Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model.
Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.
Kanno
K
K
Bio-Science Laboratory, Asahi Chemical Industry Co., Ltd, Nobeoka, Japan.
Sasaki
Y
Y
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D018260:Gelsolin; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*
D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D002118:Calcium