(for FANZCA part I)

From our previous tutorial on the adrenergic component of the ANS, you will now be thoroughly familiar with CHIME (heh)! Here's the acetylcholine molecule:

the acetylcholine molecule. CLICK HERE!

Synthesis of acetylcholine (ACh) is simple:

        acetyl-Coenzyme A     +     choline   ==>   acetylcholine

The enzyme catalysing this reaction is choline acetyltransferase , found both in mitochondria and the cytosol. There are some similarities between noradrenergic synapses (discussed in the previous web-page) and cholinergic ones. Remember that at noradrenergic synapses, about 50% of noradrenaline is taken up by a presynaptic transporter? At cholinergic synapses there is also a presynaptic transporter - the difference is that ACh is first hydrolysed back to acetate and choline, and then choline is avidly taken up. It is this presynaptic uptake of choline that is the main regulator of ACh synthesis.

Release of ACh follows the pattern we have come to know - ACh is concentrated in large amounts in presynaptic vesicles, which release their contents into the synapse when voltage-gated calcium channels open in response to membrane depolarisation. The release and effects of ACh have been characterised in minute detail, especially at the neuromuscular junction where far larger amounts of ACh are released than at other sites. (We will not discuss neuromuscular transmission in detail, as we are concentrating on the autonomic nervous system, although exam candidates would be well-advised to acquaint themselves with the minutiae)!

As we would expect, there are specific transporter proteins involved in moving both ACh and choline around:

One cannot over-emphasise the importance of cholinesterase , the enzyme that breaks down ACh at the synaptic cleft. Cholinesterase inhibitors are important both as toxins, and as therapeutic agents.

We will here concentrate on acetylcholine actions in the peripheral nervous system. Acetylcholine acts on two vastly different classes of receptors - nicotinic receptors (with two subtypes, one at the neuromuscular junction of skeletal muscle, the other within ganglia and the CNS), and muscarinic receptors (widely distributed within both peripheral and central nervous systems). Some drugs other than ACh also stimulate both nicotinic and muscarinic receptors, for example, carbamylcholine and carbachol.

Note that the effects of cholinergic agents are complex, not only because of the existence of nicotinic and muscarinic receptors (and subclasses of these) but also because of the presynaptic and postsynaptic locations of such receptors. To make things even more complex, ACh has inhibitory (heterotropic) effects on sympathetic neurones!

As an aside, the bite of the black widow spider (Latrodectus) is so toxic because of massive release of acetylcholine from neurones. This is mediated by alpha latrotoxin, which appears to conduct calcium ions into the presynaptic terminal.

Latrodectus, the black widow spider

Nicotinic receptors and drugs

The prototype drug that stimulates nicotinic receptors is of course nicotine, which stimulates the nicotinic receptors on muscle cells, those found within autonomic ganglia, and (not least) nicotinic receptors within the nervous system.

Nicotiana tabacum

the nicotine molecule. CLICK HERE!

Some drugs that stimulate nicotinic receptors also cause block, for example nicotine itself. The mechanism of this block is instructive - ongoing stimulation of the receptors causes decreased electrical excitability due to inactivation of voltage-sensitive sodium channels.

Another, even more interesting block occurs. This "Phase II block" is seen clinically in skeletal muscle with repeated administration of doses of suxamethonium - the receptors become desensitised to stimulation, one of the several reasons not to give a second dose of 'sux'. Phase II block also occurs with nicotine stimulation of receptors.

We will not dwell on the nicotinic receptors on muscle cells, concentrating instead on the neuronal type, found in both sympathetic and parasympathetic ganglia. Nicotinic receptors are ligand-gated ion channels - unlike the fairly stereotyped muscle receptor, the neuronal one is quite heterogeneous, being made up of a mixed bag of a variety of different alpha and beta subunits. (There seems to be little clinical significance of this mix, at present). ACh binds the alpha subunit and this results in opening of an ion channel.

Drugs that act at nicotinic receptors

Agonists such as nicotine are mainly toxicological curiosities, rather than clinically useful drugs. Epibatidine (from the Dendrobatid poison dart frog Epipedobates tricolor ) is a potent agonist at nicotinic receptors. Interestingly enough, in the CNS, profound analgesia is induced, but peripheral effects of course preclude its use as an analgesic - it binds not only nerve but also muscle nicotinic receptors. More nerve-specific analogues have been synthesised. Phase II trials of the epibatidine analogue ABT-594 are now being started. Here's more than you ever wanted to know about epibatidine, and here's the molecule:

poison dart frog Epipedobates tricolor, the source of epibatidine

the epibatidine molecule. CLICK HERE!

Other nicotinic agonists are lobeline (from the Lobelia plant), and dimethylphenylpiperazinium (DMPP), both of which favour ganglionic receptors, although the former is pretty nonspecific. Nicotine itself slightly favours nerve receptors over muscle ones.


Ganglionic blockers

Ganglionic transmission of impulses is insanely complex. For example, in sympathetic ganglia, physiologists have observed four distinct responses to stimulation, with different onset and duration of effect. There are several ways in which drugs can block ganglionic transmission. Here are a few:

The first ganglionic blocker was hexamethonium - it's now merely a curiosity, made more interesting by Paton's quaint description of the "hexamethonium man" - usually enthusiastically reproduced in pharmacology textbooks. We won't repeat it here. Interestingly enough, hexamethonium and related agents don't interfere with the ACh receptor directly, in fact, they block the ion channel.

the hexamethonium molecule. CLICK HERE! Here's another ganglionic blocker - trimetaphan , which has been used to transiently induce controlled hypotension:

the trimetaphan molecule. CLICK HERE!

Trimetaphan appears to work by competitive block of ACh receptors. Mecamylamine is also a blocker of neuronal nicotinic receptors, initially developed as an antihypertensive ganglionic blocker, abandoned because of postural hypotension, and now of interest because its noncompetitive voltage-dependent interference with nicotinic receptors makes it significant in the study of addiction. (And you wondered why cigarettes are addictive)! In contrast, agents such as alpha-bungarotoxin, obtained from the venom of an Indian snake called the krait, specifically block muscular nicotinic receptors.

the alpha-bungarotoxin molecule. CLICK HERE!

The krait

Alpha bungarotoxin was pretty important in the initial isolation and characterisation of muscular ACh receptors. In contrast, the original non-depolarising neuromuscular blocking agents that were used clinically were far from specific for muscular nicotinic receptors. Tubocurarine, for example, also causes significant ganglionic blockade. (As do the similarly obsolete drugs gallamine and decamethonium).

the tubocurarine molecule. CLICK HERE!

The leaf of Chondrodendron tomentosum

Presynaptic nicotinic receptors

These receptors are peculiar - one might expect them to be responsible for inhibitory feedback at nicotinic synapses, but they appear to do just the opposite! This is particularly impressive at the neuromuscular junction, where they increase acetylcholine release with repeated stimulation - and are thus important in tetanic stimulation. The central effects of nicotine may be mediated by its stimulatory effect on presynaptic nicotinic receptors.

Muscarinic receptors and drugs

ACh has similar agonist effects at both muscarinic and nicotinic receptors, but there are several other agents that favour muscarinic receptors. Muscarinic receptors all mediate their effects through G proteins. From our knowledge of muscarinic effects on various organs, we can work out the clinical manifestations of such agents — miosis, sweating, salivation, abdominal cramps, and often, bradycardia. The prototype agonist is muscarine, derived from the poisonous fly agaric, Amanita muscaria.

the muscarine molecule. CLICK HERE!

Amanita muscaria

There are many other muscarinic agonists, including pilocarpine, bethanechol, and methacholine, but they have few medical uses. Methacholine (which is simply ACh with a methy group on the choline) has been used in carefully titrated amounts to diagnose asthma. Pilocarpine is still used in glaucoma (and has been used for treatment of Sjogren's syndrome); bethanechol, the carbamate derivative of methacholine, has been used in the past to goad the lethargic bladder or gastrointestinal tract into activity.

The jaborandi (Pilocarpus sp.), source of pilocarpine

the pilocarpine molecule. CLICK HERE!

Pharmacologists like to rhapsodise about "structure-activity relationships" and these agents haven't escaped their scrutiny. The activity of ACh depends on a positively-charged quaternary ammonium group, and an atom capable of forming a hydrogen bond through an unshared pair of electrons. The distance between the two may determine whether agents are nicotinic or muscarinic. Tweaking the ester lowers hydrolysis by cholinesterase (most of the above agents are not/minimally hydrolysed by cholinesterase).

Pilocarpine, unlike many of the other (quaternary) agents which penetrate membranes poorly, has a tertiary amine group, so it can penetrate the eye when applied topically. Muscarinic agonists within the eye have several effects, causing miosis and ciliary muscle contraction, and indirectly improving drainage of aqueous humour through the canals of Schlemm.

The five muscarinic receptors

Although five muscarinic receptors have been identified, helpfully labelled M1 to M5, only three are well-characterised. (M4 receptors are confined to the CNS, and M5 are not well understood). Here are the three important ones:

  1. M1 receptors are mainly found in the nervous system - they mediate excitatory effects, lowering transmembrane potential by a decrease in K + ion conductance; as an added wrinkle, they mediate increased gastric acid secretion seen with vagal stimulation. M1 receptors work via phospholipase C, increasing IP3 and DAG levels;

  2. M2 mediate the cardiac effects of (for example) vagal stimulation - they are inhibitory (hyperpolarizing membranes by increasing potassium conductance). M2 receptors are also found presynaptically in a variety of situations - this fits in with their inhibitory nature, here exploited for negative feedback. M2 receptors lower intracellular cAMP levels (via G k );

  3. M3 receptors are responsible for all the other effects of parasympathetic stimulation, as they are the cholinergic excitatory receptors found on glands and smooth muscle. In our first tutorial, we detailed all the various effects of such receptor stimulation. M3 receptors are similar to M1 in their use of phospholipase C. Physiology is however never simple - vascular smooth muscle relaxes in some situations due to M3 receptor stimulation. This relaxation is mediated by endothelial release of nitric oxide (NO), and occurs in some vascular beds that appear devoid of parasympathetic innervation.

M5 receptors seem similar to M1 and M3 in their effects; M4 are similar to M2.

Muscarinic antagonists

Muscarinic antagonists are medically important. The traditional antagonists (atropine and hyoscine) are not specific for particular receptor subtypes. There are selective agents - the infrequently used anti-ulcer drug pirenzepine is M1 selective (you can work this out once you know it inhibits acid secretion), and gallamine, quite apart from its other effects, is M2 selective. The unpronounceable hexahydrosiladifenol is an M3 specific antagonist. (There are others: dicyclomine is M1 specific, "AF-DX 116" blocks M2).

Detailed knowledge of atropine , scopolamine (=hyoscine) and the synthetic drug glycopyrrolate is vital for all anaesthetists (and examination candidates)! The major differences between atropine and glycopyrrolate are:

One peculiar response to atropine is the bradycardia reportedly seen with small doses (eg. 0.4 mg in an adult) - the mechanism of this paradoxical effect is still not clear, if it exists at all!

The deadly nightshade, Atropa belladonna - rich in atropine

the atropine molecule. CLICK HERE!

Scopolamine has pronounced CNS sedative effects, and may also be useful in preventing nausea and vomiting, especially due to motion sickness.

The trumpet flower, Datura stramonium, a source of scopolamine

the scopolamine molecule. CLICK HERE!

Ipratropium is not without interest, as it appears to be a fairly useful agent in the management of bronchospasm, especially in those with chronic obstructive airways disease with some airway hyperreactivity. Because ipratropium is quaternary, it is very poorly absorbed when delivered as an aerosol, confining its effects to the airways. In addition, ipratropium does not inhibit mucociliary clearance (an unfortunate side-effect of atropine therapy).

the ipratropium molecule. CLICK HERE!

The effects of poisoning with antimuscarinics are largely predictable from a knowledge of muscarinic receptors and their location - dry mouth, impaired sweating, mydriasis, and tachycardia, as well as profound CNS dysfunction with delirium and hallucinations. These effects are far more dangerous in children than in adults. The mnemonic "red as a beet, blind as a bat, dry as a bone, mad as a hatter, and hot as hell" is apt.

Other agents with anticholinergic activity include tricyclic antidepressants, some major tranquillizers, and many of the older ('sedating') anti-histamines.

Muscarinic agonists

We have already listed some non-selective muscarinic agonists. There are a few muscarinic agonists that are selective for certain sub-types of muscarinic receptor, but they are of little clinical significance. Oxotremorine and "McNA343" are M1-selective.

Drugs affecting ACh release or metabolism

The main drugs of clinical importance that interfere with ACh metabolism are the anticholinesterases. There are several other drugs that interfere at other points in ACh metabolism, but apart from botulinum toxin, they have little clinical relevance. For the record, here are some:


cholinesterase. A BIIIG molecule! Acetylcholinesterase (ACHE) has two sites important in hydrolysis of ACh - the anionic site and esteratic site. There are several classes of agent that inhibit ACHE, raising ACh levels in the synapse. Cholinesterase inhibitors bind the esteratic site, usually to an acetate, carbamate or phosphate group. Clinically the most used is undoubtedly neostigmine, given as reversal of neuromuscular blocking agents administered during anaesthesia. The anticholinesterases may be:

the neostigmine molecule. CLICK HERE!      

the edrophonium molecule. CLICK HERE!      

the echothiophate molecule. CLICK HERE!

All of the organophosphates irreversibly bind ACHE. Pralidoxime has been widely used early on in organophosphate poisoning to 're-constitute' the ACHE molecule, although its effectiveness has recently been called into question. Obidoxime has similar effects:

the obidoxime molecule. CLICK HERE!

Physostigmine is a short-acting cholinesterase inhibitor that has been used in the past to manage atropine toxicity (especially in children, where atropine toxicity may be life-threatening). It has good CNS penetration, but a short half-life, and nicotinic effects should be carefully monitored.

Seeds of Physostigma venenosum, the calabar bean

the physostigmine molecule. CLICK HERE!


  1. Moss J, Craigo PA. The Autonomic Nervous System. in Miller RD [ed]. Anesthesia. 4ed. Churchill Livingstone. 1994. (An excellent review of the ANS).

  2. An excellent reference on acetylcholine from UT Neuroscience.

  3. Another good page from Washington University in St Louis.

  4. A comprehensive review of neuronal nicotinic receptors.

  5. (The source of this web-page contains hidden references to many sites which provide PDB files that can be read by CHIME)!

Thanks to Jet for picking up an error under "Muscarinic Receptors".