AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 294: H2060-H2068, 2008. First published February 22, 2008; doi:10.1152/ajpheart.00970.2007
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Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction

Hong Hui Wang,1 Hideyuki Tanaka,2 Xiaoran Qin,1,3 Tiejun Zhao,1,3 Li-Hong Ye,1,3 Tuyoshi Okagaki,4 Takeshi Katayama,1 Akio Nakamura,1 Ryoki Ishikawa,1 Sean E. Thatcher,5 Gary L. Wright,5 and Kazuhiro Kohama1,6

1Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, 2Department of Research Science, Gunma University School of Health Sciences, Gunma, 4Department of Bioresources, Mie University, Tsu, Japan; 3Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, Peoples' Republic of China; and 5Department of Physiology, The Joan Edwards School of Medicine, and 6Department 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 (IC50 = 26.1 ± 0.2 and 27.5 ± 0.5 µM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC50 = 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 Mg2+-ATPase activity of the unphosphorylated (IC50 = 12.6 ± 1.6 and 4.3 ± 0.5 µM for gizzard and bovine stomach, respectively) and phosphorylated (IC50 = 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 Mg2+-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC50 = 14.4 ± 1.6 µM) and subfragment 1 (IC50 = 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.gunma-u.ac.jp)






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