Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig

Watanabe, Masaru; Yumoto, Masatoshi; Tanaka, Hideyuki; Wang, Hong‐Hui; Katayama, Takuya; Yoshiyama, Shinji; Black, Jason Edward; Thatcher, Sean E.; Kohama, Kazuhiro

doi:10.1152/ajpcell.00269.2009

Cited by 18 publications

(21 citation statements)

References 40 publications

(71 reference statements)

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“…This agent strongly inhibited most vertebrate striated muscle- and non-muscle myosin II ATPase activities (2) as well as vertebrate smooth muscle myosin (SMM) ATPase activity (3, 4). Several groups, including us, have also found that blebbistatin inhibited the smooth muscle preparations and smooth muscle cell contraction at around 10 μM (3,4,5,6,7). The inhibitory mechanism on the actin-myosin interaction has been thought to be due to inhibition of myosin ATPase resulting in interference of cross-bridge cycling (1, 2).…”

Section: Introductionmentioning

confidence: 85%

“…The inhibitory mechanism on the actin-myosin interaction has been thought to be due to inhibition of myosin ATPase resulting in interference of cross-bridge cycling (1, 2). Also previous studies have indicated that conformational change of SMM by blebbistatin spatially interferes with the actin-myosin interaction of smooth muscle cells (4, 5, 7). Blebbistatin simultaneously inhibited F-actin-SMM interaction, force development and organization of contractile filaments in skinned smooth muscles of the guinea pig taenia cecum (5).…”

Section: Introductionmentioning

confidence: 91%

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Blebbistatin, a myosin II inhibitor, suppresses Ca<sup>2+</sup>-induced and “sensitized”-contraction of skinned tracheal muscles from guinea pig

Yumoto

Watanabe

2013

J. Smooth Muscle Res.

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Blebbistatin, a potent inhibitor of myosin II, has inhibiting effects on Ca2+-induced contraction and contractile filament organization without affecting the Ca2+-sensitivity to the force and phosphorylation level of myosin regulatory light chain (MLC20) in skinned (cell membrane permeabilized) taenia cecum from the guinea pig (Watanabe et al., Am J Physiol Cell Physiol. 2010; 298: C1118–26). In the present study, we investigated blebbistatin effects on the contractile force of skinned tracheal muscle, in which myosin filaments organization is more labile than that in the taenia cecum. Blebbistatin at 10 μM or higher suppressed Ca2+-induced tension development at any given Ca2+ concentration, but had little effects on the Ca2+- induced myosin light chain phosphorylation. Also blebbistatin at 10 μM and higher significantly suppressed GTP-γS-induced "sensitized" force development. Since the force inhibiting effects of blebbistatin on the skinned trachea were much stronger than those in skinned taenia cecum, blebbistatin might directly affect myosin filaments organization.

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Section: Introductionmentioning

confidence: 85%

Section: Introductionmentioning

confidence: 91%

Blebbistatin, a myosin II inhibitor, suppresses Ca<sup>2+</sup>-induced and “sensitized”-contraction of skinned tracheal muscles from guinea pig

Yumoto

Watanabe

2013

J. Smooth Muscle Res.

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“…Whole cell FRET analysis, as presently employed, has been used successfully to evaluate protein-protein interactions in smooth muscle cells (32,33,36). Because the donor/acceptor must be within 10-nm distance from each other for efficient energy transfer (37,38), this technique provides a measure of protein-protein distances compatible with molecular interaction.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Confocal/fret Microscopymentioning

confidence: 99%

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

et al. 2011

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Abstract. Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135-143). Myosin ATPase activity measurements indicate that the 26 -41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not β-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.

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“…Inhibitors of myosin function have been described previously (Watanabe et al, 2010). A direct inhibitor of SMM would be attractive for reducing arterial pressure through peripheral vasodilation.…”

Section: Introductionmentioning

confidence: 99%

Inhibition of Smooth Muscle Myosin as a Novel Therapeutic Target for Hypertension

Zhao¹,

Ho²,

Abarzúa³

et al. 2011

J Pharmacol Exp Ther

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We examined a novel therapeutic approach for hypertension, a small-molecule direct inhibitor of smooth muscle myosin, CK-2018448 (CK-448), which is an N,N'-alkylurea (U.S. Patent Publication 2009 -0275537 A1) in conscious dogs with renal hypertension and compared its efficacy with that of a calcium channel blocker, amlodipine. Dogs were instrumented with a miniature left ventricular pressure gauge, an aortic pressure catheter, and ultrasonic flow probes in the ascending aorta and renal and iliac arteries for measurement of cardiac output and regional blood flow. In the hypertensive state, mean arterial pressure increased from 101 Ϯ 3.8 to 142 Ϯ 1.9 mm Hg. At the doses selected, CK-448 and amlodipine increased cardiac output similarly (30 Ϯ 11% versus 33 Ϯ 6.4%) and similarly reduced mean arterial pressure (Ϫ22 Ϯ 3.6% versus Ϫ16 Ϯ 3.4%) and total peripheral resistance (Ϫ36 Ϯ 5.9% versus Ϫ37 Ϯ 5.8%). CK-448 had the greatest vasodilator effect in the renal bed, where renal blood flow increased by 46 Ϯ 9.0%, versus 11 Ϯ 3.4% for amlodipine (p Ͻ 0.01). CK-488 produced significantly less vasodilation in the limb, where iliac blood flow did not change; in contrast, it rose by 48 Ϯ 12% with amlodipine (p Ͻ 0.01). The minimal effects on limb blood flow could limit the development of peripheral edema, an adverse side effect of Ca 2ϩ channel blockers. In addition, in a rodent model of hypertension, oral administration of a smooth muscle myosin inhibitor resulted in a sustained antihypertensive effect. Thus, the smooth muscle myosin inhibitor's preferential effect on renal blood flow makes this drug mechanism particularly appealing, because many patients with hypertension have renal insufficiency, and patients with heart failure could benefit from afterload reduction coupled with enhanced renal blood flow.

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Blebbistatin, a myosin II inhibitor, suppresses contraction and disrupts contractile filaments organization of skinned taenia cecum from guinea pig

Cited by 18 publications

References 40 publications

Blebbistatin, a myosin II inhibitor, suppresses Ca<sup>2+</sup>-induced and “sensitized”-contraction of skinned tracheal muscles from guinea pig

Blebbistatin, a myosin II inhibitor, suppresses Ca<sup>2+</sup>-induced and “sensitized”-contraction of skinned tracheal muscles from guinea pig

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

Inhibition of Smooth Muscle Myosin as a Novel Therapeutic Target for Hypertension

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