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

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Abstract
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 μM. 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.