Potent Inhibition of Arterial Smooth Muscle Tonic Contractions by the Selective Myosin II Inhibitor, Blebbistatin

Abstract

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC 50 ϭ 0.5-5 M) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC 50 ϳ80 M). We found that blebbistatin potently (IC 50 ϳ3 M) inhibited the actomyosin ATPase activities of expressed "slow" [smooth muscle myosin IIA (SMA)] and "fast" [smooth muscle myosin IIB (SMB)] smooth muscle myosin II heavy-chain isoforms. Blebbistatin also inhibited the KCl-induced tonic contractions produced by rabbit femoral and renal arteries that express primarily SMA and the weaker tonic contraction produced by the saphenous artery that expresses primarily SMB, with an equivalent potency comparable with that identified for nonmuscle myosin IIA (IC 50 ϳ5 M). In femoral and saphenous arteries, blebbistatin had no effect on unloaded shortening velocity or the tonic increase in myosin light-chain phosphorylation produced by KCl but potently inhibited ␤-escin permeabilized artery contracted with calcium at pCa 5, suggesting that cell signaling events upstream from KCl-induced activation of cross-bridges were unaffected by blebbistatin. It is noteworthy that KCl-induced contractions of chicken gizzard were less potently inhibited (IC 50 ϳ20 M). Adult femoral, renal, and saphenous arteries did not express significant levels of nonmuscle myosin. These data together indicate that blebbistatin is a potent inhibitor of smooth muscle myosin II, supporting the hypothesis that the force-bearing structure responsible for tonic force maintenance in adult mammalian vascular smooth muscle is the cross-bridge formed from the blebbistatin-dependent interaction between actin and smooth muscle myosin II.

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

“…Blebbistatin inhibited the activities of unphosphorylated (IC 50 ϭ 17.5 Ϯ 0.7 and 10.1 Ϯ 0.7 M for gizzard and bovine smooth muscle myosin II, respectively) and phosphorylated (IC 50 ϭ 23.5 Ϯ 2.8 M for gizzard smooth muscle myosin II) forms of myosin II. These results suggest that blebbistatin inhibits the activity of smooth muscle myosin II at lower concentrations than previously reported (27) and agrees with the result of Eddinger et al (7).…”

“…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.…”

“…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.…”

Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle Contraction

“…In vitro biochemical studies have shown that blebbistatin is less effective on turkey smooth muscle myosin than it is on cytoplasmic myosin (40). Moreover, there is evidence that treatment with 10 -30 M blebbistatin effectively inhibits the contraction of smooth muscle tissues (41,42). Therefore, it is likely that actin polymerization, not tension, is necessary for the translocation of ␤-catenin and the recruitment of ␤-catenin to N-cadherin.…”

Evidence that actomyosin cross bridges contribute to “passive” tension in detrusor smooth muscle

“…Blebbistatin stabilizes the ADP-P i complex of the S1 myosin head that precedes the tension-generating step catalyzed by release of P i , thus inhibiting myosin from entering into the cross-bridge cycle (24). Blebbistatin inhibits mammalian smooth muscle tension and actomyosin ATPase activity (9), does not inhibit myosin light chain phosphorylation in smooth or striated muscle (9,49), and because it has no effect on action potentials or Ca 2ϩ mobilization, it can selectively uncouple excitation-contraction coupling in cardiac muscle (11).…”