Muscarinic Receptor Subtypes and ERK in the Mouse Parotid Gland

A previous study indicated that carbachol stimulated the ERK pathway independently of the EGF receptor pathway. Carbachol stimulation required protein kinase C but not calcium. Objectives: The purpose of this study was to determine the role of the 2 mouse parotid muscarinic receptor subtypes in the ERK signaling pathway. Methods: Mouse parotid gland cells were incubated with 5 µM carbachol (near EC50 value) with at least 7 different concentrations of subtype-selective muscarinic receptor antagonists as well as atropine. The cells were lysed and the supernatant subjected to electrophoresis and immunoblotting to determine the level of phosphorylated ERK relative to total ERK. Inhibition curves were analyzed by non-linear regression analysis and IC50 values were determined. Results: The following muscarinic receptor antagonists were used: pirenzepine (M1 selective), methoctramine (M2 selective), 4-DAMP (M3 selective) and atropine (non-selective). Antagonism of carbachol-stimulated ERK phosphorylation yielded near-parallel inhibition curves when comparing the 4 antagonists. The IC50 values (micromolar) for atropine, 4-DAMP, pirenzepine and methoctramine were 0.43±0.18, 0.75±0.26, 122.7±24.0, and 3527±725, respectively (n = 3 or 4 inhibition curves). This rank order of IC50 values closely matched the values for the antagonists in binding studies as well as the antagonists' effects on phosphoinositide turnover and cyclic AMP levels that we have previously reported. Conclusions: The results indicate that, just as the M3 receptor controls the calcium signaling pathway in the mouse parotid gland, the M3 receptor is the major muscarinic receptor controlling ERK. This rules out a disproportionate role in the ERK pathway for the M1/M2 muscarinic receptor which is also present in the gland. Supported by Grant DE05249 from NIDCR-NIH.