Calcium dependence of myosin light chain phosphorylation in smooth muscle cells

Smooth muscle cells grown in culture may provide a model system for studying the Ca²⁺ dependence of myosin light chain phosphorylation. Tracheal smooth muscle cells in culture had 60% of the myosin content of tracheal tissue. Western analysis with appropriate antibodies demonstrated one 20-kDa light chain and the presence of a 150-kDa myosin light chain kinase in both tracheal smooth muscle tissue and cells. Moreover, tracheal cells contained 74% of the myosin light chain kinase activity measured in tissue. Similar types of analyses of nonmuscle cells showed a much lower myosin and myosin light chain kinase content. Carbachol (10 μM) or ionomycin (10 μM) stimulation of fura-2-containing cells resulted in a rapid increase in cytosolic free Ca²⁺ concentration and in the extent of myosin light chain phosphorylation. Maximal increases in Ca²⁺ concentrations were greater with ionomycin than with carbachol (4400 versus 492 nM). Light chain phosphorylation increased after the Ca²⁺ concentration exceeded 200 nM from control values of 165 nM. Half-maximal phosphorylation (33%) occurred at 260 nM Ca²⁺. There was a similar relationship between free cytosolic Ca²⁺ concentrations and the extent of myosin light chain phosphorylation in carbachol- and ionomycin-stimulated cells. This relationship had a Hill coefficient of 2.7. These observations indicate that small changes in Ca²⁺ concentrations stimulate myosin light chain phosphorylation and thus presumably contraction in smooth muscle cells.