Blebbistatin, a myosin II inhibitor, suppresses Ca²⁺-induced and “sensitized”-contraction of skinned tracheal muscles from guinea pig

Abstract

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.

Accelerating effects of blebbistatin on relaxation process of cell membrane permeabilized trachea and taenia cecum from guinea pig

“…In smooth muscle, we previously found that blebbistatin suppressed the force development independent of Ca 2+ sensitivity for the force and MLC phosphorylation level in β escin skinned taenia cecum (7) and trachea (8), and conformational changes in SMM (7), and these results indicates direct inhibition of actin-myosin interaction through modulation of SMM ATPase activity and/or SMM structure by blebbistatin. Therefore, the present result that blebbistatin had little effects on detachment of fast cycling crossbridge (τfast-detach in Table 2) supports our previous conclusion (7,8), since the detachment of the fast cycling crossbridge depends on the dephosphorylation of MLC (13,Formula 3), and also the previous evidence that blebbistatin might not act after phosphate release from SMM (1, 2).…”

“…Also, blebbistatin at around 10 μM inhibited the contraction of intact smooth muscle preparations and smooth muscle cell (3)(4)(5)(6). Furthermore, we reported that blebbistatin inhibited the contraction of skinned (cell membrane permeabilized) taenia cecum (7) and trachea (8) from the guinea pig without affecting the phosphorylation level of myosin light chain (MLC).…”