Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction

• Published in: American journal of physiology. Heart and circulatory physiology 2008-05, Vol.294 (5), p.H2060-H2068
• Main Authors: Wang, Hong Hui, Tanaka, Hideyuki, Qin, Xiaoran, Zhao, Tiejun, Ye, Li-Hong, Okagaki, Tuyoshi, Katayama, Takeshi, Nakamura, Akio, Ishikawa, Ryoki, Thatcher, Sean E, Wright, Gary L, Kohama, Kazuhiro
• Format: Article
• Language: English
• Subjects: Actins - metabolism; Animals; Cardiovascular system; Cattle; Cell Line; Cells; Chemotaxis - drug effects; Chickens; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings - pharmacology; Inhibitor drugs; Microscopy, Confocal; Microscopy, Electron; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - enzymology; Muscle, Smooth, Vascular - metabolism; Myocytes, Smooth Muscle - drug effects; Myocytes, Smooth Muscle - enzymology; Myocytes, Smooth Muscle - metabolism; Myosin Subfragments - antagonists & inhibitors; Myosin Subfragments - metabolism; Myosin Type II - antagonists & inhibitors; Myosin Type II - metabolism; Myosin Type II - ultrastructure; Phosphorylation; Phosphorylcholine - analogs & derivatives; Phosphorylcholine - metabolism; Platelet-Derived Growth Factor - metabolism; Protein Conformation; Proteins; Proto-Oncogene Proteins c-sis; Rats; Sphingosine - analogs & derivatives; Sphingosine - metabolism; Tissues
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00970.2007

1 Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, 2 Department of Research Science, Gunma University School of Health Sciences, Gunma, 4 Department of Bioresources, Mie University, Tsu, Japan; 3 Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, Peoples' Republic of China; and 5 Department of Physiology, The Joan Edwards School of Medicine, and 6 Department of Biological Sciences, Laboratory of Molecular Physiology, Marshall University, Huntington, West Virginia Submitted 22 August 2007 ; accepted in final form 21 February 2008 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 µM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC 50 = 32.3 ± 0.9 and 31.6 ± 1.3 µM 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 µM for gizzard and bovine stomach, respectively) and phosphorylated (IC 50 = 15.0 ± 0.6 µM 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 µM) and subfragment 1 (IC 50 = 5.5 ± 0.4 µM), 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. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy Address for reprint requests and other correspondence: K. Kohama, Dept. of Molecular and Cellular Pharmacology, Faculty of Medicine, Gunma Univ. Graduate School of Medicine, 3-39-22 Showa-Machi, Maebashi, Gunma 371-8511, Japan (e-mail: kohamak{at}med.