Abstract
HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.
Highlights
The five subtypes (M1–M5) of muscarinic acetylcholine receptors are widely distributed in the body and are involved in a variety of physiological functions
Our studies have shown that stable cells lines expressing human M1 muscarinic acetylcholine receptors (mAChRs) show many of the characteristics of cells expressing the endogenous receptors – receptor internalisation with agonist exposure and phosphorylation of ERK and induction of EGR-1 after transient exposure to the agonist carbachol
The rapid carbachol-mediated induction of EGR-1 was blocked by the MEK blocker U0126, which is consistent with our previous studies [27]
Summary
The five subtypes (M1–M5) of muscarinic acetylcholine receptors (mAChRs) are widely distributed in the body and are involved in a variety of physiological functions. MAChRs mediate the majority of transmission by acetylcholine and are involved in the control of neurological functions such as movement, attention and memory processes [1]. In the central nervous system, the M1 and M3 AChR subtypes have been implicated in the survival of a variety of cell types including neuronal cells [2]. A considerable literature exists for M3 receptors and their role in cell survival [3,4,5,6] or in cell death [7]. The involvement of M1 AChR in the survival of neuronal cells has not been studied as extensively, but several reports have shown that cholinergic activity mediated through M1 AChRs modulates the survival of retinal ganglion cells [8,9,10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
