Ca2+-dependent actin remodeling in the contracting A7r5 cell.
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 alpha-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 alpha- and beta-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 alpha- and beta-actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, beta-actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca2+]i resulted in partial or complete dissolution of alpha-actin cables without further remodeling. In addition, PDBu-mediated alpha-actin remodeling was blocked in the presence of A23187. Increased [Ca2+]i also caused dispersal of alpha-actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for alpha-actin cytoskeletal structure. The incubation of cells in calcium-free media prevented alpha-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 alpha-actin cables by increased [Ca2+]i. The results suggest that the sustained elevation of [Ca2+]i beyond a threshold level initiates depolymerization of alpha-actin but not beta-actin. It further appears that PDBu-induced alpha-actin remodeling requires Ca2+ but increases of [Ca2+]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits alpha-actin dissolution in the presence of A23187/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing alpha-actin structure in the activated cell.
Li
C
C
Department of Physiology, Anatomy and Cell Biology, The Joan Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA.
Fultz
M E
ME
Parkash
J
J
Rhoten
W B
WB
Wright
G L
GL
D000199:Actins / Q000378:metabolism*; D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D019284:Thapsigargin / Q000494:pharmacology
D000199:Actins; D004791:Enzyme Inhibitors; D007476:Ionophores; D019284:Thapsigargin; D002118:Calcium