Abstract
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 microM) or ionomycin (10 microM) 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.
Highlights
Smooth muscle cells grown inculture may provide a growth phase of the cells in culture with an increase in specific model system for studying the Ca2+dependenceof my- smooth muscle contractile protein isoforms in postconfluent osin light chain phosphorylation
Carbachol (10MM) or ionomycin (10MM) stimulation icant changes in contractile protein expression and content, of fura-2-containing cellsresulted in a rapid increase but can retainsmooth muscle specific isoforms of myosin and in cytosolic free Ca2+concentration and in the extent actin
Half-maximal phosphorylation (33%) oc- though the cellular and molecular mechanisms through which curred at 260 nM Ca2+.There was a similar relation- Ca2+regulates smooth muscle contraction are still not comship between free cytosolic Ca2+concentrations and pletely understood, it is generally accepted that a primary the extent of myosin light chain phosphorylation in step involves phosphorylation of the 20-kDa light chain of carbachol- and ionomycin-stimulated cells
Summary
Half-maximal phosphorylation (33%) oc- though the cellular and molecular mechanisms through which curred at 260 nM Ca2+.There was a similar relation- Ca2+regulates smooth muscle contraction are still not comship between free cytosolic Ca2+concentrations and pletely understood, it is generally accepted that a primary the extent of myosin light chain phosphorylation in step involves phosphorylation of the 20-kDa light chain of carbachol- and ionomycin-stimulated cells.
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