Background: This study is to investigate the roles of muscarinic receptor 3 (M3 receptor) in the effect of penehyclidine hydrochloride (PHC) upregulated beta-arrestin-1 expression in lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cell (HPMVEC). Methods: HPMVECs were transfected with a shRNA-containing plasmid that specifically targets M3 receptor mRNA. Cells were collected to measure F-actin contents, levels of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), as well as changes of F-actin cytoskeleton arrangement by Laser scanning confocal. Beta-arrestin-1 protein expressions were determined by Western blot and beta-arrestin-1 mRNA expressions were measured by Real-time PCR. Results: Similar to normal cells, PHC could also increase F-actin contents and beta-arrestin-1 expressions, reduce ICAM-1 and VCAM-1 expressions, and inhibit LPS-stimulated reorganization of F-actin and formation of stress fiber in M3 receptor shRNA group. Compared with normal cells, F-actin cytoskeleton was neat, ICAM-1 and VCAM-1 expressions were decreased, as well as F-actin contents were increased in M3 receptor shRNA group. However, there were no differences in beta-arrestin-1 expressions between normal cell groups and M3 receptor shRNA groups. Conclusion: These results indicate that M3 receptor plays an important role in pulmonary microvascular endothelial barrier function, and knock-out of M3 receptor could attenuate LPS-induced pulmonary microvascular endothelial injury. However, upregulative effect of PHC on beta-arrestin-1 expression is independent with presence of M3 receptor.
Keywords: F-actin; ICAM-1; M3 receptor; VCAM-1; beta-arrestin-1; penehyclidine hydrochloride.