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Event: 1602

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Activation, Muscarinic Acetylcholine Receptors

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Activation, Muscarinic Acetylcholine Receptors

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Molecular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
eukaryotic cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). 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 signalling 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. More help
Process Object Action
G-protein coupled acetylcholine receptor binding muscarinic acetylcholine receptor increased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
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

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. 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

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
Embryo Moderate
Juvenile Moderate

Sex Applicability

No help message 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. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. 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 Gi pathway (Haga, 2013). Gi 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

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. 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).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

           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

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  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. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). 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 Gq-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.