blister and sphingosine-phosphorylcholine

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.

Topics: Actins; Animals; Becaplermin; Cattle; Cell Line; Chemotaxis; Chickens; Dose-Response Relationship, Drug; Enzyme Inhibitors; Fluorescence Resonance Energy Transfer; Fluorescent Antibody Technique; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; Microscopy, Confocal; Microscopy, Electron; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Myosin Subfragments; Myosin Type II; Phosphorylation; Phosphorylcholine; Platelet-Derived Growth Factor; Protein Conformation; Proto-Oncogene Proteins c-sis; Rats; Sphingosine

We prepared a cell-populated collagen-gel fiber including GbaSM-4 cells derived from the basilar artery of guinea pigs. This fiber tended to be a differentiated contractile phenotype in electron-microscope observations. Sphingosylphosphorylcholine (SPC) can induce contraction of the fiber (EC50 = 0.70 +/- 0.05 microM), and blebbistatin can inhibit the SPC-induced contraction (IC50 = 22.8 +/- 1.26 microM). Phosphorylation of the 20 kD myosin light chain (MLC20) significantly increased in GbaSM-4 cells provided with 1 microM SPC (P<0.05), which was maintained in the presence of 1 to 100 microM blebbistatin. These results suggest that vascular smooth muscle can relax even if MLC20 is phosphorylated.

Topics: Animals; Cells, Cultured; Collagen; Gels; Guinea Pigs; Heterocyclic Compounds, 4 or More Rings; Microscopy, Electron; Muscle Contraction; Muscle, Smooth, Vascular; Myosin Light Chains; Phosphorylcholine; Sphingosine