Ca2+-dependent contraction of human lung fibroblasts treated with Triton X-100: a role of Ca2+-calmodulin-dependent phosphorylation of myosin 20,000-dalton light chain.
Human lung fibroblast MRC-5 cells treated with Triton X-100 (MRC-5 cell models) were able to contract in the presence of MgATP and Ca2+ of more than 1 microM. Immunofluorescence microscopy with antibodies to actin and myosin 20,000-dalton (20 Kd) light chain revealed that stress fibers were prominent in MRC-5 cell models. Use of a fluorescent actin probe, 7-nitrobenz-2-oxa-1,3-diazole-phallacidin permitted visualization of contraction of the stress fibers in the presence of MgATP and Ca2+. Of the proteins in MRC-5 cell models, only a myosin 20 Kd light chain was phosphorylated in a Ca2+-dependent manner. This Ca2+-dependent phosphorylation of the 20 Kd light chain closely corresponded with the contraction of MRC-5 cell models: 1) Both phosphorylation of the 20 Kd light chain and contraction of MRC-5 cell models were inhibited by calmodulin antagonists such as N-(6-aminohexyl)5-chloro-1-napthalene sulfonamide. 2) The threshold Ca2+ concentration for phosphorylation of the 20 Kd light chain was similar to that for contraction of MRC-5 cell models. Both were lowered by exogenous calmodulin in a concentration-dependent manner. 3) The 20 Kd light chain was thiophosphorylated by incubation of MRC-5 cell models with an ATP analogue, adenosine 5'-0-(3-thiotriphosphate) only in the presence of Ca2+. After this treatment, MRC-5 cell models lost the Ca2+-dependence for contraction. These results indicate that Ca2+-calmodulin-dependent phosphorylation of myosin 20 Kd light chain is required for contraction of MRC-5 cell models.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism; D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D009119:Muscle Contraction; D009218:Myosins / Q000378:metabolism*; D017830:Octoxynol; D010766:Phosphorylation; D011092:Polyethylene Glycols / Q000494:pharmacology*
D002147:Calmodulin; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D009218:Myosins; D002118:Calcium