MAPK antibody (1:200 dilution) and counterstained with Haematoxylin. The figure shows representative photographs from three independent experiments performed with similar results. Immunoreaction deposits are visualised with Fast Red chromogen. Bar, 20 µm.
|
|
View larger version (113K):
[in a new window]
|
Fig. 8. ANP immunolocalisation in the ventricle of isolated amphibian heart perfused under various hyperosmotic conditions. Hearts were perfused under normal conditions (C) or subjected to various forms of hyperosmotic stress such as 0.5 mol l–1 sorbitol for 15 min in the absence (S) or presence of 1 µmol l–1 SB203580 (S+SB), 206 mmol l–1 NaCl for 2 min (NaCl) and 16 mmol l–1 KCl for 30 s (KCl), respectively. (A) Paraffin- or (B) cryo-sections were incubated with an antibody specific for human ANP (1–28) (1:500 dilution) and counterstained with Haematoxylin. In sections incubated only with secondary antibody (negative control, NC) no immunoreactivity was detected. The figure shows representative photographs from three independent experiments performed with similar results. Immunoreaction deposits are visualised with Fast Red chromogen. Bar, 20 µm.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2002
