title: Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction.
abstract: Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC(50) = 26.1 +/- 0.2 and 27.5 +/- 0.5 microM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC(50) = 32.3 +/- 0.9 and 31.6 +/- 1.3 muM for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg(2+)-ATPase activity of the unphosphorylated (IC(50) = 12.6 +/- 1.6 and 4.3 +/- 0.5 microM for gizzard and bovine stomach, respectively) and phosphorylated (IC(50) = 15.0 +/- 0.6 microM for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg(2+)-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC(50) = 14.4 +/- 1.6 microM) and subfragment 1 (IC(50) = 5.5 +/- 0.4 microM), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Faculty of Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-Machi, Maebashi, Gunma, Japan.
authors.forename: Hong Hui
authors.initials: HH
authors.lastname: Wang
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Xiaoran
authors.initials: X
authors.lastname: Qin
authors.forename: Tiejun
authors.initials: T
authors.lastname: Zhao
authors.forename: Li-Hong
authors.initials: LH
authors.lastname: Ye
authors.forename: Tuyoshi
authors.initials: T
authors.lastname: Okagaki
authors.forename: Takeshi
authors.initials: T
authors.lastname: Katayama
authors.forename: Akio
authors.initials: A
authors.lastname: Nakamura
authors.forename: Ryoki
authors.initials: R
authors.lastname: Ishikawa
authors.forename: Sean E
authors.initials: SE
authors.lastname: Thatcher
authors.forename: Gary L
authors.initials: GL
authors.lastname: Wright
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kohama
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D000077214:Becaplermin; D002417:Cattle; D002460:Cell Line; D002633:Chemotaxis / Q000187:drug effects*; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology*; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015879:Myosin Subfragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D024462:Myosin Type II / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011487:Protein Conformation; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D000199:Actins; D004791:Enzyme Inhibitors; D006576:Heterocyclic Compounds, 4 or More Rings; D015879:Myosin Subfragments; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D000077214:Becaplermin; C472645:blebbistatin; D024462:Myosin Type II; D013110:Sphingosine