Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line.
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 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 epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of 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 Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate 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 small interfering RNA. 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.
Lin
Alan L
AL
Department of Dental Diagnostic Science, University of Texas Health Science Center, San Antonio, TX, USA.
Zhu
Bing
B
Zhang
WanKe
W
Dang
Howard
H
Zhang
Bin-Xian
BX
Katz
Michael S
MS
Yeh
Chih-Ko
CK
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010306:Parotid Gland / Q000187:drug effects / Q000201:enzymology; D010766:Phosphorylation; D010862:Pilocarpine / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3 / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012469:Salivary Glands / Q000187:drug effects* / Q000201:enzymology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C086088:CHRM1 protein, human; D002614:Chelating Agents; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D047428:Protein Kinase Inhibitors; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D010862:Pilocarpine; D002217:Carbachol; C512478:EGFR protein, human; D066246:ErbB Receptors; D019061:src-Family Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium