By Duy Thai, 1997 Pharmacology Semester 1 page 1 of 4

CHOLINERGIC PHARMACOLOGY I

• The major areas where Ach acts as a neurotransmitter are:

• In the presynaptic nerve fibres (both parasympathetic and sympathetic)
• In the postganlionic parasympathetic fibres
• In the postganlionic sympathetic fibres going to the sweat glands of the skin
• In the somatic fibres innervating skeletal muscle

Acetylcholine synthesis
• Very few drugs of therapeutic significance act to interfere with Ach synthesis
• Unlike NA, Ach synthesis is relatively easy

Choline acetyltransferase
Acetyl CoA + choline Coenzyme A + Ach

Obtained from the

Choline breakdown of Ach and also
from the diet (lecithin)

Choline acetytransferase CoA

Acetyl CoA Acetyl choline

ATP citrate ligase

Citrate

Acetyl CoA does not pass

Acetyl CoA through the mitochondrial
membrane, therefore, it is Vesicle storage
converted to citrate first

Ways to interfere with the synthesis

• Provide cofactor deficiencies
• Deficiencies in Fe, O2 and glucose (not a therapeutic possibility)
• Choline acetyltransferase inhibition
• 4 Napthylvinylpyridine (4 NVP)
• Not very effective at inhibiting synthesis because there are also stores of Ach.
• You need to inhibit the synthesis of Ach for a very long time and completely
reduce the stores of Ach. There is a very large safety factor - not much Ach is
required to get a response
• Choline uptake inhibition (active, carrier mediated transport process)
• Hemoccholinium 3 (HC3)
• Can get a slowly, progressive failure of transmission
• A cholinergic nerve which is simulated faster, will increase the loss from stores,
without replenishement

Storage of Ach
• Ach is stored in vesicles in the nerve terminal.
• The vescicle is 40 to 50nm in diameter.
• Unlike the storage vesicles of NA (which are dark due to the presence of chromogranins), the storage vesicles
of Ach are clear.
• The vesicles also contain ATP, and vesiculin (a specific protein - unkown function)
• The drug which blocks the storage of Ach is vesamicol

Exocytotic release
• Ca2+ dependent
By Duy Thai, 1997 Pharmacology Semester 1 page 2 of 4

• Drugs affecting the release:

• Drugs which increase Ach release (mainly venom toxins)
• β bungarotoxin
• Banded krait venom
• Redback spider venom
• These toxins cause a massive release of Ach which results in fasciculations of muscle
followed by paralysis as all of the Ach is drained from the nerve terminal
• Drugs which decrease Ach release
• Botulinum toxin
• Produced by the Clostridium botulinum
• This bacterium lives in unsterilised canned foods.
• It is very potent, only a few bacteria are needed to have heaps of poison present
• It acts solely on the release process.
• The toxin interacts irreversibly with the lipids inside the nerve terminal. This
explains why, once poisoned by the toxin, it takes months to receover (since lipids
have a slow turnover rate). The effects of the toxin can be attenuated by using
substances containing sialic acid residues (e.g. gangliosides)
• Surprisingly, there is a therapeutic use of this toxin (in small doses)
• When injected into muscles of the eye, it causes prolonged paralysis,
which can be used to treat squint. By the time the effects wear off (in
months) the condition would have corrected itself, preventing the need for
surgery.

Ach receptors
• Early evidence suggested the presence of 2 types of recepotors
• If Ach was given intravenously, you would get a fall in blood pressure
• If atropine was then given, you would get a rise in blood pressure.
• What atropine did was to block the M receptors (which was responsible for the
parasympathetic effects). By blocking the M receptors, it allowed unrestricted action of Ach
on the N receptors, which are present on the sympathetic ganglia.
• Nicotinic receptors are present in:
• All ganglia
• On skeletal muscle
• On the adrenal gland (a specialied ganglia)
• The nicotinic receptor is a ligand gated ion channel designed for very rapid (within millisec)
response times.
• Muscarinic receptors are found in:
• Parasympathetic postganglionic target tissues
• Sympathetic cholinergic target tissues
• The muscarinic receptor is a G protein coupled, 7 transmembrane

Muscarinic receptors
• 7 transmembrane spanning receptor
• Has an intracellular domain which is responsible for coupling with the G protein complex
• Has an extracellular ligand binding unit where Ach binds to
• The intracellular terminal carboxyl tail is responsible for controlling the sensitivity of the receptor. If it is
phosphorylated, then the receptor becomes desensitised.
• Evidence for subtypes:
• There are regional differences in the affinity of antagonists
• The affinity of antagonists can be measured by a Schild plot to find the value of KA.
• If we have exactly the same receptor, no matter where it is found, it will have the same
affinity for an antagonist. (Remember that affinity for a receptor is different to response.
You can have exactly the same receptor, with the same affinity but have a different
response due to different transduction mechanisms.
• The development of selective drugs
• Pirenepine is selective for M1 receptors which control gastric acid secretion
• Molecular biologists have isolated and cloned 5 different subtypes of M receptors
By Duy Thai, 1997 Pharmacology Semester 1 page 3 of 4

Molecular biology of M receptors

• 5 subtypes (of which the first 3 are important)
• All are 7 transmembrane, G protein coupled receptors (hence have an intermediate response time - seconds)
• There is clear homology (the are clearly related)
• There are specific areas of the receptor which are related to specific functions (structure function
relationship)
• Areas related to G protein coupling
• Areas regulating sensitivity
• Here goes! The receptor subtypes are:
• M1, M3, M5 (the odd receptors)
• Use Gq/G11 G proteins
• The G protein activates the phophoinositol pathway
• The active intermediate is IP3 which stimulates the release of Ca2+
• Closes K+ channel
• M2, M4 (the even receptors)
• Use Gi/Go
• This G protein inhibits adenylate cyclase, hence reducing the levels of cAMP

• The M1 receptor is found in the stomach, controlling gastric acid secretion

• The M3 receptor is found on the endothelium of blood vessels and in the gut (causing increased gut motility)
and exocrine glands.
• The M2 receptor is found predominantly in the heart
• slows the HR
• opens K+ channels
• reduces Ca2+ conductance

The only important, therapeutical muscarinic antagonist is pirenzepine (M1 blocker)

• From all these effects, I make the conclusion that M1 and M3 receptors are the “activator” receptors -
causing acid release and intestinal motility. M2 receptors are the “slowing down” receptors, causing reduced
heart activity.

Structure activity relationships at muscarinic receptors

• Acetylcholine requires the following to be active:
• A quaternary N to form a cationic head
• If the N+ is replaced by a non charged C, then it is inactive
• The size of the cationic head is important:
• If the methyl (-CH3) groups are replaced by ethyl (-CH2CH3) groups, then the following
happens:
• One ethyl substitution (monoethyl substitution) has no effect, the compound
behaves similar to Ach
• Diethyl substitution markedly reduces the activity of the compound at the M
receptor
• Triethyl substitution results in no activity at the M receptor
• The ester link (-CH2 - O - C) has a role in regulating the activity and selective
• If the -O- is replaced with a -CH2- then the selectivity of the compound to M receptors is
reduced (the compound becomes more nicotinic selective)
• If the position of the -O- is changed, then there is not much difference in activity. The
distance is thus more important
• The alkylamine chain regulates the selectivity
• If a methyl group is placed on the β carbon, the compound becomes β methyl choline and is
M selective
• If a methyl group is placed on the α carbon, the compound is α methyl choline and is N
selective.
• The acetyl group is important in the activity of the compound.
By Duy Thai, 1997 Pharmacology Semester 1 page 4 of 4

• If you lack the acetyl group and only have the choline present, then you will have the same
pharmacological action as Ach, only with 1/1000 of the activity. (That means for every one
molecule of Ach, you need 1000 molecules of choline to get the same effect)
• The -CH3 tail of the acetyl group is important to determine whether the compound can be degraded
by acetylcholinesterase (ACE). If the -CH3 is replaced by a NH2, then the compound will no longer
be a substrate for ACE.
• The design of specific agonists makes use of the previous key structural requirements.
• Bethanechol has the following structual modifications:
• It has a β methyl substitution
• The -CH3 tail of the acetyl group is replaced by a -NH2
• Therefore, bethanechol is a muscarinic selective agonist which has a long lasting activity
(since it is not degraded by ACE)