A myosin phosphatase modulates contractility in skinned smooth muscle

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 (A_0.5; 0.26 M) 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 A_0.5 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 M) 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.