Differential actin isoform reorganization in the contracting A7r5 cell

In the present study, we investigated the reorganization of α- and β-actin in the contracting A7r5 smooth muscle cell. The remodeling of these actin variants was markedly different in response to increasing concentrations of phorbol 12, 13-dibutyrate (PDBu). At the lowest concentrations (≤10−7 mol/L), cells showed an ~70% loss in α-actin stress fibers with robust transport of this isoform to podosomes. By comparison, β-actin remained in stress fibers in cells stimulated at low concentrations (≤10−7 mol/L) of PDBu. However, at high concentrations (≥10−6 mol/L) ~50% of cells showed transport of β-actin to podosomes. Consistent with these findings, staining with phalloidin indicated a significant decrease in the whole-cell content of F-actin with PDBu treatment. However, staining with DNase I indicated no change in the cellular content of G-actin, suggesting reduced access of phalloidin to tightly packed actin in the podosome core. Inhibition of protein kinase C (staurosporine, bisindolymaleimide) blocked PDBu-induced (5 × 10−8 mol/L) loss in α-actin stress fibers or reversed podosome formation with re-establishment of α-actin stress fibers. By comparison, these inhibitors caused partial loss of β-actin stress fibers. The results support our earlier conclusion of independent remodeling of α- and β-actin cytoskeletal structure and suggest that the regulation of these structures is different.

Actin filament disruption inhibits L-type Ca2+ channel current in cultured vascular smooth muscle cells

To clarify interactions between the cytoskeleton and activity of L-type Ca2+ (CaL) channels in vascular smooth muscle (VSM) cells, we investigated the effect of disruption of actin filaments and microtubules on the L-type Ca2+ current [ I Ba(L)] of cultured VSM cells (A7r5 cell line) using whole cell voltage clamp. The cells were exposed to each disrupter for 1 h and then examined electrophysiologically and morphologically. Results of immunostaining using anti-α-actin and anti-α-tubulin antibodies showed that colchicine disrupted both actin filaments and microtubules, cytochalasin D disrupted only actin filaments, and nocodazole disrupted only microtubules. I Ba(L) was greatly reduced in cells that were exposed to colchicine or cytochalasin D but not to nocodazole. Colchicine even inhibited I Ba(L) by about 40% when the actin filaments were stabilized by phalloidin or when the cells were treated with phalloidin plus taxol to stabilize both cytoskeletal components. These results suggest that colchicine must also cause some inhibition of I Ba(L) due to another unknown mechanism, e.g., a direct block of CaLchannels. In summary, actin filament disruption of VSM cells inhibits CaL channel activity, whereas disrupting the microtubules does not.