Cholinomimetics

Question 1

Describe the synthesis, release and metabolism of acetylcholine.

Answer 1

Synthesis

Acetyl coA + choline → Ach + coA

• Catalysed by choline acetyltransferase (CAT)

Release

• ACh packaged into vesicles
• AP triggers Ca²⁺ influx into pre-synaptic terminal
• This stimulates vesicle exocytosis and release of ACh into synpatic cleft

Metabolism

• Acetylcholinesterase, present in the synaptic cleft, breaks down ACh:
  
  • ACh → choline + acetate
• Choline and acetate can be recycled back into the pre-synaptic nerve terminal so they can be used to make more ACh

Question 2

What is the difference between muscarinic and nicotinic effects?

Answer 2

Muscarinic effects are those that can be replicated by muscarine (muscarinic receptor agonist)

Nicotinic effects are those that can be replicated by nicotine (nicotinic receptor agonist)

NOTE: ACh acts on both muscarinic and nicotinic receptors

Question 3

What can be given to abolish muscarinic effects?

Answer 3

Atropine (competitive muscarinic antagonist)

Question 4

After atropine blockade of muscarinic actions, what can large doses of ACh induce?

Answer 4

Large doses of ACh can induce similar effects to those caused by nicotine

• Because the muscarinic receptors are blocked so therefore you get more ACh binding to the nicotinic receptors
• These nicotinic receptors also respond to nicotine therefore nicotine and ACh would have similar effects in this case

Question 5

Which branch of the ANS do muscarinic actions correspond to?

Answer 5

Parasympathetic nervous system

• Muscarinic receptors are present on parasympatheitc effector organs
• EXCEPTION: sweat glands have muscarinic ACh receptors but have sympathetic stimulation (i.e. post-ganglionic neurone terminated in sympathetic trunk)

Question 6

State where you would find the different muscarinic receptor subtypes.

Answer 6

M1

• Salivary glands
• Stomach
• CNS

M2

• Heart

M3

• Salivary glands
• Bronchial/visceral smooth muscle
• Sweat glands
• Eye

M4 - CNS

M5 - CNS

NOTES:

• M1, M2 and M3 are the main receptor subtypes
• Muscarinic receptors are generally excitatory (stimulates muscle contraction, secretion) except for on the heart (decreases heart rate and contractlilty)

Question 7

What type of receptor are all muscarinic receptors?

Answer 7

G-protein coupled receptors

Question 8

What is the difference in the G-protein receptors of M1, M3 and M5 compared to M2 and M4?

Answer 8

M1, M3 and M5 = G_q protein linked receptors (odds)

• They stimulate phospholipase C (PLC) which converts PIP₂ increases IP₃ and DAG
• Second messengers: IP₃ and DAG - increased

M2 and M4 = G_i protein linked receptors (evens)

• They inhibit adenyl cyclase which would convert ATP to cAMP
• Second messenger: cAMP - decreased

Question 9

Describe the structure of nicotinic receptors. What determines its ligand binding properties?

Answer 9

Nicotinic receptors are ligand-gated ion channels (i.e. ion channel opens when ligand binds)

Nicotinic receptors consist of 5 subunits:

• α = alpha
• β = beta
• γ = gamma
• δ = delta
• ε = epsilon

The combination of subunits determines its ligand binding properties.

Question 10

What are the two main types of nicotinic receptor? Describe their subunit composition.

Answer 10

Two main nACh receptor types:

• Muscle
• Autonomic ganglion

Muscle - 2α, β, δ, ε

Ganglion - 2α 3β (similar structure to this for nACh receptors found in the CNS)

Question 11

What is the relevance of having two nicotinic receptor subtypes in terms of pharmacology?

Answer 11

• This difference in subunit combination gives slightly different ligand-binding properties
• This means that drugs can be developed which are more selective for one receptor subtype than the other (e.g. drugs which act on the NMJ)

REMEMBER: The drugs are never 100% selective so they also have the potential to affect the other (unwanted) receptor subtype

Question 12

How do the effects of acetylcholine on nicotinic receptors compare to its effects on muscarinic receptors?

Answer 12

The effects of acetylcholine are relatively weak on nicotinic compared to muscarinic

• Relatively weak - i.e. you need more ACh to stimulate nicotinic receptors
  
  • This is probably because ACh has a lower affinity for nicotinic receptors so you need more collisions between receptor ligand in order for proper binding to take place
  • Reduced chance of proper binding per collision

Question 13

What three effects does muscarinic stimulation have on the eye?

Answer 13

Contraction of the ciliary muscle

• Accommodates for near vision
• It does this by making the lens thicker so there is more refraction of light from nearby objects onto the retina

Contraction of sphincter pupillae (circular muscle of the iris)

• This constricts the pupil (miosis) and increases drainage of intraocular fluid

Lacrimation (tears)

Question 14

What is glaucoma?

Answer 14

Sustained raised intraocular pressure – this can cause damage to the optic nerves and retina which can lead to blindness

Question 15

How can a muscarinic agonist be used to treat glaucoma?

Answer 15

• In angle-closure glaucoma, the angle between the iris and cornea is too small (closed)
• This which blocks the flow of the aqueous humour out of the iris
• Contraction of sphincter pupillae (cicular muscle of iris) opens up this angle
• This provides a pathway for aqueous humour drainage via the canals of Schlemm → reduced intraocular pressure

REMEMBER: This is only relevant for angle-closure glaucoma (there are other types of glaucoma which have a different cause for the raised intraocular pressure)

NOTE: Aqueous humour drains through the trabecular meshwork into the canals of Schlemm

Question 16

Describe, in detail (including the mechanism), the muscarinic effects on the heart.

Answer 16

• Decrease in intracellular Ca²⁺ in heart muscle decreases myocardial contractility
  
  • ↓ contractility → ↓ force of contraction → ↓ CO
  • This is because Ca²⁺ is required to bind to the muscle fibre to stimulate it to contract
  • Less Ca²⁺ binding means less actin-myosin cross bridges and hence reduced force of contraction
• Increasing K⁺ efflux from the conductive tissue in the heart decreases heart rate
  
  • This is because it hyperpolarises (more -ve) the cells meaning that it takes longer to reach the threshold potential for generating an AP
  • Reduced AP frequency = reduced HR

NOTE: receptors present in atria (i.e. atrial muscle) and nodes (i.e. SAN and AVN)

Question 17

Describe the muscarinic effects on the vasculature.

Answer 17

• Most blood vessels do NOT have parasympathetic innervation
• Acetylcholine acts on vascular endothelial cells to stimulate NO release via M3 ACh receptor
• NO induces vascular smooth muscle relaxation → vasodilation
• Result is a decrease in TPR

NOTE: This mechanism is more relevant to the clinical use of cholinomimetics than normal physiology

Question 18

Summarise the muscarinic effects on the cardiovascular system.

Answer 18

• Decreased heart rate (bradycardia)
• Decreased cardiac output
  
  • Due to decreased atrial contraction
  • Reduced atrial contraction → reduced blood volume emptied from atria into ventricles → reduced blood volume ejected from ventricles - SV
• Vasodilaation and reduced TPR
  
  • Due to stimulation of NO production

All of these combined can lead to a sharp drop in blood pressure

Question 19

Describe the muscarinic effects on non-vascular smooth muscle.

Answer 19

It is the opposite of muscarinic effects on vascular smooth muscle

It causes CONTRACTION of non-vascular smooth muscle

• Lungs – bronchoconstriction → diffuculty breathing
• GI tract – increased peristalsis (motility) → GI pain
• Bladder – increased bladder emptying

Question 20

Describe the muscarinic effects on exocrine glands.

Answer 20

Increases secretions from exocrine glands

• Increased salivation
• Increased lacrimation (tears)
• Increased bronchial secretions (e.g. mucus) → diffuculty breathing
• Increased GI secretions (including gastric HCl production) Increased sweating (SNS-mediated)

Question 21

What are the two types of cholinomimetic drug?

Answer 21

Directly Acting – muscarinic receptor agonists

Indirectly Acting – acetylcholinesterase inhibitors (anticholinesterases)

Question 22

State two types of muscarinic receptor agonists and give an example of each.

Answer 22

Choline Esters – Bethanechol

Alkaloids - Pilocarpine

Question 23

Describe the selectivity of pilocarpine.

Answer 23

Non-selective muscarinic receptor agonist

• It stimulates ALL muscarinic receptor subtypes

Question 24

What is the half-life of pilocarpine?

Answer 24

Approximately 3-4 hours

• Relatively long half life
• This is due to its good lipid solubility
  
  • This makes it harder to be cleared from the plasma (undergo renal excretion)
  • Its lipid solubililty means that it is reabsorbed back into the blood from the urine via the kidney tubular cells (lipid membranes)

Question 25

What is pilocarpine used to treat?

Answer 25

Particularly useful in ophthalmology as a _local_ treatment for **glaucoma** * Local *(in this case)* = eyedrops

Question 26

State some side effects of pilocarpine.

Answer 26

* Blurred vision * *PNS allows for near vision (accommodation)* * *This means that distant things look blurry* * Hypotension * Sweating * Respiratory difficulty * GI disturbance and pain * *Due to excessive motility and over-exertion*

Question 27

Describe the selectivity of bethanechol.

Answer 27

M3 selective agonist NOTE: * Bethanechol has a very similar structure to ACh * Only a minor modification to ACh which is non-selective creates an agonist which is selective *(i.e. more selective to M3 than the others but can still act on the others)*

Question 28

What is the half-life of bethanechol?

Answer 28

Approximately 3-4 hours * Resistant to degradation * *This is probably what gives it a longer half-life* * *Not broken down by ACh so can diffuse back into plasma once its had its effect (takes longer to be cleared from plasma)* * Orally active *(i.e. taken orally)* * Limited access to the brain *(as not lipid soluble)*

Question 29

What is bethanechol mainly used to treat?

Answer 29

* Assist bladder emptying * Enhance gastric motility

Question 30

State some side-effects of bethanechol.

Answer 30

* Sweating * Impaired *(blurred)* vision * Bradycardia * Hypotension * Respiratory difficulty

Question 31

What are the two types of anticholinesterase? Give examples of each.

Answer 31

Reversible – **physostigmine**, neostigmine, donepezil Irreversible – **ecothiopate**, dyflos, sarin NOTE: Cholinesterase metabolises ACh into _choline_ and _acetate_

Question 32

What are the two types of cholinesterase?

Answer 32

* Acetylcholinesterase * *True or specific cholinesterase - specific to ACh* * Butyrylcholinesterase * *Pseudocholinesterase* * *Not specific to ACh therefore 'pseudo'* They differ in distribution, substrate specificity and function NOTE: Technically both are true cholinesterases as they both metabolise choline esters but just different types

Question 33

Describe the properties of acetylcholinesterase.

Answer 33

* Found in **all** cholinergic synapses * Peripheral AND central * Very rapid action * \>10 000 reactions per second * Highly **selective** for acetylcholine * Works by hydrolysis reactions * It has a serine on its active site with an -OH group * *R group = CH₂OH* * *​*This OH group is required to initate the hydrolysis of the ester bond

Question 34

Describe the properties of butyrylcholinesterase.

Answer 34

* Found in **plasma** and **most tissues** but NOT cholinergic synapses * **Broad substrate specificity** * Hydrolyses other esters *(e.g. suxamethonium)* * Is principal reason for low plasma acetylcholine * Shows genetic variation

Question 35

State the effects of low, moderate and high doses of cholinesterase inhibitors.

Answer 35

Low dose: * Enhanced muscarinic activity Moderate dose: * Further enhancement of muscarinic activity * Increased transmission at ALL autonomic ganglia * because nicotinic **ACh** receptors are present at all autonomic ganglia High dose (toxic): * Depolarising block at autonomic ganglia & NMJ * *The nicotinic receptors get over-stimulated, so they shut down*

Question 36

Describe the mechanism of action of reversible anticholinesterases.

Answer 36

EXAMPLES: **Physostigmine**, neostigmine Mechanism of action: * Competes with acetylcholine for active site on the cholinesterase enzyme * Donates a carbamyl group to the enzyme, blocking the active site and preventing acetylcholine from binding * Carbamyl group removed by slow hydrolysis (minutes rather than milliseconds) * *Spontaneous hydrolysis - just slow* * Increase duration acetylcholine remains in synapse * *As ACh cannot be broken down until the carbamyl group is removed which takes time*

Question 37

Describe the structure of physostigmine.

Answer 37

It is a naturally occurring tertiary amine.

Question 38

Where does physostigmine primarily act?

Answer 38

At the postganglionic parasympathetic synapse

Question 39

What is the half-life of physostigmine?

Answer 39

30 mins

Question 40

What is physostigmine used to treat?

Answer 40

Glaucoma * Aids in introcular fluid drainage Atropine poisoning * Atropine is a _competitive muscarinic antagonist_ meaning that it is surmountable *(i.e. effect can be overcome)* * Physostigmine increases the concentration of acetylcholine at the synapse so that the acetylcholine can outcompete the atropine

Question 41

What type of compound are irreversible anticholinesterases?

Answer 41

_Organophosphates_, e.g: * **Ecothiopate** * Dyflos * Parathion * Sarin

Question 42

Describe the mechanism of action of irreversible anticholinesterases. Describe some properties of ecothiopate.

Answer 42

They rapidly react with the enzyme active site, leaving a large blocking group The blocking group is stable and resistant to hydrolysis so recovery requires the production of new enzymes *(takes days or weeks)* Ecothiopate: * **Potent** inhibitor of acetylcholinesterase * Slow reactivation of the enzyme by hydrolysis takes **several days** * **​***Essentially it can be removed from the enzyme by hydrolysis so this takes a very long time* * *Therefore it is more efficient to just make new enzymes* * *Based on this it is considered to be an _irreversible_ inhibitor*

Question 43

Which is the only organophosphate which is in clinical use? What are the others used for?

Answer 43

**Ecothipate** in clinical use Others commonly used as: * Insecticides * *Affects NS of insects* * *And becuase they are much smaller the dose is much more toxic to them than larger animals such as humans* * Nerve gas (agent) *= organic chemicals which disrupt neural transmission mechanisms, specifically at synapses by irreversibly blocking acetylcholinesterase*

Question 44

What is ecothipate used to treat?

Answer 44

Glaucoma * Used as eye drops * Acts to increase intraocular fluid drainage * Prolonged duration of action

Question 45

State some side effects of ecothiopate.

Answer 45

**Systemic** side effects: * Blurred vision * Sweating * Respiratory difficulty * Hypotension * GI disturbance and pain * Bradycardia

Question 46

What type of anticholinesterases can cross the blood-brain barrier and give some examples?

Answer 46

Non-polar * Physostigmine * Nerve agents (organophosphates)

Question 47

Describe the effects of low and high doses of anticholinesterase drugs on CNS activity.

Answer 47

Low doses: * CNS excitation with the possibility of convulsions High doses: * Unconsciousness * Respiratory depression * Death

Question 48

Describe what happens in organophosphate poisoning.

Answer 48

Accidental exposure to organophosphates used in insecticides, or deliberate use as nerve agents can cause severe toxicity resulting in: * Increased muscarinic activity * CNS excitation * Depolarising NM block

Question 49

Describe the treatment of organophosphate poisoning.

Answer 49

**IV atropine:** * This is a competitive muscarinic receptor agonist so blocks the muscarinic receptors * Therefore it reduces the effect of the raised synaptic acetylcholine concentration * Patient is put on artifical respiration because of the respiratory depression * *Caused by the excess acetylcholine at the synapse → depolarising block* If found within the first few hours, the patient should be given **IV pralidoxime** * This can unblock the enzyme * BUT phosphorylated enzyme ages within a few hours * *i.e. Essentially the whole ezyme-organophosphate compound undergoes a conformational change (ageing) meaning that the enzyme is _irreversibly resistant_ to being unblocked by pralidoxime*