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.
AJP Heart and Circulatory Physiology 06/2008; 294(5):H2060-8. · 3.71 Impact Factor