Effects of acetylcholine receptor activation

This chapter is relevant to Section M1 (i) of the 2023 CICM Primary Syllabus, which expects the exam candidates to "describe the autonomic nervous system, including anatomy, receptors, subtypes and transmitters (including their synthesis, release and fate)."  This seems to occupy a dominant position in the thinking of the CICM examiners, as many of the autonomic SAQs have been directed to the pharmacology of cholinergic and anticholinergic drugs. Examples include:

  • Question 19 from the second paper of 2019 (atropine)
  • Question 14 from the first paper of 2018 (anticholinesterase drugs)
  • Question 21 from the first paper of 2015 (anticholinesterase drugs)
  • Question 3 from the first paper of 2011 (atropine vs. glycopyrrolate) 
  • Question 8(p.2) from the first paper of 2009 (organophosphate poisoning)

Historical evidence supports the cynical remarks of any reader who may point out that these are in fact pharmacology questions and can be answered by rote-memorising large tables. Even though the 

physiology of parasympathetic neurotransmission was a key feature in the stems of Question 4 from the second paper of 2014 and Question 1 from the first paper of 2017, the examiners commented that "some candidates described the cellular basis of Nicotinic, Muscarinic and M1-M5 receptors which didn't attract marks" because  "detail concerning receptor physiology was not required". As such, what follows is surplus to need, and the time-poor exam candidate is redirected back to the repetitive droning of their spaced repetition. 

Acetylcholine receptor effects in brief summary:

  • Nicotinic: numerous subtypes, a pentameric ligand-gated sodium channel
    • Operates the ganglionic neurotransmission in the autonomic nervous system
  • M1- Gq protein coupled – second messenger is IP3
    • Involved in cognitive function, eg. memory
    • Increased seizure activity
  • M2- Gi protein coupled – decreases cAMP
    • Miosis (contraction) of the pupillary sphincter muscle
    • Contraction of the ciliary muscle for far vision
    • Lacrimal gland secretion
    • Significant reduction in heart rate
    • Significant reduction in atrial contractility, and shortened action potential duration
    • Significant reduction in the conduction velocity of the AV node
    • Slight decrease in ventricular contractility
    • Increased motility and tone of the stomach
    • Relaxation of gastric sphincters
    • Stimulation of gastric secretion
    • Contraction of the gallbladder
    • Relaxation of the intestinal sphincters, and increased intestinal motility
  • M3- Gq protein coupled – second messenger is IP3
    • ​​​​​​​Miosis (contraction) of the pupillary sphincter muscle
    • Contraction of the ciliary muscle for far vision
    • Lacrimal gland secretion
    • Salivation and dilation of the salivary ducts
    • Greatly increased nasal mucus secretion
    • Increased production of nitric oxide synthase by the vascular endothelium
    • Increased motility and tone of the stomach
    • Relaxation of gastric sphincters
    • Stimulation of gastric secretion
    • Contraction of the gallbladder
    • Relaxation of the intestinal sphincters, and increased intestinal motility
    • Bladder detrusor muscle contraction, and relaxation of the trigone sphincter
    • Erection
    • Generalised secretion of the sweat glands (not just sweaty palms, but all over)
    • Increased secretion of the pancreatic juice
  • M4- Gi protein coupled – decrease cAMP
    • Inhibition of neurotransmitter release in the CNS
    • Facilitates Dopamine release
  • M5- Gq protein coupled – second messenger is IP3
    • ​​​​​​​Facilitates dopamine release

Caulfield (1993) is probably the best reference for muscarinic receptors, and Skok (2002) for nicotinic ganglionic ones, though in all honesty most people would be happy with a table such as this one from Katzung. More information than can be contained in a single Powerpoint slide is probably in excess to what is required, considering how little attention this has received in the exams. The trainee interested in cholingergic biochemistry will likely be coming at the subject from a toxicology angle, and will benefit more from a discussion of cholinergic and anticholinergic drugs.

Classification of acetylcholine receptors

Like the adrenergic group of receptors, the acetylcholine receptor families were discovered by people who were mostly experimenting with the effects of different toxins. Unlike the adrenergic agents, the names of the toxins ended up incorporated into the names of the receptors, rather than being given Greek letters. Muscarine, an active principle of Amanita muscaria which the fungus appears to have intended as an insecticide, was implicated as an important stimulant of cholinergic neurotransmission by Henry Dale before there was even such a concept as a "receptor" (in 1914), and the term "muscarinic" remained in use even though it was not until the 1960s that the receptors were identified and characterised properly. "Nicotinic" neurotransmission was also the observation of the effect of a toxin, this time nicotine on the sympathetic nerves of cats (Langley, 1901). The reason nobody uses the term "acetylcholinic receptors" is because nobody was using acetylcholine to do these experiments, and its importance as a neurotransmitter was not recognised until Feldberg & Gaddum (1934), by which stage everybody was already using "nicotinic" and "muscarinic" to describe the different physiological effects, though admittedly "cholinergic" was also in play since 1933. 

Nobody cares, of course, as the febrile hour of the exam approaches, and the CICM First Part candidate will surely be more interested in the modern nomenclature, and ideally in the shortest way possible.

Thus, from the IUPHAR/BPS,  the following subtypes are recognised:

  • Muscarinic receptors:
    • M1- Gq protein coupled – second messenger is IP3
    • M2- Gi protein coupled – decreases cAMP
    • M3- Gq protein coupled – second messenger is IP3
    • M4- Gi protein coupled – decrease cAMP
    • M5- Gq protein coupled – second messenger is IP3
  • Nicotinic receptors:

This "N1" and "N2" classification system is actually a necessary oversimplification designed to shield the reader from the nightmarish reality, that in fact these receptors are not just two types, but a near-infinite range of possible combinations from a pool of 17 homologous polypeptides (α1 to 10, β1–4, γ, δ, and ε). These modules combine into pentamers to form the protein pore, and the specific configuration we usually see in the ganglia are the α3-type receptors, although there are many others present as well. The abundant varieties of nicotinic receptor subtypes gives rise to different functional characteristics and drug affinities, of which the most obvious is nicotine itself. Muscle acetylcholine receptor bind nicotine with only the greatest reluctance, whereas ganglionic and CNS subtypes are much more interested. 

For the neuromuscular junction receptors, the five subunits are beta, delta epsilon and two alpha-1

The two alpha-1 subunits act as the binding sites of acetylcholine as well as the curare toxins.  For the autonomic ganglia receptors, the five subunits are three beta and two alpha-3 The two alpha-3 subunits also act as binding sites for acetylcholine, but they will not bind neuromuscular blocking agents.

nicotinic receptor structure

Not everyone uses N1 and N2, and even college-recommended texts occasionally call them something else. For example, they are "muscle type" and "neuronal type" in Goodman & Gilman, and Ganong  merely subdivide them into "those found in muscle at neuromuscular junctions" and "those found in autonomic ganglia and the central nervous system", whereas Katzung doesn't even bother to classify them in any way. The "N1" and N2" thing comes from StatPearls, but the reference they offer in support does not actually use those terms, which means it may not exist anywhere other than StatPearls (and, now, apparently, also here).   

For the muscarinic receptors, the situation is somewhat less murky. They are defined very clearly on the basis of antagonists selective for each receptor. For example, M1 receptors are defined by their affinity for pirenzepine, which is ironic because pirenzepine cannot cross the blood-brain barrier and M1 receptors are almost entirely limited to the CNS. The distribution of these receptor types is mostly based on immunofluorescence studies which target each receptor subtype with very specific antibodies and then observe which parts of the sliced rat glow brightest for the camera. Using these techniques X-filled tables such as this one from Skok:

M1, M3 and M5 muscarinic receptor signalling pathway

M1, M3 and M 5 are Gq protein coupled: the second messenger is IP3, and the result is calcium release. 

In general, the rule of thumb for these receptors is the excitation of excitable tissue, and the activation of various glandular and secretory function. Most glandular cells, posed with a massive influx of diacylglycerol and IP3 will begin to secrete stuff. Similarly, smooth muscle will contract when there is a calcium influx.

M1 M3 and M5 receptor intracellular signalling pathway

 

Effects of activating M1, M3 and M5 muscarinic receptors

M1: 

  • Involved in cognitive function, eg. memory
  • Increased seizure activity

M3:

  • Miosis (contraction) of the pupillary sphincter muscle
  • Contraction of the ciliary muscle for far vision
  • Lacrimal gland secretion
  • Salivation and dilation of the salivary ducts
  • Greatly increased nasal mucus secretion
  • Increased production of nitric oxide synthase by the vascular endothelium
  • Increased motility and tone of the stomach
  • Relaxation of gastric sphincters
  • Stimulation of gastric secretion
  • Contraction of the gallbladder
  • Relaxation of the intestinal sphincters, and increased intestinal motility
  • Bladder detrusor muscle contraction, and relaxation of the trigone sphincter
  • Erection
  • Generalised secretion of the sweat glands (not just sweaty palms, but all over)
  • Increased secretion of the pancreatic juice

M5:

  • Facilitates dopamine release

M2 and M4 muscarinic receptor signalling pathway

M2 and M4 are Gi protein coupled: they deactivate adenylyl cyclase, and decrease the levels of cAMP.

Broadly speaking, these receptors are membrane stabilizers. Excitable tissues hyperpolarize, inward rectifying potassium currents start flowing, voltage-gated calcium channels are inhibited.

M2 and M4 receptor intracellular signalling pathway

Effects of activating M2 and M4 muscarinic receptors

M2:

  • Miosis (contraction) of the pupillary sphincter muscle
  • Contraction of the ciliary muscle for far vision
  • Lacrimal gland secretion
  • Significant reduction in heart rate
  • Significant reduction in atrial contractility, and shortened action potential duration
  • Significant reduction in the conduction velocity of the AV node
  • Slight decrease in ventricular contractility
  • Increased motility and tone of the stomach
  • Relaxation of gastric sphincters
  • Stimulation of gastric secretion
  • Contraction of the gallbladder
  • Relaxation of the intestinal sphincters, and increased intestinal motility

M4:

  • Inhibition of neurotransmitter release in the CNS
  • Facilitates Dopamine release

References

Bowden, K., and G. A. Mogey. "The story of muscarine." Journal of Pharmacy and Pharmacology 10.1 (1958): 145-156.

Wang, Pen-Chung, and Madeleine M. Joullié. "Muscarine alkaloids." The Alkaloids: Chemistry and Pharmacology. Vol. 23. Academic Press, 1984. 327-380.

Feeney, K., and T. Stijve. "Re-examining the Role of Muscarine in the Chemistry of Amanita muscaria. Mushroom.the Journal of Wild Mushrooming 106 (2011): 32-36.

Schmiedeberg, Oswald. Das Muscarin: das giftige Alkaloid des Fliegenpilzes (Agaricus muscarius L.): seine Darstellung, chemischen Eigenschaften, physiologischen Wirkungen, toxicologische Bedeutung und sein Verhältniss zur Pilzvergiftung im allgemeinen. FCW Vogel, 1869.

Dale, Henry Hallett. "The action of certain esters and ethers of choline, and their relation to muscarine." Journal of Pharmacology and Experimental Therapeutics 6.2 (1914): 147-190.

SIMONART, ANDRÉ. "ON THE ACTION OF CERTAIN ETHERS OF β-ALKYL CHOLINE DERIVATIVES." Journal of Pharmacology and Experimental Therapeutics 50.1 (1934): 1-14.

Langley, John Newport. "On the stimulation and paralysis of nerve-cells and of nerve-endings: Part I." The Journal of physiology 27.3 (1901): 224.

Kuffler, S. W., and D. O. J. U. Yoshikami. "The number of transmitter molecules in a quantum: an estimate from iontophoretic application of acetylcholine at the neuromuscular synapse." The Journal of physiology 251.2 (1975): 465-482.

Feldberg, Wilhelm, and John Henry Gaddum. "The chemical transmitter at synapses in a sympathetic ganglion." The Journal of physiology 81.3 (1934): 305.

Dale, Henry Hallett. "Nomenclature of fibers in the autonomic system and their effects." The Journal of Physiology (1933).

Colquhoun, David, et al. "Nicotinic acetylcholine receptors." Drug Discovery and Drug Development (2003): 357-405.

Kalamida, Dimitra, et al. "Muscle and neuronal nicotinic acetylcholine receptors: structure, function and pathogenicity." The FEBS journal 274.15 (2007): 3799-3845.

Papke, Roger L. "Merging old and new perspectives on nicotinic acetylcholine receptors." Biochemical pharmacology 89.1 (2014): 1-11.

Skok, Vladimir I. "Nicotinic acetylcholine receptors in autonomic ganglia.Autonomic Neuroscience 97.1 (2002): 1-11.

Caulfield, Malcolm P. "Muscarinic receptors—characterization, coupling and function." Pharmacology & therapeutics 58.3 (1993): 319-379.