Myosin phosphorylation during contraction and relaxation of tracheal smooth muscle

The role of myosin phosphorylation in regulating smooth muscle contraction has been investigated by quantitating the myosin phosphate content of tracheal smooth muscle frozen during contraction or relaxation. Myosin was purified from quick-frozen muscle samples with the aid of antibodies prepared against tracheal smooth muscle myosin, and the phosphate content was determined after separation of nonphosphorylated and phosphorylated myosin subunits by isoelectric focusing. The myosin phosphate content increased from an initial value of 0.50 to 1.1 mol of phosphate/mol of myosin within 3 min after the addition of 100 microM methacholine to resting tracheal smooth muscle. Myosin phosphorylation coincided temporally with the increase in isometric tension. Tracheal smooth muscles relaxed and the phosphate content decreased from 1.2 to 0.50 mol of phosphate/mol of myosin upon the addition of 10 microM atropine to muscles which had been previously contracted with 100 microM methacholine. Incubating methacholine-contracted muscles in a calcium-free Krebs buffer relaxed tracheal smooth muscles and reduced the phosphate content to 0.2 mol of phosphate/mol of myosin. Addition of 3 mM CaCl2 to these muscles elicited an increase in isometric tension concomitant with phosphorylation of myosin. These results support the hypothesis that myosin phosphorylation is important in regulating smooth muscle contraction.