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

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 (IC50 ϭ 26.1 Ϯ 0.2 and 27.5 Ϯ 0.5 M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC 50 ϭ 32.3 Ϯ 0.9 and 31.6 Ϯ 1.3 M 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 2ϩ -ATPase activity of the unphosphorylated (IC 50 ϭ 12.6 Ϯ 1.6 and 4.3 Ϯ 0.5 M for gizzard and bovine stomach, respectively) and phosphorylated (IC 50 ϭ 15.0 Ϯ 0.6 M 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 2ϩ -ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC 50 ϭ 14.4 Ϯ 1.6 M) and subfragment 1 (IC50 ϭ 5.5 Ϯ 0.4 M), 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 CELL MIGRATION IS DRIVEN through complex processes, such as extension of the leading membrane edge, with formation of adhesive contacts and stress fibers (26, 52). Myosin II is widely believed to be one of the main components producing the forces required in this process (12). However, the details of the regulatory mechanism of myosin II in cell migration remain to be established. The general understanding is that cell migration is regulated not only by pathways of signal transduction involving myosin light chain (MLC) phosphorylation, but also by MLC phosphorylation-independent pathways (2,18,28,30). The exact role of myosin II in cell migration and the mechanism whereby myosin II contributes force generation for cell migration remain unanswered.Blebbistatin was recently identified as a specific inhibitor of myosin II-dependent cell processes (49). Its membrane-permeable characteristic and effect on various myosin II isoforms make this agent an invaluable tool in research of myosin II-involved cellular events, including cell motility (5, 23), cell shape maintenance (50), muscle contraction (7, 15), and cytokinesis (49). Blebbistatin has been shown to preferentially bind to the myosin-ADP-phosphate complex with high affinity and prevent phosphate release (24, 37), resulting in inhibition of the actin-myosin interaction. Blebbistatin also inhibited the ability of skeletal muscle and nonmuscle myosin II to move actin f...

Inhibiting nonmuscle myosin II impedes inflammatory infiltration and ameliorates progressive renal disease

“…MCP-1-stimulated macrophages showed an evident physical association between actin and myosin ( Figure 1b). Blebbistatin, a 1-phenyl-2-pyrrolidinone derivative (Figure 1a), at the concentration of 50 mM, drastically abrogated the co-precipitation of nonmuscle myosin heavy chain IIA with actin in all cells studied (Figure 1b), consistent with a previous work 33 showing that inhibition of NMII by blebbistatin induces actinomyosin complex disassembly.…”

Perivascular Delivery of Blebbistatin Reduces Neointimal Hyperplasia after Carotid Injury in the Mouse

“…Previous studies have shown that blebbistatin inhibits PDGF-BB-induced migration of SMC derived from the brain basilar artery of guinea pigs and an immortalized cell line of aortic smooth muscle cells obtained from Sprague-Dawley rat embryos (Wang et al, 2008). Effects of blebbistatin on SMC proliferation and migratory responses to injury are largely unknown; thus, additional studies exploring mechanisms of blebbistatin effects were performed with cultured RASMC.…”

“…Blebbistatin disrupts directed cellular motility and cytokinesis in vertebrate cells and inhibits contraction of contractile ring assembly (Straight et al, 2003). Exposure to blebbistatin has profound effects on SMC ex vivo, including disruption of actin-myosin interactions (Wang et al, 2008), inhibition of contraction of cultured SMC (Katayama et al, 2006), inhibition of ATPase activity of smooth muscle myosin (Eddinger et al, 2007), inhibition of KCl-induced tonic contractions produced by rabbit femoral and renal arteries (Eddinger et al, 2007), and inhibition of chemotaxis of SMC toward sphingosylphosphorylcholine and platelet-derived growth factor-BB (PDGF-BB) (Wang et al, 2008).…”

“…Because myosin expression is differentially regulated after vascular injury and has been implicated in the control of important reparative processes such as smooth muscle cell proliferation (Simons and Rosenberg, 1992) and migration (Wang et al, 2008), we sought to test the hypothesis that inhibition of myosin activity would limit neointimal formation after vascular injury and thereby potentially represent a novel therapeutic option for preventing restenosis after revascularization.…”

Myosin Light Chain Kinase / Actin Interaction in Phorbol Dibutyrate–Stimulated Smooth Muscle Cells

“…Inclusion of nuclear staining of MLCK in the A7r5 cell did not affect the significance of the results presented herein (data not shown). The quantification by FRET using this experimental protocol was described previously (31,32).…”