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Cellular stress inhibits vascular smooth muscle relaxation

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Presentation on theme: "Cellular stress inhibits vascular smooth muscle relaxation"— Presentation transcript:

1 Cellular stress inhibits vascular smooth muscle relaxation
Louis Knoepp, MD, Arthur Beall, PhD, David Woodrum, BS, J.Sheppard Mondy, MD, Ellen Shaver, MD, Mary Dickinson, BS, Colleen M. Brophy, MD  Journal of Vascular Surgery  Volume 31, Issue 2, Pages (February 2000) DOI: /S (00) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

2 Fig. 1 Contractile responses of vascular smooth muscle after arsenite: Strips of vascular smooth muscle were treated with the protocols described. Treatment of strips of bovine carotid artery smooth muscle with sodium arsenite (As) did not change the basal tension of the strips. The magnitude of the contractile response to serotonin (5HT ; 10–6 mmol) and to potassium chloride (KCl ; 110 mmol/L) was the same before and after arsenite treatment. Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

3 Fig. 2 Cyclic nucleotide-dependent vasorelaxation is impaired after arsenite treatment: Strips of vascular smooth muscle were treated with the protocols described. The contractile responses to serotonin (10–6 mmol) were normalized to the initial high extracellular potassium chloride contraction (%KCl contraction , A ) and the relaxation responses to forskolin (10–5 mmol) were determined based on the maximal contraction elicited by serotonin (%relaxation , B ). The strips were equilibrated in buffer alone (C) or treated with arsenite (A) , arsenite and SB (A/S) , or SB alone (S) as described in the experimental protocols (n = 6-10 animals; *P <.05 compared with control). Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

4 Fig. 3 Characterization of phosphorylation state–specific antibodies: Strips of vascular smooth muscle were incubated in the presence of 32P-[orthophosphate], treated with arsenite (0.5 mmol) and homogenized. The proteins were separated on two-dimensional gels and transferred to Immobilon. An autoradiograph was obtained (A ), and the blots were probed with antibodies that recognize all isoforms of HSP27 (B ),29 followed by the phosphorylation-state specific antibodies (C ). The different phosphor-ylated isoforms of HSP27 (A, B, C, and D ) and the nonphosphorylated isoform (NP) are shown. The relative mobility of the phosphorylated myosin light chains (m) is shown in A. Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

5 Fig. 4 Sensitivity of phosphorylation state–specific antibodies: Recombinant HSP27 was phosphorylated in vitro by MAPKAP kinase II. The proteins were separated by SDS-PAGE and transferred to Immobilon. The blots were probed with the phosphorylation state–specific HSP27 antibodies. The relative mobility of molecular weight markers is on the left of the panel, and the amount of recombinant HSP27 (in micrograms) is on the bottom. Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

6 Fig. 5 Quantitation of HSP27 phosphorylation: Strips of vascular smooth muscle were equilibrated in buffer alone (C) or treated with arsenite (A) , arsenite and SB (A/S) , or SB alone (SB) as described in the experimental protocols. The strips were homogenized, separated on SDS-PAGE gels, transferred to Immobilon, and probed with the phosphorylation state specific antibodies (A ). The relative mobility of molecular weight markers is shown to the left of A and of phosphorylated HSP27 by the arrow. Densitometric analysis (relative densitometric units) of multiple experiments is depicted in B (n = 6 animals; *P <.05, compared with control). Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

7 Fig. 6 Whole cell phosphorylation: Strips of bovine carotid artery smooth muscle were treated with buffer alone (A ), buffer alone followed by forskolin (B ), arsenite (0.5 mmol/L for 30 minutes followed by equilibration in buffer for 2 hours; C ), or arsenite (0.5 mmol/L for 30 minutes followed by equilibration in buffer for 2 hours) followed by forskolin (10 μmol/L for 10 minutes; D ). HSP phosphorylation was quantitated with the use of whole cell phosphorylation and two-dimensional electrophoresis. The relative mobility of the myosin light chains (m) , the three phosphorylated isoforms of HSP27 (A, B, C) , and the three phosphorylated isoforms of HSP20 (8, 3, 4) are labeled on the blots. The relative mobility of molecular weight markers is shown on the right of each panel, and the isoelectric-focusing gradient is shown on the top of A. Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

8 Fig. 7 Immunohistochemical analysis of HSP27 in arse-nite-treated vascular smooth muscle: Strips of bovine carotid artery smooth muscle were treated with buffer alone (A ), arsenite (0.5 mmol/L for 30 minutes followed by equilibration in buffer for 2 hours; B ), or SB (100 μmol/L) for 15 minutes and then sodium arsenite (0.5 mmol/L) for 30 minutes; the strips were washed three times in buffer; SB (100 μmol/L) was added, and the strips were equilibrated for 2 hours (C ; original magnification, 63×). The strips were fixed and stained with the affinity-purified phosphorylation state–specific anti-HSP27 antibodies. Journal of Vascular Surgery  , DOI: ( /S (00) ) Copyright © 2000 Society for Vascular Surgery and International Society for Cardiovascular Surgery, North American Chapter Terms and Conditions

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