Myosin light chain phosphorylation in smooth muscle and nonmuscle cells as a probe of calmodulin function

The function of Ca²⁺-calmodulin-dependent phosphorylation of phosphorylatable myosin light chain (P-light chain) is currently an area of immense interest and research. In the smooth muscle, P-light chain phosphorylation is a requisite for the interaction of myosin with actin. One major problem in working with intact smooth muscle or nonmuscle cells has been the lack of a sensitive technique for quantitating the extent of P-light chain phosphorylation in preparations that are routinely used in physiological and pharmacological investigations. This chapter discusses methodology for quantitating P-light chain phosphorylation during changes in isometric tension development of the bovine tracheal smooth muscle. The applicability of the methodology for quantitating P-light chain phosphorylation in various types of vascular smooth muscles, cardiac muscle, and skeletal muscles has also been determined in the chapter. This methodology should also be adaptable to other tissues or nonmuscle cells, such as platelets and macrophages, where myosin P-light chain phosphorylation may be involved in the regulation of motile processes. The combined use of pyrophosphate gel electrophoresis, isoelectric focusing, and ammoniacal-silver staining offers a relatively rapid, accurate, and sensitive technique for quantitating the extent of P-light chain phosphorylation during changes in contractile activity in the smooth muscle.