Submitted on April 11, 2007
Accepted on March 25, 2008
Distinct pathways of extracellular signal-regulated kinase (ERK) activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line
Alan L Lin1, Bing Zhu1, WanKe Zhang2, Howard Dang3, Bin-Xian Zhang2, Michael S. Katz4, and Chih-Ko Yeh5*
1 Dental Diagnostic Science, University of Texas Health Science Center at San Antonio, San , Texas, United States
2 Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States
3 Community Dentistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States
4 Medicine-Division of Geriatrics and Gerontology, UTHSCSA, GREEC Dir, Audie L Murphy Memorial Veterans Hospital, San Antonio, Texas, United States
5 Geriatric Reseach, Educational, & Clinical Center, South Texas Veterans Health Care System, San Antonio, Texas, United States
* To whom correspondence should be addressed. E-mail: yeh{at}uthscsa.edu.
Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of extracellular signal-regulated kinases 1/2 (ERK1/2) by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the EGF receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG1478. Down- regulation of protein kinase C (PKC) by prolonged treatment of cells with the phorbol ester PMA diminished carbachol induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular calcium ([Ca2+]i) by EGTA did not affect ERK activation by either agent. Unlike carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist 4-DAMP decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3 but not M1 receptor siRNA. The Src inhibitor PP2 blocked pilocarpine induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.
