HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: 294/5/H2060    most recent 00970.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Wang, H. H. Articles by Kohama, K. Search for Related Content PubMed PubMed Citation Articles by Wang, H. H. Articles by Kohama, K.

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

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