Autonomic pharmacology

Question 1

Synthesis of catecholamines

Answer 1

• Autonomic pharmacology = part of the peripheral nervous system, conveys all outputs from CNS to rest of body, except motor innervation of skeletal muscle and largely outside voluntary control, not protected by blood brain barrier
• Most Ach as neurotransmitter, noradrenaline released from post ganglionic neurones of sympathetic NS, but also uses ATP as co-transporter
• Catecholamine: variety of naturally occurring amines, function as neurotransmitter and hormones in body, characterised by catechol group (benzene ring + 2 OH groups) attached to amine group
• Synthesised from L-tyrosine: tyrosine → dihydroxyphenyalanine → dopamine → norepinephrine → epinephrine
• Enzymes involved: tyrosine hydroxylase (rate-limiting step), DOPA decarboxylase, dopamine-ß-hydroxylase, and PNMT
• Drugs affecting noradrenaline synthesis:
  1. A-methyl tyrosine = inhibits hydroxylase, useful in phaechromocytoma
  2. Carbidopa = inhibits DOPA-decarboxylase, works in periphery (not CNS) useful in Parkinson’s disease, reduces unwanted peripheral actions of administered L-dopa
  3. Methyldopa = utilised by sympathetic neurones, converted to α-methyl noradrenaline, displaces noradrenaline from vesicles, acts as ‘false transmitter’, useful in hypertension
  4. 6-OH dopamine = neurotoxin, has experimental uses only
• Catecholamines synthesised in brain, adrenal medulla, and by some sympathetic nerve fibres, particular catecholamine synthesised by nerve cell/neurone depends on which enzymes are present

Question 2

Packaging of noradrenaline and adrenaline

Answer 2

• Not free in cytoplasm, stored in subcellular membrane-limited particles (chromaffin granules)
• Sympathetic terminals: large dense-core vesicles = cell body, axon and varicosities, small dense-core vesicles = varicosities
• Epinephrine = produced and stored primarily in adrenal glands, norepinephrine = stored in small amounts in adrenal tissue, major storage and release at neurons of sympathetic NS (requires optimal electrolyte milieu) rate of synthesis varies with rate of utilisation of catecholamine
• Noradrenalin in sympathetic NS stored in heterogenous fashion, O-methylation can serve as mechanism for the rapid removal from extra neural pool
• Noradrenaline metabolism via deamination by monoamine oxidase and O-methylation by catechol O-methyltransferase
• Endogenous noradrenaline: localised in component in splenic nerves, uptake + binding by granulated vesicles help in temporary inactivation of catecholamine
• Methods of getting noradrenaline into vesicles:
  1. Import dopamine, then convert, using enzyme, to noradrenaline
  2. In noradrenaline recycling, it can move through VMAT into vesicles
• VMAT = secondary active transporter, needs energy from H+ pumped into vesicles (increase acidity), hydrogen gradient used to drive dopamine entry
• Reserpine: inhibits vesicular amine transporter, vesicular stores of catecholamine run down slowly as amines leak out (in past treated hypertension)
• Noradrenergic neuron blockers inhibits noradrenaline release, they enter nerve terminals through NET, inhibit action potential (via ion channels) or exocytic proteins
• Indirectly acting amines (amphetamine, ephedrine and tyramine), structurally related to noradrenaline, transported into nerve terminal, into vesicles, displaces noradrenaline (similar effect of dopamine and 5HT in CNS)

Question 3

Prejunctional regulation of exocytosis

Answer 3

• Exocytosis = arrival of action potential causes depolarisation of varicosity → opening of voltage-gated Ca2+ channels, CA2+ entry causes an increase in concentration of free calcium in varicosity → activates Ca2+ sensitive protein that initiate process of exocytosis (e.g fusion of vesicles with the nerve terminal membrane and release of contents)
• Variety of substances act on prejunctional receptors to regulate release and inhibition of noradrenaline (presynaptic modulation by endogenous mediators)
• Autoinhibition of noradrenaline release: noradrenaline can act locally on presynaptic receptors (α2- adrenoceptors, negatively coupled to adenylyl cyclase) to inhibit its own release and ATP release, clonidine = selective α2-adrenoceptor agonist, used to treat hypertension
• Regulation of release:
  1. Ach = inhibits release via muscarinic receptors, produces lateral inhibition from parasympathetic nerve, faciliatory nicotinic receptors also
  2. Adenosine = inhibits release via A1 receptors
  3. Opioids inhibits release via μ receptors
  4. Angiotensin II - facilitates release via AT1 receptors

Question 4

Uptake and degradation

Answer 4

• Catecholamines uptake: neuronal + non-neuronal, both = saturable active transport
• Neuronal: due to secondary active transporter NAT (noradrenaline transporter), main mechanism for terminating actions of noradrenaline
• High affinity for noradrenaline, low maximal rate of uptake, relatively selective for noradrenaline (noradrenaline > adrenaline > isoprenaline)
• Cotransports Na+, Cl- and catecholamine, inhibited by cocaine, tricyclic antidepressants (desipramine), and phenoxybenzamine
• NAT inhibitors enhance effects of sympathetic activity, NAT closely related to dopamine and serotonin transporters
• Desipramine: tricyclic antidepressant, major action = CNS, adverse effects = tachycardia and dysrhythmia
• Cocaine: CNS action → tachycardia + ↑ blood pressure (peripheral effects), used as local anaesthetic
• Non-neuronal: cardiac, smooth muscle and endothelium, low affinity noradrenaline, high maximal rate of uptake, not selective for noradrenaline (adrenaline > noradrenaline > isoprenaline), inhibited by normetanephrine, steroid hormones, and phenoxybenzamine
• Degradation by monoamine oxidase: bound to surface membrane of mitochondria, found in a many tissues, including nerve terminals
• Converts catecholamines to aldehydes → metabolised by aldehyde dehydrogenase (at periphery) or aldehyde reductase (CNS), inhibited by drugs used therapeutically for effects on CNS (most inhibitors block irreversibly, clinical use antidepressants)
• Effects: ↑ noradrenaline, dopamine and 5-HT in brain and peripheral tissues
• Adverse effects: postural hypotension, atropine-like effects, weight gain, restlessness, insomnia, cheese reaction
• By catechol-O-methyl transferase: found in tissues, including nerve terminals, is cytoplasmic, converts catecholamines to methoxy derivatives by transferring methyl group to one of catechol -OH groups
• Acts on catecholamines and products of other degenerative reactions
  metabolic degradation of catecholamine: VMA
• 3-methoxy,4-hyroxy mandelic acid = final metabolite of adrenaline + noradrenaline (excreted in urine)
• Urinary VMA levels used as diagnostic test for phaeochromocytoma, (rare tumour of chromaffin tissue, secretes high levels of catecholamines → elevated blood pressure)

Question 5

Structure and classification of adrenoceptors

Answer 5

• Physiological effects of activation of sympathetic NS: brought about by action of transmitter substances on specific cell membrane receptors (adrenoceptors), convey message from outside to inside of cell
• Sympathetic nerves release noradrenaline, adrenal medulla release adrenaline and noradrenaline
• Subtypes of receptors catecholamines bind: α-adrenoceptors and β-adrenoceptors
• α-adrenoceptors divided into α1 (noradrenaline ≥ adrenaline > isoprenaline) and α2 (adrenaline > noradrenaline > isoprenaline)
• β-adrenoceptors divisions: β1 (isoprenaline > noradrenaline > adrenaline), β2 (isoprenaline > adrenaline > noradrenaline), β3 (isoprenaline > noradrenaline = adrenaline)
• Structure of adrenoceptors: adrenergic receptors, part G-protein couple receptors family (metabotropic receptors), recruit intracellular, integral membrane proteins (G-protein -each has 3 subunits of α,β,γ), to produce cellular effects
• Agonist, e.g noradrenaline, binds to receptor → conformation change, protein unstable
• GDP phosphorylated to GTP making G protein more unstable, α subunit separates from β + γ subunits to interact with target proteins
• Single polypeptide chain of around 400-500 AA, extracellular N-terminus, and intracellular C-terminus with 7 transmembrane α-helices
• Binding site buried in cleft between α helices (in plane of membrane)
• α and β adrenoceptors recruit different intracellular G-proteins, α and β adrenoceptors utilise different intracellular messaging systems for cellular effects
• Site directed mutagenesis experiments show long third cytoplasmic loop = region of
  receptor coupled to G protein (C terminus may also be involved)
• Differences in AA sequence of long third cytoplasmic loop = basis of α and β adrenoceptors interaction with different G proteins (produce different cellular effects)

Question 6

What are some second messenger systems?

Answer 6

• α1 adrenoceptors: couple to phospholipase C-β, couple to G protein Gq, α-subunit of Gq activates phospholipase C-β (PLC)
• PLC acts on membrane of phospholipid PIP2, produces 2 products: inositol trisphosphate (IP3) and diacylglycerol (DAG)
• IP3 releases Ca2+ from ER and DAG activates protein kinase C → phosphorylates various cellular proteins
• α2 adrenoceptors: inhibition of adenylyl cyclase, coupled to G protein Gi, α-subunit of Gi inhibits adenylyl cyclase (AC)
• AC converts cellular ATP to cAMP, cAMP levels ↓ since cAMP activates protein kinase A, there is reduced activity of PKA → reduced phosphorylation of certain intracellular protein producing effect, e.g inhibition of transmitter release from autonomic nerve terminals
• β-adrenoceptors: stimulation of adenylyl cyclase, coupled with G protein Gs, α-subunit of Gs stimulates adenylyl cyclase
• AC converts cellular ATP to cyclic AMP, cAMP levels are increased, and since cAMP activates protein kinase A, there is increased activity of PKA
• Enhanced phosphorylation of certain intracellular protein and this produces effect, e.g relaxation of smooth muscle

Question 7

Uses of adrenoceptor agonists/ antagonists

Answer 7

Adrenoceptor agonists
- Adrenaline: mixed α and β receptor agonist, used for cardiac arrest (help restore cardiac rhythm), acute anaphylaxis (type I hypersensitivity), severe asthma (emergency treatment), prolongation of local anaesthetic action, poorly absorbed by GIT (not given orally - given intramuscularly or subcutaneously), removed by tissues through uptake 1 &2, metabolised by MAO + COMT, plasma half-life = order of minutes
- Dobutamine: selective β1 receptor agonist, used for cardiogenic shock (acute heart failure), intravenously, plasma half-life of around 2 minutes, side effects of arrhythmia
- Salbutamol: selective β2 receptor agonists, used for asthma or delay of premature labour, given as inhaled aerosol/powder, can be given either orally/intravenously, mainly excreted unchanged and a plasma half-life of around 4 hours
- Terbutaline: same as salbutamol but not given orally
- Salmeterol: selective β2 receptor agonists, used for asthma, given by aerosol, long lasting (half-life of around 8 hours)
- Mirabegron: prodrug for selective β3 receptor agonist, treatment of overactive bladder syndrome
- Phenylephrine: selective α1 receptor agonist, for nasal decongestion, given intranasally, metabolised by MAO with a short plasma half-life
- Clonidine: α2 receptor agonist, hypertension + migraine, given orally half-life of around 12 hours
- Dexmedetomidine: α2 receptor agonist, for intravenous sedation without respiratory depression, inhibition of Na+ and K+ channel is of greater importance
Adrenoceptor α antagonists
- Hypertension: α1 selective antagonists, prazosin = short acting, doxazosin = long acting, used for reducing blood pressure
- Benign prostatic hypertrophy: selective α1A receptor antagonist, involves tamsolusin
- Phaeochromocytoma: non-selective α antagonist, involves phenoxybenzamine and used prior to surgery
Adrenoceptor β antagonists
- cardiovascular uses: hypertension, angina pectoris, used following MI
- glaucoma uses to improve drainage of aqueous humour
- thyrotoxicosis used as pre-operatively as an adjunct
- anxiety stress to control tremor/ palpitations
- adverse effects include: bronchoconstriction, worsening of pre-existing cardiac failure, bradycardia, heart block, hypoglycaemia, physical fatigue, cold extremities

Question 8

Cholinergic nerve approach

Answer 8

1. Supply
   - Nerve cannot make enough choline (taken up from blood), comes from diet + liver
   - Uptake into nerve endings via high-affinity carrier (Na+-dependent process)
   - Hemicholinium = competitive inhibitor of choline carrier, causes activity-dependent block of cholinergic transmission, due to depletion of Ach stores
2. Synthesis
   - Choline + acetyl CoA → Ach + CoA, catalysed by choline acetyltransferase (ChAt)
   - ChAt occurs in nerve cytoplasm, inhibitors are not used clinically
   - Triethyl choline also a substrate, gives acetyl triethyl chlorine
3. Storage
   - Storage maintained by energy-dependent pump, inhibition of pump by vesamicol leads to depletion of stores
   - Uptake mechanism of storage pump not very specific: acetyl triethyl choline, formed from triethyl choline by ChAt, can be stored and released as a ‘false transmitter’
   - ‘False transmitter’ has weak effect on postsynaptic receptors, illustrates unusual type of drug action: feed a precursor that is not itself active; converted to false transmitter, stored and released from nerves, functional effects depends on how potent it is at postsynaptic receptors compared with natural transmitter
4. Release
   - Requires entry of Ca2+ into nerve ending, occurs by exocytosis, drugs that affect release of Ach: black widow spider venom α-latrotoxin (causes ↑ release and depletion of vesicles) and botulinum toxin (blocks release, clinically treats blepharospasm, salivary drooling, axillary hyperhidrosis, achalasia)
5. Inactivation
   - Diffusion not important, unless cholinesterase is inhibited, main mechanism is hydrolysis by tissue AchE: Ach → acetate and choline (irreversible reaction)
   - Sarin: AchE inhibitor blocks skeletal neuromuscular transmission, augments parasympathetic effects
6. Feedback
   - Cholinergic nerves have presynaptic (or prejunctional receptors)
   - Ach (muscarinic) inhibits release of Ach (enteric), Ach (nicotinic) ↑ release of Ach
   - ATP converted to adenosine → inhibits release at α1 receptors, morphine inhibits release at μ-receptors (constipation), and noradrenaline inhibits release at adrenoceptors by sympathetic cross-inhibition in tissues

Question 9

Nicotinic vs Muscarinic receptors

Answer 9

Nicotinic receptors
- Ligand gated ion channel, permeable to cations with 5 subunits (2 α-subunits)
- Initially characterised pharmacologically by response to various agonists, in autonomic ganglia, different order of potency of different agonists
- Skeletal muscle order = nicotine, carbachol, DMPP, methacholine muscarine
- Autonomic ganglia order = nicotine, DMPP, carbachol, methacholine muscarine
- Transiently stimulate ganglia + motor end-plate if given briefly at very high conc, but, receptors rapidly desensitise, agonists can inhibit ganglionic transmission if given at low conc for long time, ganglia no longer targeted clinically
- Hexamethonium = blocker of transmission within ganglia, drops sympathetic tone to cardiovascular system so drops blood pressure, works by blocking ion channel (non-competitive antagonist)
- Problems: poorly absorbed from gut (given by injection instead), blocks sympathetic ganglia, and parasympathetic ganglia, give unpredictable side-effects
- Tubocurarine blocks skeletal + autonomic ganglia receptors (non-specific antagonist)
- Galantamine acts on nicotinic receptors ↑ activity (allosteric potentiating ligand or ‘positive allosteric modulator’), AchE activity, peripheral side effect changes gut motility
Muscarinic receptors
- Parasympathetic NS, relative potency series for agonists when cholinesterase is inhibited: methacholine, muscarine, carbachol, nicotine
- Classed in M1 (Pirenzepine - antagonist), M2 (Gallamine - antagonist), M3 subclasses
- Clinical uses of antimuscarinic drugs: asthma, bradycardia, ↓ gut motility, during operations, AchE side-effects, dilate pupils, urinary incontinence, and motion sickness
- Muscarinic agonists: Ach, carbachol, muscarine, pilocarpine
- Effects of parasympathomimetic: cardiovascular (↓ heart rate, ↓ CO), smooth muscle (contracts, vascular dilates via endothelium), and exocrine glands (secrete sweat, lacrimation, salivation, and bronchial secretion)
- Signalling pathway: GQ produces phosphatidylinositol, glycerol and IP3 → Ca2+ release from intracellular stores