Teaching an Old Drug New Tricks: Agonism, Antagonism, and Biased Signaling of Pilocarpine through M3 Muscarinic Acetylcholine Receptor

Abstract

Pilocarpine is a prototypical drug used to treat glaucoma and dry mouth and is classified as either a full or partial muscarinic agonist. Here, we report several unexpected results pertaining to its interaction with muscarinic M3 receptor (M3R). We found that pilocarpine was 1000 times less potent in stimulating mouse-eye pupil constriction than muscarinic agonists oxotremorin-M (Oxo-M) or carbachol (CCh), although all three ligands have similar values for M3R. In contrast to CCh or Oxo-M, pilocarpine does not induce Ca mobilization via endogenous M3R in human embryonic kidney cell line 293T (HEK293T) or mouse insulinoma (MIN6) cells. Pilocarpine also fails to stimulate insulin secretion and, instead, antagonizes the insulinotropic effect of Oxo-M and CCh-induced Ca upregulation; however, in HEK293T or Chinese hamster ovary-K1 cells overexpressing M3R, pilocarpine induces Ca transients like those recorded with another cognate G protein-coupled muscarinic receptor, M1R. Stimulation of cells overexpressing M1R or M3R with CCh resulted in a similar reduction in phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast to CCh, pilocarpine stimulated PIP2 hydrolysis only in cells overexpressing M1R but not M3R. Moreover, pilocarpine blocked CCh-stimulated PIP2 hydrolysis in M3R-overexpressing cells, thus, it acted as an antagonist. Pilocarpine activates extracellular regulated kinase 1/2 in MIN6 cells. The stimulatory effect on extracellular regulated kinase (ERK1/2) was blocked by the Src family kinase inhibitor PP2, indicating that the action of pilocarpine on endogenous M3R is biased toward -arrestin. Taken together, our findings show that pilocarpine can act as either an agonist or antagonist of M3R, depending on the cell type, expression level, and signaling pathway downstream of this receptor.

Cholinergic modulation inhibits cortical spreading depression in mouse neocortex through activation of muscarinic receptors and decreased excitatory/inhibitory drive

“…The experimental system in these studies was different in comparison with our study (anesthetized rats vs acute mouse brain slices). However, we could speculate that possible causes of the discrepancies might be linked to differential activation of muscarinic receptor subtypes and cell-specific biased signaling obtained with different cholinergic agonists; for instance, pilocarpine shows more pronounced selectivity for the M3 vs M1 receptors in comparison with McN-A-343 (Mitchelson, 2012;Pronin et al, 2017). Additionally, this could be coupled to different modifications of excitability, with more sustained and profound hyperexcitability that could induce remodeling of neuronal circuits, leading, for example, to increased extracellular K + buffering that would inhibit CSD (Guedes and Cavalheiro, 1997;Koroleva and Bures, 1980).…”

G Protein-Coupled Receptors (GPCRs)-Mediated Calcium Signaling in Ovarian Cancer: Focus on GPCRs activated by Neurotransmitters and Inflammation-Associated Molecules

“…Additionally, M1 receptors were detected in the thecal cells of rat ovarian follicles as well as in cells of the corpus luteum on proestrus day, and it was demonstrated that ovulation demands the activation of these receptors in the left ovary [40]. In vitro studies performed on Chinese hamster ovary (CHO)-K1 cells indicated no response to cholinergic stimulation [41]. Meanwhile, overexpressing M1 receptors or M3 receptors in CHO cells determined a similar intracellular calcium increase in response to pilocarpine, a partial agonist for muscarinic receptors, or to carbachol [41].…”

“…In vitro studies performed on Chinese hamster ovary (CHO)-K1 cells indicated no response to cholinergic stimulation [41]. Meanwhile, overexpressing M1 receptors or M3 receptors in CHO cells determined a similar intracellular calcium increase in response to pilocarpine, a partial agonist for muscarinic receptors, or to carbachol [41]. The study of Pronin and colleagues [41] is very important to understand that studies on non-tumorigenic ovarian cell lines should be considered with caution, as some of them do not respond to cholinergic stimulation.…”

Role of Muscarinic Acetylcholine Signaling in Gastrointestinal Cancers

“…Muscarinic ACh receptors are widely expressed throughout the body, including in the gut, brain, eye, heart, vasculature, etc. [20,21,22,23,24,25,26,27,28]. In particular, CHRM1 and CHRM3 are robustly expressed in the gastrointestinal (GI) tract.…”