Inhibition of sustained and initial actin polymerization prevents ACh-induced sustained and initial smooth muscle contraction in muscle cells.

Muscle contraction was measured by scanning micrometry. A: Pre-treatment of dispersed smooth muscle cells with the MLCK inhibitor ML-9 (10 μM, 10 min), RhoA inhibitor Clostridium botulinum C3 exoenzyme (2μg/ml, 1 h), Rho kinase inhibitor Y27632 (10 μM, 10 min), FAK inhibitor PF573228 (10 μM, 1 h) or Arp2/3 inhibitor CK-666 (200 μM, 1h), actin polymerization inhibitors (cytochalasin D, 10 μM; latrunculin, 1 μM, 1 h) was followed by 1 μM ACh for 10 min. B: Pre-treatment of dispersed smooth muscle cells with MLCK inhibitor, ML-9 (10 μM, 10 min), or Arp2/3 complex inhibitor, CK-666 (200 μM, 1h), and actin polymerization inhibitors (cytochalasin D, 10 μM; latrunculin, 1 μM, 1h) followed by 1 μM ACh for 1 min. The results are expressed as percentage decrease in cell length. Insert: MLC20 phosphorylation was measured by western blot using phospho specific antibody. Pre-treatment of cultured smooth muscle cells with MLCK inhibitor, ML-9 (10 μM, 10 min) and actin polymerization inhibitor (cytochalasin D, 10 μM, 1h) followed by 1 μM ACh for 1 min. Values are means ± SE of three experiments. **P<0.001.