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₁- and ß-adrenoceptor stimulation differentially activate p38-MAPK and atrial natriuretic peptide production in the perfused amphibian heart

Ioanna-Katerina S. Aggeli¹, Catherine Gaitanaki¹, Antigone Lazou² and Isidoros Beis^1,*

¹ Department of Animal and Human Physiology, School of Biology, Faculty of Sciences, University of Athens, Panepistimioupolis, Athens, Greece 157 84
² Laboratory of Animal Physiology, Department of Zoology, School of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece 54 006

View larger version (41K): [in a new window]   Fig. 1. Time course of MAPK phosphorylation by phenylephrine (PHE) in the amphibian heart. ERK (Ai), JNK (Bi) and p38-MAPK (Ci) phosphorylation was assessed by western immunoblotting in samples (50 µg, 100 µg and 100 µg of protein, respectively) from control (C) Rana ridibunda hearts and hearts perfused with 50 µmol l-1 PHE for the times indicated, using the respective phospho-specific antibodies. Total (phosphorylation state-independent) levels of ERK, JNK1 or p38-MAPK were also assessed using respective antibodies, as described in Materials and methods (Aii-Cii). Western blots are representative of three independent experiments. Bands were quantified by laser scanning densitometry and plotted (Aiii-Ciii). Values are means ± S.E.M. for the relative time points (N=3 separate heart perfusions at each time point). Values significantly different from controls: *P<0.05, **P<0.01, P<0.001.

View larger version (28K): [in a new window]   Fig. 2. Effect of SB203580 on p38-MAPK activation by phenylephrine (PHE). SB203580 (1 µmol l-1) was added after a 15 min equilibration period and was present throughout the experiment. Samples (100 µg of protein) from control Rana ridibunda hearts, hearts perfused with 0.5 mol l-1 sorbitol and hearts perfused with 50 µmol l-1 PHE in the presence or absence of SB203580 were assayed for p38-MAPK phosphorylation (Ai) as well as for total p38-MAPK immunoreactivity (Aii). The experiment was repeated on two further occasions with similar results. Bands were quantified by laser scanning densitometry and plotted (B). **Values significantly different from controls (P<0.01).

View larger version (30K): [in a new window]   Fig. 3. Effect of - and ß-adrenergic antagonists on p38-MAPK stimulation by phenylephrine (PHE). (A) p38-MAPK phosphorylation was assessed by western immunoblotting in samples (100 µg of protein) from control Rana ridibunda hearts and hearts perfused with 50 µmol l-1 PHE in the presence or absence of 1 µmol l-1 prazosin (PZ) (Ai), 1 µmol l-1 phentolamine (PHT) (Aii), 1 µmol l-1 yohimbine (YOH) (Aiii) and 1 µmol l-1 propranolol (PRO) (Aiv). Western blot analysis was performed as described in Materials and methods. Identical samples were also assayed for total p38-MAPK immunoreactivity (B). Each experiment was repeated on two further occasions with similar results. Bands were quantified by laser scanning densitometry and plotted (C). Asterisks indicate values significantly different from controls: *P<0.05, **P<0.01.

View larger version (34K): [in a new window]   Fig. 4. Time course of p38-MAPK activation by isoproterenol (ISO). p38-MAPK phosphorylation was assessed by western immunoblotting in samples (100 µg of protein) from control (C) Rana ridibunda hearts, hearts perfused with 0.5 mol l-1 sorbitol (S) and hearts perfused with 50 µmol l-1 ISO for the times indicated at 25 ° (A) and 18 °C (Bi), using a phospho-specific p38-MAPK antibody. Total kinase levels were also assayed (Bii). Bands were quantified by laser scanning densitometry and plotted (C). Values are means ± S.E.M. for the relative time points (N=3 separate heart perfusions at each time point). Values significantly different from controls: *P<0.05, **P<0.01, P<0.001.

View larger version (30K): [in a new window]   Fig. 5. Effect of - and ß-AR antagonists on p38-MAPK stimulation by isoproterenol (ISO). p38-MAPK phosphorylation was assessed by western immunoblotting in samples (100 µg of protein) from control Rana ridibunda hearts and hearts perfused with 50 µmol l-1 ISO in the presence or absence of either 1 µmol l-1 propranolol (PRO) (Ai) or 10 µmol l-1 prazosin (PZ) (Aii). Western blot analysis was performed as described in Materials and methods. Identical samples were also assayed for total p38-MAPK immunoreactivity (Aiii). Values are means ± S.E.M. Each experiment was repeated on at least two further occasions with similar results. Bands were quantified by laser scanning densitometry and plotted (B). Values significantly different from controls: P<0.001.

View larger version (139K): [in a new window]   Fig. 6. Immunohistochemical localisation of phosphorylated p38-MAPK in the ventricle of isolated amphibian heart perfused under normal conditions (A) and with 50 µmol l-1 of phenylephrine (PHE) for 0.5 min in the absence (B) or presence of either 1 µmol l-1 prazosin (C), 1 µmol l-1 propranolol (D) or 1 µmol l-1 SB203580 (E). Cryosections were incubated with phospho-p38 MAPK specific antibody (1:200 dilution) and counterstained with Haematoxylin. Representative photographs from three independent experiments performed with similar results are shown. Immunolocalisation deposits are visualised with Fast Red chromogen. Bar, 20 µm.

View larger version (140K): [in a new window]   Fig. 7. Immunohistochemical localisation of phosphorylated p38-MAPK in the ventricle of isolated amphibian heart perfused under normal conditions (A) and with 50 µmol l-1 of isoproterenol (ISO) for 5 min in the absence (B) or presence of either 10 µmol l-1 prazosin (C), 1 µmol l-1 propranolol (D) or 1 µmol l-1 SB203580 (E). Cryosections were incubated with phospho-p38 MAPK specific antibody (1:200 dilution) and counterstained with Haematoxylin. Representative photographs from three independent experiments performed with similar results are shown. Immunolocalisation deposits are visualised with Fast Red chromogen. Bar, 20 µm.

View larger version (138K): [in a new window]   Fig. 8. ANP immunolocalisation in the ventricle of isolated amphibian heart perfused with phenylephrine (PHE) and various adrenoceptor antagonists or SB203580. Hearts were perfused under normal conditions (A) and with 50 µmol l-1 of PHE for 0.5 min in the absence (B) or presence of either 1 µmol l-1 prazosin (C), 1 µmol l-1 propranolol (D) or 1 µmol l-1 SB203580 (E). Cryosections were incubated with an antibody specific for human atrial natriuretic peptide (ANP 1-28) (1:500 dilution) and counterstained with Haematoxylin. Immunoreaction deposits for ANP were visualised with Fast Red Chromogen. Representative photographs from three independent experiments are shown. Bar, 20 µm.

View larger version (135K): [in a new window]   Fig. 9. ANP immunolocalisation in the ventricle of isolated amphibian heart perfused with isoproterenol (ISO) and various adrenoceptor antagonists or SB203580. Hearts were perfused under normal conditions (A) and with 50 µmol l-1 of ISO for 5 min in the absence (B) or presence of either 10 µmol l-1 prazosin (C), 1 µmol l-1 propranolol (D) or 1 µmol l-1 SB203580 (E). Cryosections were incubated with an antibody specific for human ANP (1-28) (1:500 dilution) and counterstained with Haematoxylin. Immunoreaction deposits for ANP were visualised with Fast Red chromogen. Representative photographs from three independent experiments are shown. Bar, 20 µm.