Abstract
Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the α-actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775–781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca2+]i by A23187 and thapsigargin on α- and β-actin remodeling. The effects of A23187 and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the α- and β-actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, β-actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca2+]i resulted in partial or complete dissolution of α-actin cables without further remodeling. In addition, PDBu-mediated α-actin remodeling was blocked in the presence of A23187. Increased [Ca2+]i also caused dispersal of α-actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for α-actin cytoskeletal structure. The incubation of cells in calcium-free media prevented α-actin dissolution by A23187/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of α-actin cables by increased [Ca2+]i. The results suggest that the sustained elevation of [Ca2+]i beyond a threshold level initiates depolymerization of α-actin but not β-actin. It further appears that PDBu-induced α-actin remodeling requires Ca2+ but increases of [Ca2+]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits α-actin dissolution in the presence of A23187/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing α-actin structure in the activated cell.
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Li, C., Fultz, M., Parkash, J. et al. Ca2+-dependent actin remodeling in the contracting A7r5 cell. J Muscle Res Cell Motil 22, 521–534 (2001). https://doi.org/10.1023/A:1015026530258
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DOI: https://doi.org/10.1023/A:1015026530258