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 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.
Taylor
D A
DA
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
Stull
J T
JT
D000818:Animals; D001572:Benzofurans; D002118:Calcium / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004987:Ethers / Q000494:pharmacology; D016257:Fura-2; D015759:Ionomycin; D007700:Kinetics; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D015879:Myosin Subfragments; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D012701:Serotonin / Q000494:pharmacology; D014132:Trachea / Q000166:cytology / Q000201:enzymology
D001572:Benzofurans; D004987:Ethers; D015879:Myosin Subfragments; D010446:Peptide Fragments; D012701:Serotonin; D015759:Ionomycin; D002217:Carbachol; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium; D016257:Fura-2