Cytoplasmic Ca²⁺ is a primary determinant for myosin phosphorylation in smooth muscle cells

Initiation of smooth muscle contraction is associated with Ca²⁺/calmodulin activation of myosin light chain kinase which catalyzes the phosphorylation of the 20-kDa light chain of myosin. In tracheal smooth muscle cells in culture, the extent of myosin light chain phosphorylation is less than 10% at basal cytosolic free Ca²⁺ concentrations of 150 nM. Stimulation of these cells with serotonin, histamine, carbachol, or the Ca²⁺ ionophore, ionomycin, increases free cytosolic Ca²⁺ concentrations and the extent of myosin light chain phosphorylation. Light chain phosphorylation reaches a maximal value of 67% at Ca²⁺ concentrations below 1 μM. The relationship between the extent of light chain phosphorylation and cytosolic free Ca²⁺ concentration is apparently independent of the source of free intracellular Ca²⁺ or the agent used to stimulate the cells and is not altered by pre-exposure of the contractile apparatus to high concentrations of free Ca²⁺. Pretreatment of cells with 8-bromo-cyclic GMP or forskolin decreases free cytosolic Ca²⁺ concentrations and the extent of myosin light chain phosphorylation in response to histamine or ionomycin. Pretreatment with 8-bromo-cyclic GMP also decreases the maximal extent of light chain phosphorylation. These results indicate that cytosolic free Ca²⁺ concentration, per se, is a primary determinant for myosin light chain phosphorylation in tracheal smooth muscle cells.