Objective: To investigate the role of Rho-kinase signaling pathway in human airway smooth muscle cell (ASMCs) migration and cytoskeletal reorganization induced by endothelin-1 (ET-1).
Methods: Primary cultured human ASMCs obtained by tracheal explant culture method were examined for cell migration in response to ET-1 treatment using modified Boyden chambers. The changes in actin cytoskeletal reorganization were observed under confocal laser scanning microscope, and the phosphorylation of myosin-phosphatase target 1 (p-MYPT1) was examined using Western blot analysis.
Results: At the concentration of 0.1, 1, 10, and 100 nmol/L, ET-1 induced migration of the ASMCs, and 10 nmol/L ET-1 produced the most obvious effect (P<0.01). Rho-kinase inhibitor Y-27632 showed a dose-dependent inhibitory effect on ET-1-induced ASMC migration, and in cells exposed to 10 nmol/L ET-1, Y-27632 at 10 micromol/L significantly blocked ASMC migration (P<0.01). ET-1 (10 nmol/L) exposure resulted in reorganization of actin cytoskeleton and formation of stress fibers in the ASMCs, which were obviously inhibited by Y-27632. Compared with the control group, the AMSCs showed significant enhancement of p-MYPT1 protein expression after ET-1 exposure for 15 and 30 min (P<0.01), but prolonged exposure failed to result in the expression enhancement (P>0.05).
Conclusion: Rho-kinase signaling pathway may play an important role in ET-1-induced ASMC migration and reorganization of actin cytoskeleton.