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CARDIOVASCULAR

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

Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin (T.J.E., J.M.); Department of Biological Sciences, Cardinal Stritch University, Milwaukee, Wisconsin (D.P.M.); Departments of Biochemistry and Pediatrics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia (A.S.M., P.H.R.); and University of Vermont, Department of Molecular Physiology and Biophysics, Burlington, Vermont (A.S.R.)

Blebbistatin is reported to be a selective and specific small molecule inhibitor of the myosin II isoforms expressed by striated muscles and nonmuscle (IC₅₀ = 0.5–5 µM) but is a poor inhibitor of purified turkey smooth muscle myosin II (IC₅₀ 80 µM). We found that blebbistatin potently (IC₅₀ 3 µM) 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 (IC₅₀ 5 µM). 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 (IC₅₀ 20 µM). 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.

Address correspondence to: Dr. Paul H. Ratz, Departments of Biochemistry and Pediatrics, Virginia Commonwealth University, School of Medicine, P.O. Box 980614, 1101 East Marshall Street, Richmond, VA 23298-0614. E-mail: phratz{at}vcu.edu

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