Session 6 and 7 Groupwork

Question 1

What are four distinct general classes of synapse in the ANS ACh acts as the major neurotransmitter on?

Answer 1

Post-ganglionic sympathetic innervation of sweat glands

Pre-ganglionic sympathetic synapses

Pre-ganglionic parasympathetic synapses

Post-ganglionic parasympathetic fibre-target tissue (neuroeffector junction)

Question 2

What is ACh synthesised from? What enzyme is involved? Where does synthesis occur? What is the rate limiting factor?

Answer 2

• Synthesied from Choline and Acetyl CoA
• Enzyme: Choline Acetyltransferase
• Synthesis occurs in the cytoplasm of cholinergic neurones in the axon terminal
• Rate limiting factor: availability of the choline which depends on uptake of choline into the neurone.

Question 3

How is ACh packaged for release?

Answer 3

• After synthesis, ACh is transported into synaptic vesicles for storage via ACh/H+ antiport channel. An ATPase that pumps protons into the vesicle (V-ATPase) provides the energy necessary for this process.
• Cholinergic vesicles also contain ATP and heparan sulphate, which serve as counter ions for positively charged ACh.
• The increase in intracellular Ca2+ facilitates the binding of Synaptotagmin to the SNARE-Complex proteins, which together mediate vesicle-membrane attachment and fusion.

Question 4

What are the events that occur to cause release of ACh by pre-ganglionic cholinergic fibres synapses?

Answer 4

Action potential arrives at the axon.

Depolarisation opens the voltage-gated Ca2+ channels in the presynaptic membrane.

Ca2+ enters down its diffusion gradient binding to synaptotagmin.

This binding brings the vesicle close to the membrane.

The vesicel binds to the snare complex to make a fusion pore.

ACh is released and diffuses into the cleft.

ACh binds to post-synaaptic receptors - nACHr

Ach is removed from synaptic cleft by Acetylcholinesterase.

Question 5

Give an example of an agent which interferes with release of Acetylcholine

Answer 5

Cholistridium Botulinum:

Cleaves specifc part of SNARE complex.

Impaired targeting to synapse

Reduced ACh release

Results in paralysis

Clinically used to treat a variety of conditions such as muscle spasm.

Also known as botox.

Question 6

What mechanisms does the parasympathetic synapse have that rapidly terminate the action of ACh?

Answer 6

• Acetylcholinesterase breaks down ACh into choline and acetate.
• Butyrylcholinesterase also responsible for degrading ACh.
• Acetylcholinesterase located on the post-synaptic membrane hydrolyses ACh to acetate and choline.
• Inactivation of NA+ channels on the post-synaptic membrane.

Question 7

What happens to free choline and acetate present in synapses?

Answer 7

• Choline and acetate diffuse back into the presynaptic neurone.
• Acetate is converted into Acetyl CoA in the mitochondria.
• Acetyl CoA and Choline are recombined in a reaction catalysed by choline acetyltransferase.

Question 8

Briefly list some of the important sites of parasympathetic innervation

Answer 8

• Salivary glands - watery,high enzyme
• Heart - decreased heart rate, decreased conduction and velocity
• Stomach - digestion
• Penis - erection (erectile tissue has no sympathetic innervation)
• Bladder- urination
• Descending colon - peristalsis
• Preganglionic axons are myelinated and Postganglionic axons are not.

Question 9

What is the effect of increased parasympathetic discharge in the heart?

Answer 9

• Parasympathetic innervation to the heart is via the Vagus Nerve (10th Cranial Nerve)
• Parasympathetic action on the:
• SA Node: decreases HR
• AV node and Purkinje fibres: decreases contractile velocity
• Atria and ventricles: decreases contractility (Contractility = force of contraction-stroke volume)

Question 10

ACh is employed as the neurotransmitter at all parasympathetic nerve synapses. Are there any other synapses in the ANS that use ACh as a neurotransmitter?

Answer 10

• Most of the PNS
• All preganglionic sympathetic neurones.
• Sympathetic innervation of sweat glands
• Neuromuscular junctions

Question 11

What naturally-occuring substances, which each either mimic or prevent some of the actions of ACH, were first used to show that two distinct classes of ACh receptor exist?

Answer 11

• ACh agonists: Nicotine and Muscarine
• ACh antagonists: Scopolamine and Atropine (both are anti-muscarinic)
• E.g. in bradycardia, you give atropine which blocks parasympathetic innervation.

Question 12

Briefly, explain how the receptor subtype found at the ganglionic junction causes a post-ganglionic response when activated by ACh?

Answer 12

Nicotinic

• 2 ACh receptors bind to the alpha-subunit of nAChRs.
• This causes Na+ influx and K+ efflux.
• Depolarisation
• Vm reaches -10mV threshold causing opening of voltage-gated Na+ Channels

Question 13

What is the class of ACh receptor found at the parasympathetic neuroeffector junction?

Answer 13

• Muscarinic - this is the slow part of transmission (seconds to hours).
• These are G protein coupled receptors.
• Act directly via second messengers or act indirectly by kinases.
• There are different types of muscarinic receptors (M1-M5)

Question 14

Name the Predominant G-Protein and effectors involved in transducing signals from M1, M2 and M3

Answer 14

Question 15

Briefly describe the series of events that couple activation of M2 muscarinic receptors to the activation of the effectors.

Answer 15

Question 16

For the tissues: SA node, Bronchi, Bladder, Glands and Parasympathetic nerve terminals at neuro-effector junctions i.e. ‘pre-synaptic receptors’, list the major physiological actions of ACh

Answer 16

Question 17

What might be the advantage of synthesising cholinoceptor agonists which only interact with a particular receptor subtype? And are such agents available?

Answer 17

Certain cholinceptors such as M2 are primarly located within the heart therefore drugs targeted to this receptor can be specific without affecting other cholinoceptor pathways.

Direct agonists produce a pharmacological effect by receptor activation. Indirect agonists inhibit Acetylcholinesterase and therefore increase the levels of ACh causing an increased cholinergic response. Indirect agonists can be reversible or irreversible.

The need for specific drugs is that the more specific the drug, the less unwanted side effects occur in other areas of the body since the receptors are found in many areas of the body.

Yes, such agents are available

Question 18

Are cholinoceptor agonists used clinically?

Answer 18

They are used clinically however as a group they show little specificity in their actions which limits their clinical usefulness.

Question 19

One clinical use of muscarinic cholinoceptor antagonists is to treat gastrointestinal disorders. Also these agents are frequently given as a premedication for general anaesthesia. Why are muscarinic cholinoceptor antagonists used under these circumstanes?

Answer 19

• GI disorders e.g. IBS, Gi hypermobility and peptic ulcers: antagonism of smooth muscle contraction and exocrine secretion means there is reduced activity of the GI tract. Acts on M1 and M3 receptors. e.g. Scopolamine is a smooth muscle relacant.
• Premedication for Anaesthesia; e.g. atropine and glycopytrolate are used to suppress respiratory secretion prior to surgery, reduce anxiety and pain, causes tachycardia because drugs block the vagus nerve (anaesthetics causes bradycardia and hypotension from vasodilation so tachycardia is useful). Act on M1 - M5 receptors.

Question 20

What unwanted side effects limit the usefulness of some muscarinic cholinoceptor antagonists?

Answer 20

• Atropine causes urinary retention, blurred vision, dry mouth, sandy eyes, constipation, confusion, restlessness, hallucinations, delirium (CNS toxicity)and this can progress to collapse of the circulatory and respiratory systems leading to death. Children are particularly sensitive.
• Scopolamine: dry mouth, tachycardia, dyshidrosis (type of eczema)

Question 21

Draw a simple diagram to illustrate the anatomy of the human eye relevant to sites of sympathetic and parasympathetic innervation.

Answer 21

• Ciliary muscle innervated by both
• Lacrimal gland is parasympathetic
• Iris sphincter muscle is parasympathetic
• Pupil dilation is sympathetic
• Pupil constriction is parasympathetic

Question 22

An abnormally raised intraocular pressure is termed glaucoma. Untreated this can lead to irreversible damage of the eye and blindness. What are the most likely causes of this condition?

Answer 22

• Dietary
• Ethnicity (greater risk in Africans, East Asians and Inuit)
• Sex (higher in women)
• Genetics
• Steroid induced
• Restricted blood flow to eye (diabetic retinopathy, central retinal vein occlusion)
• Ocular trauma

Question 23

What are the consequences of increasing parasympathetic tone in the eye?

Answer 23

Increasing the parasympathetic tone in the eye increases the cillary muscle contraction which drains the fluid of Schlemm’s canal, decreasing intraocular pressure and helps stop the progression of glaucoma

Question 24

What receptor ligands are used clinically to treat glaucoma?

Answer 24

• Pilocarpine is muscarinic receptor agonist (M3) - activates mAChR
• Alpha1 agonist stimulates Phoslipase C
• Alpha agonist inhibits adenylyl cyclase
• Beta adrenergic antagonists decrease production of aqueous fluid by ciliary body and increase drainage to relieve pressure in the eye

Question 25

If the desired effect is to increase the stimulation of muscarinic receptors, what alternative non-receptor strategy can be adopted? Which agents are used clinically?

Answer 25

Carbonic anydrase inhibitors e.g. acetazolamide and dorzolamide decrease secretion of aqueous humour.

Question 26

What are the effects of increasing sympathetic tone in the eye?

Answer 26

Causes dilation of the pupil which increases aqueous humour production –> increased intraocular (in the eye) pressure

Question 27

Which agents, active at adrenoceptors, are used clinically in the treatment of Glaucoma?

Answer 27

• Beta-adrenergic receptor antagonists decrease aqueous humour production by ciliary body.
• Alpha2-Adrenergic agonists decrease aqueous humour production and increase drainage.
• Alpha-adrenergic agonists decrease aqueous humour production through ciliary body blood vessels.

Question 28

Explain the following statemnt: Noradrenaline is the major neurotransmitter at the neuroeffector junction of sympathetic post-ganglionic fibres.

Answer 28

In the sympathetic pathway:

Preganglionic neurones release ACh.

Postganglionic neurones release NA which binds to the alpha and beta adrenergic receptors on the effector organ.

Question 29

Are there any post-ganglionic synapses in the sympathetic nervous system at which NA is not the transmitter?

Answer 29

• Innervation of merocrine sweat gland; neurotransmitter is ACh, receptor is M3
• Renal cortex: neurotransmitter is Dopamine, receptor is D1

Question 30

The “biogenic amines” (dopamine, noradrenaline and adrenaline) are all synthesised from the amino acid tyrosine: outline the synthetic pathway

Answer 30

• Tyrosine is transported in sympathetic nerve axon.
• Converted to DOPA by tyrosine hydroxylase.
• DOPA is converted to dopamine by DOPA decarboxylase
• Dopamine is packaged into vesicles containing dopamine beta-hydroxylase and is converted into NA
• NA is converted into adrenaline by phenylethanolamine N-methyl transferase (PNMT)
• (Dopamine is produced in neuronal cell bodies in the brainstem)

Question 31

What determines whether a nerve terminal is “dopaminergic” (releases dopamine) or “(nor)adrenergic” (releases noradrenaline)?

Answer 31

• Depends on expression of an enzyme - how far tyrosine is broken down.
• Noradrenergic neurones contain dopamine beta-hydroxylase meaning they are able to convert doapmine to noradrenaline.
• Dopaminergic neurones lack dopamine beta-hydroxylase.
• A deficiency in this enzyme causes Ptosis (drooping of the upper eyelid), postural hypotension, inhibits digestive motility, hypoglycaemia.

Question 32

How is Noradrenaline packaged for release?

Answer 32

Dopamine transported into synaptic vesicles

Dopamine –> noradrenaline

The vesicles are ready for release (once AP arrives –> Ca2+ influx –> exocytosis of Noradrenaline)

Packaging is accomplished by VMAT in lipid bilayer. Chromaffin veiscles are formed (in the adrenal medulla).

Question 33

Briefly outline the sequence of events which occur following release of ACh by the pre-ganglionic fibre which lead to noradrenaline release.

Answer 33

The action potential opens voltage sensitive calcium channels causing an influx of calcium

Vesicles then travel towards the surface membrane

They fuse releasing noradrenaline and ATP

This process involves SNAPs and VAMPs

Noradrenaline is released into the nerve terminal and binds to adrenoreceptors on the post synaptic cell.

Adrenoceptors are GPCR and induce a secondary messenger response.

Question 34

What are Important Sites of Sympathetic Innervation?

Answer 34

Eyes

Heart

Airways of Lungs

Stomach and Intestine

Gall Bladder

Skin

Adipose Tissue (Lipolysis)

Question 35

Explain how increased NA has a Positive Inotropic and Chronotropic effect in the heart

Answer 35

NA binds to B1-adrenoceptors.

Noradrenergic post-ganglionic nerve fibres innervate SA Node, AV node and myocardium

Increases Heart Rate by increasing the production of cAMP which speeds up the pacemaker potential – makes the pacemaker potential slope steeper and therefore threshold is reached sooner. This reduces time between APs and increases HR.

Increases Force of Contraction by causing an increase in cAMP which phosphorylates Ca2+ channels so there is increased Ca2+ entry during AP.

This leads to increased uptake of Ca2+ in sarcoplasmic reticulum → increased sensitivity of contractile machinery to Ca2+ → increased force of contraction.

No opposite effect by parasympathetic system which only changes HR.

Question 36

What are the different Adrenoceptor Subtypes?

Answer 36

They are all GPCRs.

Question 37

How is NA removed from the Synaptic Cleft?

Answer 37

Reuptake occurs via a Norepinephrine Transporter (NET).

Reuptake is Sodium-Chloride Dependent; NET is a Cotransporter of Na+ and Cl-

This occurs at 1:1:1 ratio

The ion gradients of Na+ and Cl- make this reuptake energetically favourable.

The ion gradient which drives this mechanism is generated by the Na+/K+-ATPase (Sodium pump) which is ATP-dependent (active transport)

Question 38

What is the Fate of NA after its Removal?

Answer 38

2 different processes - 2 different transporters

Uptake-1 is the process in which NA is recycled through active transport reuptake into the presynaptic neurone.

Part of the NA is then metabolized to a biologically inactive metabolite (DOPGAL) by mitochondrial monoamine oxidase (MAO).

Part of the NA is sequestered in the storage vesicles for re-release.

Uptake-2 operates extracellularly when there is high [NA]. COMT methylates the meta hydroxyl group of NA to biologically inactive Normetanephine

Question 39

What are the major enzymes reponsible for inactivation of NA?

Answer 39

MOA: Monoamine Oxidase – widely distributed }

COMT: Catechol-O-Methyltransferase – also widely distributed, particular in the liver, kidneys and smooth muscles.

NA is oxidised and methylated to metabolites known as O-methylated derivatives.

Question 40

What are Useful Metabolites that can be used as an indirect index measurement of sympathetic activity?

Answer 40

Measurement of the O-methylated derivatives normetanephrine and metanephrine in the urine is a good index of the rate of secretion of NA and adrenaline and therefore an indirect index of sympathetic activity.

Other O-methylated derivates that are not excreted are largely oxidised.

Vanillylmandelic acid (VMA) is the most plentiful oxidised metabolite in the urine.

Question 41

What are the major physiological effects of NA?

Answer 41

Question 42

Does the hormone adrenaline contribute to, or exert the predominant effect, for any adrenoceptor activation?

Answer 42

• Alpha1; NA > Adrenaline
• Alpha2: Adrenaline greater than or equal to NA
• Beta1: NA = Adrenaline
• Beta2: Adrenaline MUCH > NA
• Beta3: NA > Adrenaline
• e.g. vasculature of coronary arteries and skeletal muscle: adrenaline has an effect on the smooth muscle within the wall of arterioles which have Alpha 1 and Beta 2 receptors. Adrenaline has a higher affinity for Beta2. Low dose - B2 dominates. High dose - Alpha1 activated too (more Alpha1 receptors present so dominates). Therefore vasoconstriction is the overall effect when [adrenaline] is high.

Question 43

Outline some therapeutic uses of different adrenoceptor agonists…

Answer 43

Question 44

Why are different agents required for these different therapeutic interventions?

Answer 44

Different subtypes –> Different effects

Therefore, different agents for different therapeutic interventions

Question 45

Give an example of the clinical use of an alpha and beta adrenoceptor antagonist, + their side effects

Answer 45

Question 46

Alpha-methyltyrosine , alpha-methyl DOPA and guanethidine have been used clinically to inhibit sympathetic neurotransmission.

Which enzyme is specifically inhibited by alpha-methyltyrosine? Why is this important with respect to noradrenaline synthesis?

Answer 46

• Competitive inhibitor of tyorsine hydroxylase - competes with tyrosine at the tyrosine binding site.
• Tyrosine hydroxylase hydroxlyses tyrosine (gained from diet) to L-DOPA which is a precursor for dopamine.
• Dopamine –> NA –> Adrenaline
• Alpha-methyl tyrosine is minimally metabolised in the body - absorbed well; bioavailability is high.

Question 47

What particular type of cancer is treated by administration of alpha-methyl tyrosine?

Answer 47

• Alpha-methyl tyrosine is used for pheochromocytoma (a neuroendocrine tumour of the adrenal gland medulla).
• This tumour increases release of NA and some adrenaline due to its sympathetic nervous system hyperactivity.
• Pheochromocytoma is familial and has a young adult-mid life onset.
• Alpha-methyl tyrosine clinical usage has declined due to its side effects including: increased sleepiness, increased anger,tension, withdrawl symptoms of insomnia, sedation, parkinson’s like effects (due to lack of dopamine), possible cysteinuria/crystalluria

Question 48

Instead of acting as an inhibitor, alpha-methyl DOPA acts as a “false substrate” for which biosynthetic enzyme?

What product of alpha-methyl DOPA metabolism accumulates in noradrenergic terminals?

If released what is the majro actions of this “false transmitter”?

Answer 48

Alpha-methyl DOPA is an agonist of a2 receptors. It is also an inhibitor of DOPA decarboxylase enzyme (L-DOPA –> Dopamine).

Alpha-methyl-DOPA competitively inhibits DOPA decarboxylase whcih results in reduced DOpaminergic and Adrenergic neurotransmission in the PNS.

It is also an alpha-adrenergic agonist (selective for alpha2 Adrenergic receptors).

Alpha-methyl DOPA is converted to alpha-methylnoradrenaline by dopamine beta-hydroxylase (DBh). Alpha-methylnoradrenaline is an agonist of presynpatic CNS alpha2-adrenergic receptors.

Activation of these receptors in the brainstem appears to inhibit sympathetic nervous system output and lower blood pressure, acts on the effects of the sympathetic system.

Question 49

Alpha-methyl DOPA has been adopted as one clinical strategy for the treatment of hypertension; explain the theory behind this therapeutic approach

Answer 49

• Alpha-methylnorepinephrine is an agonist of presynaptic CNS alpha2-adrenergic receptors.
• Activation of these receptors in the brainstem appears to inhibit sympathetic nervous system output and lower blood pressure.
• Alpha-methyl DOPA clinical usage has declined due to its side effects including decreased blood pressure, depression, anxiety, apathy, anhedonia (inability to experience pleasure), Parkinson’s like effects.

Question 50

Guanethidine is a noreadrenergic neurone blocking drug - what does this mean? Briefly outline the hypotheses put forward to explain how guanethidine inhibits noradrenaline release.

Answer 50

• Antihypertensive drug used in hypertensive emergies - inhibits NA
• Guanethidine is transported into the synapse in the same way as NA in the CNS (NA transporter - NET) so replaces NA and leads to its gradual decrease.
• When Guanethidine enters the nerve, it becomes concentrated in vesicles (it replaces NA).
• Its uptake is essential for its function.
• Lowers blood pressure.

Question 51

Why is Guanethidine no longer used clinically?

Answer 51

Unwanted side effects:

Hypotension

Sexual Dysfunction (delayed or retrograde ejaculation)

Diarrhoea

Question 52

Generally, the preferred tools for inhibiting noradrenergic transmission act at adrenoceptors - why do you think adrenoceptor agonists and antagonists have become the drugs of choice?

Answer 52

• Re-uptake of noradrenaline can only be inhibited whereas the adrenergic receptors can be both stimulated and inhibited.
• Functional selectivity
• The extent of the effect can therefore be more precisely controlled.