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Event: 1602

Key Event Title

A descriptive phrase which defines a discrete biological change that can be measured. More help

Activation, Muscarinic Acetylcholine Receptors

Short name

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Biological Context

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Level of Biological Organization
Molecular

Cell term

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Cell term
eukaryotic cell

Organ term

Key Event Components

The KE, as defined by a set structured ontology terms consisting of a biological process, object, and action with each term originating from one of 14 biological ontologies (Ives, et al., 2017; https://aopwiki.org/info_pages/2/info_linked_pages/7#List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling).Biological process describes dynamics of the underlying biological system (e.g., receptor signaling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signaling by that receptor). Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. Further information on Event Components and Biological Context may be viewed on the attached pdf. More help

Process	Object	Action
G-protein coupled acetylcholine receptor binding	muscarinic acetylcholine receptor	increased

Key Event Overview

AOPs Including This Key Event

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AOP Name	Role of event in AOP	Point of Contact	Author Status	OECD Status
AChE Inhibition Leading to Neurodegeneration	KeyEvent	Karen Watanabe (send email)	Under development: Not open for comment. Do not cite

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KE.In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help

Term	Scientific Term	Evidence	Link
zebrafish	Danio rerio	Moderate	NCBI
mice	Mus sp.	Moderate	NCBI

Life Stages

An indication of the the relevant life stage(s) for this KE. More help

Life stage	Evidence
Embryo	Moderate
Juvenile	Moderate

Sex Applicability

An indication of the the relevant sex for this KE. More help

Term	Evidence
Unspecific	Moderate

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. More help

Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors (GPCRs) with five different subtypes (M1, M2, M3, M4, and M5). GPCRs are transmembrane receptors that detect extracellular signals and activate internal pathways which modulate a variety of processes such as locomotion, learning and memory, thermoregulation and epileptic seizures (Gainetdinov and Caron, 1999). Subtypes M1, M3, and M5 are Gq- coupled receptors that activate phospholipase C enzyme resulting in two secondary messengers, inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). Subtypes M2 and M4 are inhibitory and signal using the G_i pathway (Haga, 2013). G_i protein activation inhibits adenylyl cyclase, and reduces the conversion of ATP to cAMP (Jett and Lein, 2011).

In its resting state, the mAChR G-protein subunits (alpha, beta and gamma) are clustered together and the alpha subunit is bound to GDP. Once a ligand binds to an mAChR, the receptor undergoes a conformation change that allows the alpha subunit to exchange its bound GDP with GTP, then the alpha subunit dissociates from the beta and gamma subunits. Once the alpha subunit is free of the beta and gamma subunits, it moves along the cell membrane to affect its target enzyme, which typically sends out secondary messenger signals (Kandel et al., 2013)

How It Is Measured or Detected

A description of the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements.These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA). Do not provide detailed protocols. More help

Most studies investigating the function of mAChRs involve blocking signaling from these receptors through use of selective antagonists like atropine or scopolamine, or the use of gene targeted knockout specimens (Bymaster et al. 2003; Faria et al. 2017). The distribution and density of mAChRs can be measured using radiolabeled agonists that bind to the mAChR binding site. The receptor activity can be measured by detecting secondary-messengers regulated by the G-protein.

Use mAChR agonist [³H] quinuclidinyl benzilate (QNB) to label mAChRs (all subtypes; see Fonnum and Sterri (2011) and measure binding levels as described by Fitzgerald and Costa (1993) and Gazit et al. (1979)
Determination of the relative levels of specific mAChR subtypes in tissues has been found through the use of subtype-specific antisera as described by Dörje et al. (1991)
Kinetic measurements of DAG production and IP3 release can be obtained through fluorescent reporters as in Falkenburger et al. (2013) and Dickson et al. (2013).
Changes in the activity and quantity of cAMP and the cAMP-dependent protein kinases can serve as an indicator of the activity of mAChRs bound to Gi-proteins (M2 and M4). cAMP content can be determined using a radioimmunoassay (RIA) kit (Heikkilä et al., 1991).
Adenylyl cyclase activity can be determined through an assay as described by Salomon et al. (1974) and used by Raheja and Dip Gill (2007).

Domain of Applicability

A description of the scientific basis for the indicated domains of applicability and the WoE calls (if provided). More help

Taxa:

mAChRs are found in most vertebrates, many of the studies cited are conducted using zebrafish and mice. Zebrafish are frequently used for high-throughput assays as they have well-conserved neurotransmitter structures, including acetylcholine transmitters (Garcia et al., 2016). This can provide valuable data regarding the activation of mAChRs in mammalian systems. Knockout mice also help to elucidate the functions of specific mAChR subtypes (Gainetdinov and Caron, 1999).

Life stage:

mAChRs signal neurons throughout all life stages (Miller and Yeh, 2016). They do not only affect individuals during developmental stages, but there have been some studies conducted specifically on the developmental effects of chemicals that affect acetylcholine signaling (Burke et al., 2017). Most of the whole animal experimental data are from younger specimens, but there have also been experiments on adult individuals (Fitzgerald and Costa, 1993).

Sex:

mAChRs are found in both males and females, with similar functions (Burke et al., 2017).

References

List of the literature that was cited for this KE description. More help

Burke, R. D., S. W. Todd, E. Lumsden, R. J. Mullins, J. Mamczarz, W. P. Fawcett, R. P. Gullapalli, W. R. Randall, E. F. R. Pereira and E. X. Albuquerque (2017), "Developmental neurotoxicity of the organophosphorus insecticide chlorpyrifos: from clinical findings to preclinical models and potential mechanisms”, Journal of Neurochemistry 142: 162-177. DOI: 10.1111/jnc.14077.

Dickson, E. J., B. H. Falkenburger and B. Hille (2013), "Quantitative properties and receptor reserve of the IP3 and calcium branch of Gq-coupled receptor signaling”, Journal of General Physiology 141(5): 521-535. DOI: 10.1085/jgp.201210886.

Dörje, F., A. I. Levey and M. R. Brann (1991), "Immunological detection of muscarinic receptor subtype proteins (m1-m5) in rabbit peripheral tissues”, Molecular Pharmacology 40(4): 459-462.

Falkenburger, B. H., E. J. Dickson and B. Hille (2013), "Quantitative properties and receptor reserve of the DAG and PKC branch of G_q-coupled receptor signaling”, The Journal of General Physiology 141(5): 537-555. DOI: 10.1085/jgp.201210887.

Faria, M., Prats, E., Padrós, F., Soares, A. M., & Raldúa, D. (2017). Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication. Archives of toxicology, 91(4), 1891-1901.

Fitzgerald, B. B. and L. G. Costa (1993), "Modulation of Muscarinic Receptors and Acetylcholinesterase Activity in Lymphocytes and in Brain Areas Following Repeated Organophosphate Exposure in Rats”, Fundamental and Applied Toxicology 20(2): 210-216. DOI: 10.1006/faat.1993.1028.

Fonnum, F. and S. H. Sterri (2011), “Tolerance Development to Toxicity of Cholinesterase Inhibitors”, in Toxicology of organophosphate and carbamate compounds, R. C. Gupta, Ed., Academic Press: 257-267.

Gainetdinov, R. R. and M. G. Caron (1999), "Delineating muscarinic receptor functions”, Proceedings of the National Academy of Sciences of the United States of America 96(22): 12222-12223. DOI: 10.1073/pnas.96.22.12222.

Garcia, G. R., P. D. Noyes and R. L. Tanguay (2016), "Advancements in zebrafish applications for 21st century toxicology”, Pharmacology and Therapeutics 161: 11-21. DOI: 10.1016/j.pharmthera.2016.03.009.

Gazit, H., I. Silman and Y. Dudai (1979), "Administration of an organophosphate causes a decrease in muscarinic receptor levels in rat brain”, Brain Research 174(2): 351-356. DOI: 10.1016/0006-8993(79)90861-8.

Haga, T. (2013), "Molecular properties of muscarinic acetylcholine receptors”, Proceedings of the Japan Academy Series B: Physical and Biological Sciences 89(6): 226-256. DOI: 10.2183/pjab.89.226.

Heikkilä, J., C. Jansson and K. E. O. Åkerman (1991), "Differential coupling of muscarinic receptors to Ca2+ mobilization and cyclic AMP in SH-SY5Y and IMR 32 neuroblastoma cells”, European Journal of Pharmacology: Molecular Pharmacology 208(1): 9-15. DOI: 10.1016/0922-4106(91)90045-J.

Jett, D. A. and P. J. Lein (2011), “Noncholinesterase Mechanisms of Central and Peripheral Neurotoxicity: Muscarinic Receptors and Other Targets”, in Toxicology of organophosphate and carbamate compounds, R. C. Gupta, Ed., Academic Press: 233-245.

Kandel, E., J. Schwartz, T. Jessell, S. Siegelbaum and A. J. Hudspeth (2013), “Modulation of Synaptic Transmission: Second Messengers”, in Principles of Neural Science, Fifth Edition, Blacklick, United States, McGraw-Hill Publishing: 236-259.

Miller, S. L. and H. H. Yeh (2016), “Neurotransmitters and Neurotransmission in the Developing and Adult Nervous System”, in Conn's Translational Neuroscience: 49-84.

Raheja, G. and K. Dip Gill (2007), "Altered cholinergic metabolism and muscarinic receptor linked second messenger pathways after chronic exposure to dichlorvos in rat brain”, Toxicology and Industrial Health 23(1): 25-37. DOI: 10.1177/0748233707072490.

Salomon, Y., C. Londos and M. Rodbell (1974), "A highly sensitive adenylate cyclase assay”, Anal Biochem 58(2): 541-548. DOI: 10.1016/0003-2697(74)90222-x.