Roles of M₃ receptor in the effect of penehyclidine hydrochloride upregulated beta-arrestin-1 expression in LPS-stimulated HPMVEC

Abstract

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:

• M3 receptor
• beta-arrestin-1
• penehyclidine hydrochloride
• F-actin
• ICAM-1
• VCAM-1

Acknowledgements

The authors thank the English writing center at the Ohio State University for their excellent assistances in manuscript revision.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by grants from National Natural Science Foundation of China (Grant No. 81571291), National Natural Science Foundation of China (Grant No. 81101408), and the Youth Science Plan for Light of the Morning Sun of Wuhan City (Grant No. 2016070204010150).