A myosin phosphatase modulates contractility in skinned smooth muscle

Abstract

The influence of a purified holoenzyme form of polycation-modulable (PCM-) myosin phosphatase on Ca2+-dependent actin-myosin interactions was studied in detergent-skinned smooth muscle fibers from chicken gizzard. The concentration of Ca2+ required for half maximal isometric contraction (A0.5; 0.26 microM) of fibers incubated in the absence of phosphatase was increased 2-fold when PCM-phosphatase (13 U/ml) was included in the medium. Removal of the phosphatase restored A0.5 to control level showing that the enzyme-mediated decrease in Ca2+-sensitivity was reversible. Two-dimensional electrophoresis of fiber homogenates revealed that PCM-phosphatase decreased Ca2+-sensitivity for phosphorylation of the regulatory myosin light chains in parallel fashion. Ca2+-dependent increases in isometric force were directly correlated to increases in the extent of light chain phosphorylation up to about 0.35 mol PO4/mol light chain; further increases in phosphorylation were not associated with further increases in force. Addition of PCM-phosphatase to fibers which had been contracted with a suboptimal concentration of Ca2+ (0.35 microM) resulted in rapid relaxation. Unloaded shortening velocity, reflecting cross-bridge cycling rate, was reduced by 92% in the presence of PCM-phosphatase and light chain phosphorylation was decreased by 50%. These data show that both tension and unloaded shortening velocity may be related to Ca2+-dependent phosphorylation of the light chains. The results indicate that the level of phosphorylation attained in the fiber preparations studied probably reflects the ratio of myosin kinase to phosphatase activities. Since protein phosphatases are regulated enzymes the results also suggest that modulation of phosphatase activity may participate in control of smooth muscle contractility.

Effects of gomisin A on vascular contraction in rat aortic rings

“…3), the relaxant action of GA in the rat aorta did not /calmodulin-dependent manner (Kamm et al 1985). Conversely, MLCP mediates dephosphorylation of phosphorylated MLC causing relaxation (Bialojan et al 1987;Somlyo et al 1994). Furthermore, activation of Rhokinase by NaF or U46619 inhibits the activity of MLCP through phosphorylation of regulatory MYPT1 Thr855 , leading to an increased MLC 20 phosphorylation and contraction (Wang et al 2001;Wilson et al 2005;Yang et al 2009).…”

Protein phosphatase type-1, not type-2A, modulates actin microfilament integrity and myosin light chain phosphorylation in living nonmuscle cells.

“…Protein phosphatases reactive with MLC have been isolated from smooth muscle (22, 26, 29, 37-39, 45, reviewed 16, 40), as well as from cardiac (36,37,42) and skeletal muscle (14,15, reviewed in reference 16) and appear to be related to either type-1 (14,15,42, reviewed in reference 16) or type 2A enzymes (8,10,26,30). Both a polycation-modulated phosphatase holoenzyme (9) and the catalytic subunit of a skeletal protein phosphatase (41) have been found to decrease isometric tension of skinned smooth muscle and skeletal muscle fibers respectively.…”

The Frequency Response of Smooth Muscle Stiffness during Ca2+-Activated Contraction

“…In smooth muscle, contraction is primarily initiated through the phosphorylation of the 20-kDa myosin light chain (MLC) by Ca 2ϩ -calmodulin-dependent MLC kinase (Hartshorne, 1987;Kamm and Stull, 1985), whereas relaxation occurs via the dephosphorylation of the MLC by a MLC phosphatase (Bialojan et al, 1987;Haeberle et al, 1985). However, for many types of stimuli, force is maintained after intracellular Ca 2ϩ and MLC phosphorylation fall from the high level reached during force development.…”