ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase.
When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000469:Alkaline Phosphatase / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D004820:Epidermolysis Bullosa / Q000473:pathology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D006801:Humans; D009219:Myosin-Light-Chain Kinase; D011494:Protein Kinases / Q000378:metabolism*; D012867:Skin / Q000166:cytology* / Q000378:metabolism / Q000473:pathology; D014268:Trifluoperazine / Q000494:pharmacology
D014268:Trifluoperazine; D003571:Cytochalasin B; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D000469:Alkaline Phosphatase; D000251:Adenosine Triphosphatases