Figure 5. The NM myosin II cross‐bridge cycling inhibitor, blebbistatin suppresses ACh‐induced tension development and the assembly and activation of membrane adhesome complexes, although it does not inhibit NM myosin II assembly.
A, blebbistatin significantly inhibited force generation in response to 10−5 m ACh stimulation in tracheal smooth muscle (n = 8). B, immunoblot of cytosolic (soluble, S) and cytoskeletal (pellet, P) fractions from extracts of tracheal smooth muscle tissues treated with 10 μm blebbistatin or untreated (Control) and stimulated with ACh or unstimulated (US). Blebbistatin did not significantly affect the ACh‐induced increase in ratio of NM myosin II in the cytoskeletal (P) vs. soluble (S) fractions in response to 5 min of stimulation with 10−5 m ACh (n = 7). C, increase in NM myosin II Ser1943 phosphorylation in tracheal muscle tissues in response to 5 min of stimulation with 10−5 m. ACh was not significantly affected by blebbistatin (n = 12). D, blebbistatin had no significant effect on the increase in the number of NM myosin IIA and IIB complexes at the cell membrane in response to ACh (Control: US cells, n = 18; ACh stimulated cells, n = 28. Blebbistatin treated cells: US, n = 20; ACh, n = 29). Images show in situ PLA fluorescence (top) and PLA fluorescence merged with phase contrast (bottom) from freshly dissociated smooth muscle cells US or stimulated with ACh. PLA spots show interactions between NM myosin IIA and NM myosin IIB in freshly dissociated cells (left). E, PLA spots show interactions between talin and vinculin in freshly dissociated cells (left). ACh stimulation resulted in a significant increase in the number of talin‐vinculin complexes at the cell membrane in control cells (US, n = 24; ACh, n = 24). By contrast, few spots were observed in blebbistatin treated cells (US, n = 24; ACh, n = 25). Very few spots are observed in the unstimulated control cells and blebbistatin treated cells. F, representative immunoblot from extracts of two control muscle tissues and two blebbistatin treated tissues stimulated with ACh or not stimulated (US) (left). The increase in vinculin Tyr1065 phosphorylation and paxillin Tyr118 phosphorylation in response to 10−5 m ACh was significantly inhibited in tissues treated with blebbistatin (n = 7). Values are the mean ± SEM.*Significant difference between treatments (P < 0.05). ns, not significantly different.