Submitted on August 22, 2007
Accepted on February 21, 2008
Blebbistatin Inhibits the Chemotaxis of Vascular Smooth Muscle Cells through Breaking the Interaction between Myosin II and Actin
Hong Hui Wang1, Hideyuki Tanaka2, Xiaoran Qin3, Tiejun Zhao3, Lihong Ye3, Tuyoshi Okagaki4, Takeshi Katayama1, Akio Nakamura1, Ryoki Ishikawa1, Sean E Thatcher5, Gary L Wright5, and Kazuhiro Kohama6*
1 Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan
2 Department of Research Science, Gunma University School of Health Sciences, Maebashi, Gunma, Japan
3 Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, China; Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan
4 Department of Bioresources, Mie University, Tsu, Mie, Japan
5 Department of Physiology, The Joan Edwards School of Medicine, Marshall University School of Medicine, Huntington, West Virginia, United States
6 Department of Molecular and Cellular Pharmacology, Gunma University Graduate School of Medicine, Maebashi, Japan; Department of Biological Sciences, Laboratory of Molecular Physiology, Marshall University, Huntington, West Virginia, United States
* To whom correspondence should be addressed. E-mail: kohamak{at}med.gunma-u.ac.jp.
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 both sphingosylphosphorylcholine (IC50 =26.1 ±0.2µM for GbaSM-4 cells and 27.5±0.5 µM for A7r5 cells) and platelet-derived growth factor BB (IC50=32.3±0.9µM for GbaSM-4 cells and 31.6±1.3µM for A7r5 cells) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated that blebbistatin caused a disruption in the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg2+-ATPase activity of both the unphosphorylated (IC50 =12.6±1.6µM for gizzard and 4.3±0.5µM for bovine stomach) and phosphorylated (IC50=15.0±0.6µM 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 mero-myosin (IC50=14.4±1.6µM) and subfragment 1 (IC50 = 5.5 ±0.4µM) 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 (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.
