Mitogen-activated protein kinase activation: An alternate signaling pathway for sustained vascular smooth muscle contraction Aaron M. Epstein, MD, Douglas.

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Presentation transcript:

Mitogen-activated protein kinase activation: An alternate signaling pathway for sustained vascular smooth muscle contraction Aaron M. Epstein, MD, Douglas Throckmorton, MD, Colleen M. Brophy, MD Journal of Vascular Surgery Volume 26, Issue 2, Pages 327-332 (August 1997) DOI: 10.1016/S0741-5214(97)70196-4 Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions

Fig. 1 Strips of bovine carotid artery smooth muscle were equilibrated in a muscle bath. Strips were then preincubated in the presence (+) or absence (−) of the tyrosine kinase inhibitor genestein (50 μmol/L) for 15 minutes followed by treatment with the specific vasoactive agonists, high extracellular KCl (110 mmol/L), phorbol dibutyrate (10−7 mol/L), serotonin (10−6 mol/L), angiotensin II (10−6 mol/L), or endothelin-1 (10−7 mol/L). Genestein significantly inhibited contractions induced by phorbol dibutyrate, serotonin, angiotensin II, and endothelin-1 (*p < 0.05, analysis of variance, three different arteries for each agonist) but did not inhibit contractions induced by high extracellular KCl. Journal of Vascular Surgery 1997 26, 327-332DOI: (10.1016/S0741-5214(97)70196-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions

Fig. 2 Strips of bovine carotid artery smooth muscle were equilibrated in a muscle bath and preincubated with buffer (closed circles) or genestein 50 μmol/L (closed squares) for 15 minutes. Strips were then treated with serotonin (1 μmol/L), and the contractile responses were recorded. Pretreatment with genestein significantly inhibited the maximal contractile response induced by serotonin (*p < 0.05, three separate experiments) and abolished the sustained phase of contraction. Journal of Vascular Surgery 1997 26, 327-332DOI: (10.1016/S0741-5214(97)70196-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions

Fig. 3 Strips of bovine carotid artery smooth muscle were treated with serotonin (1 μmol/L; A). Some strips were pretreated with genestein (50 μmol/L; B). Strips were homogenized at the various time points indicated, and immunoblots were performed with antiphosphotyrosine antibodies. Treatment with serotonin led to an increase in tyrosine phosphorylation of the 42 kDa isoform of MAP kinase (arrows). Densitometric analysis demonstrated a significant increase in tyrosine phosphorylation, which peaked at 3 minutes and remained elevated over baseline for 30 minutes (C; closed circles; *p < 0.05 analysis of variance, three separate experiments). Preincubation with genestein inhibited the increase in tyrosine phosphorylation of MAP kinase (C; open circles). Journal of Vascular Surgery 1997 26, 327-332DOI: (10.1016/S0741-5214(97)70196-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions

Fig. 3 Strips of bovine carotid artery smooth muscle were treated with serotonin (1 μmol/L; A). Some strips were pretreated with genestein (50 μmol/L; B). Strips were homogenized at the various time points indicated, and immunoblots were performed with antiphosphotyrosine antibodies. Treatment with serotonin led to an increase in tyrosine phosphorylation of the 42 kDa isoform of MAP kinase (arrows). Densitometric analysis demonstrated a significant increase in tyrosine phosphorylation, which peaked at 3 minutes and remained elevated over baseline for 30 minutes (C; closed circles; *p < 0.05 analysis of variance, three separate experiments). Preincubation with genestein inhibited the increase in tyrosine phosphorylation of MAP kinase (C; open circles). Journal of Vascular Surgery 1997 26, 327-332DOI: (10.1016/S0741-5214(97)70196-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions

Fig. 3 Strips of bovine carotid artery smooth muscle were treated with serotonin (1 μmol/L; A). Some strips were pretreated with genestein (50 μmol/L; B). Strips were homogenized at the various time points indicated, and immunoblots were performed with antiphosphotyrosine antibodies. Treatment with serotonin led to an increase in tyrosine phosphorylation of the 42 kDa isoform of MAP kinase (arrows). Densitometric analysis demonstrated a significant increase in tyrosine phosphorylation, which peaked at 3 minutes and remained elevated over baseline for 30 minutes (C; closed circles; *p < 0.05 analysis of variance, three separate experiments). Preincubation with genestein inhibited the increase in tyrosine phosphorylation of MAP kinase (C; open circles). Journal of Vascular Surgery 1997 26, 327-332DOI: (10.1016/S0741-5214(97)70196-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions

Fig. 4 Strips of bovine carotid artery smooth muscle were phosphorylated and remained in buffer (control, C) or were treated with serotonin (S, 1 μmol/L, 15 minutes). Strips were homogenized, and immunoprecipitation with anticaldesmon antibodies was performed. Autoradiographs demonstrate an increase in caldesmon phosphorylation with serotonin stimulation (A). Densitometric analysis demonstrated that this increase in caldesmon phosphorylation was significant (B, *p < 0.05, analysis of variance, five separate experiments). Journal of Vascular Surgery 1997 26, 327-332DOI: (10.1016/S0741-5214(97)70196-4) Copyright © 1997 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North America Chapter Terms and Conditions