Blebbistatin Inhibits Sphingosylphosphorylcholine-Induced Contraction of Collagen-Gel Fiber Populated by Vascular Smooth-Muscle Cells

Katayama, Takuya; Yoshiyama, Shinji; Tanaka, Hideyuki; Wang, Hong‐Hui; Nakamura, Akio; Kohama, Kazuhiro

doi:10.1254/jphs.sc0060104

Cited by 15 publications

(20 citation statements)

References 13 publications

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“…Eddinger et al (7) recently reported that the ATPase activity of smooth muscle myosin was inhibited by blebbistatin at a concentration low enough to explain our data (15) and aroused our interest in the effect of blebbistatin on the migration of vascular smooth muscle cells (VSMCs). In the present study, we analyzed the mechanism of inhibition of blebbistatin on VSMC migration on the basis of the motor activity of smooth muscle myosin II.…”

mentioning

confidence: 57%

“…However, inhibition of contraction has been reported in smooth muscle at significantly lower concentrations of blebbistatin (7,15,39). Because IC 50 for nonmuscle myosin II is 1-5 M (27), the increased inhibition in these studies might be explained in terms of the nonmuscle myosin II (7,39).…”

Section: Discussionmentioning

confidence: 77%

“…GbaSM-4 cells, a VSMC line derived from the brain basilar artery of guinea pigs (33) and characterized elsewhere (15), were maintained in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin. A7r5 cells, a VSMC line derived from embryonic rat aorta, were purchased from American Type Culture Collection and then cultured in the same way as GbaSM-4 cells.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Wang¹,

Tanaka²,

Qin³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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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...

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mentioning

confidence: 57%

Section: Discussionmentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Wang¹,

Tanaka²,

Qin³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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“…The dried preparations were then incubated in urea-sample buffer (containting 20 mM Trizma base, 10 mM DTT, 8M urea) for 8 hrs at 4°C. The extracts were subjected to glycerol-PAGE coupled with Western blot (Sakurada et al, 1998;Katayama et al, 2006). MLC20 was detected with anti-MLC20 antibody given by Dr. S. Yoshiyama of Gunma University and visualized with horseradish preoxidase-conjutated anti rabbit IgG (GE Healthcare UK, Buckinghamshire, UK) and enhanced chemiliminescence (ECL) Western blotting detection system (ECL+plus, GE Healthcare, UK).…”

Section: Mlc20 Phosphorylationmentioning

confidence: 99%

Differential effects of an expected actin-tropomyosin binding region of heat shock protein 20 on the relaxation in skinned carotid artery and taenia cecum from guinea pig

Yumoto

Watanabe²,

Konishi³

et al. 2009

J. Smooth Muscle Res.

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To explore the possible role of heat shock protein 20 (HSP20) -linked regulation of actin-myosin interaction in living vascular smooth muscle contraction, we studied the effects of HSP20p and TnIp, synthetic peptides originating from an actin tropomyosin binding region of human heat shock protein 20 [residues 110-121; GFVAREFHRRYR] and that of rabbit cardiac troponin I [residues 136-147; GKFKRPTLRRVR], respectively, on the active stress and phosphorylation level of myosin regulatory light chain (MLC20) during relaxation of skinned (cell membrane permeabilized) preparations from "tonic" carotid artery and "phasic" taenia cecum from guinea pig. Active stress of the skinned preparations, resulting from actin-myosin interaction, biphasically decayed following Ca 2+ removal (relaxation). Decay of MLC20 phosphorylation level by Ca 2+ removal was much faster than active stress in an exponential manner. In skinned carotid artery, HSP20p did neither affect relaxation time course nor MLC20 dephosphorylation, whereas, in skinned taenia cecum, the peptide slowed relaxation time course through inhibition of MLC20 dephosphorylation and slowing "latch"-bridge dissociation. On the other hand, TnIp accelerated relaxation time course without affecting MLC20 dephosphorylation in both skinned carotid artery and skinned taenia cecum. Our present results suggest that, HSP20p slows the relaxation processes through intracellular regulatory mechanisms such as Rho A/Rho-kinase mediated pathways, which are known to be dominant in "phasic" smooth muscles but to be recessive in "tonic" smooth muscles.

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The signaling mechanism of the sphingosylphosphorylcholine-induced contraction in cat esophageal smooth muscle cells

et al. 2007

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We investigated the signaling pathway on sphingosinephosphorylcholine (SPC) -induced contraction in cat esophageal smooth muscle cells. SPC induced in a dose-dependent manner contractile effect. We have previously shown that lysophospholipid (LPL) receptor subtypes including the S1P1, S1P2, S1P3, and S1P5 receptor are present in esophageal smooth muscle. Only EDG-5 (S1P2) receptor antibody penetration into permeablilized cells inhibited the SPC-induced contraction. Pertussis toxin (PTX) and specific antibodies to G(i1), G(i2), G(i3) and G(o) inhibited the contraction, implying that SPC-induced contraction depends on PTX-sensitive G(i1), G(i2), G(i3), and G(o) protein. A phospholipase inhibitor U73122 and incubation of permeabilized cells with PLC-beta3 antibody inhibited SPC-induced contraction. The PKC-mediated contraction may be isozyme specific since only PKCepsilon antibody inhibited the contraction. Preincubation with MEK inhibitor PD98059 blocked the SPC-induced contraction, but p38 MAPK inhibitor SB202190 did not. Cotreatment with GF109203X and PD98059 did not show synergistic effects, suggesting that these two kinases are involved in the same signaling pathway in the SPC-induced contraction. The data suggest that S1P-induced contraction in feline esophageal smooth muscle cells depends on activation of the G(i1), G(i2), G(i3) and G(o) proteins and the PLCbeta3 isozyme via the S1P2 receptor, leading to stimulation of a PKCE pathway, which subsequently activates a p44/p42 MAPK pathway.

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Blebbistatin Inhibits Sphingosylphosphorylcholine-Induced Contraction of Collagen-Gel Fiber Populated by Vascular Smooth-Muscle Cells

Cited by 15 publications

References 13 publications

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

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

Differential effects of an expected actin-tropomyosin binding region of heat shock protein 20 on the relaxation in skinned carotid artery and taenia cecum from guinea pig

The signaling mechanism of the sphingosylphosphorylcholine-induced contraction in cat esophageal smooth muscle cells

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