Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery.
Increases in cyclic nucleotide levels induce smooth muscle relaxation by deactivation [reductions in myosin regulatory light chain (MRLC) phosphorylation (e.g., by reduced [Ca(2+)])] or force suppression (reduction in force without reduction in MRLC phosphorylation). Ser(16)-heat shock protein 20 (HSP20) phosphorylation is the proposed mediator of force suppression. We evaluated three potential hypotheses whereby Ser(16)-HSP20 phosphorylation could regulate smooth muscle force: 1) a threshold level of HSP20 phosphorylation could inactivate a thin filament as a whole, 2) phosphorylation of a single HSP20 could fully inactivate a small region of a thin filament, or 3) HSP20 phosphorylation could weakly inhibit myosin binding at either the thin- or thick-filament level. We tested these hypotheses by analyzing the dependence of force on Ser(16)-HSP20 phosphorylation in swine carotid media. First, we determined that swine HSP20 has a second phosphorylation site at Ser(157). Ser(157)-HSP20 phosphorylation values were high and did not change during contractile activation or forskolin-induced relaxation. Forskolin significantly increased Ser(16)-HSP20 phosphorylation. The relationship between Ser(16)-HSP20 phosphorylation and force remained linear and was shifted downward in partially activated muscles relaxed with forskolin. Neither forskolin nor nitroglycerin induced actin depolymerization as detected using the F/G-actin ratio method in smooth muscle homogenates. These results suggest that force suppression does not occur in accordance with the first hypothesis (inactivation of a thin filament as a whole). Our data are more consistent with the second and third hypotheses that force suppression is mediated by full or partial inhibition of local myosin binding at the thin- or thick-filament level.
Meeks
Melissa K
MK
Cardiovascular Division, Department of Internal Medicine, University of Virginia Health System, Charlottesville, VA 22908-1395, USA.
Ripley
Marcia L
ML
Jin
Zhicheng
Z
Rembold
Christopher M
CM
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002339:Carotid Arteries / Q000187:drug effects* / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology*; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000502:physiology*; D009126:Muscle Relaxation / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology* / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D013314:Stress, Mechanical; D013552:Swine
D000199:Actins; D050886:HSP20 Heat-Shock Proteins; C494382:HSPB6 protein, human; D006360:Heat-Shock Proteins; D009712:Nucleotides, Cyclic; D010750:Phosphoproteins; D005576:Colforsin; D012694:Serine