Pharmacology of Peripheral NT Flashcards
D-tubocurarine (major constituent of curare)
Competitive non-depolarising blocking agent of postsynaptic nAChR
Induces histamine release from mast cells so associated with transient hypotension.
Alpha-latrotoxin
PROMOTES ACh release:
Causes massively depolarisation/Ca2+-independent discharge of synaptic vesicles
Binds to 2 different types of presynaptic proteins that may mediate its action: neurexins and CIRL1 (adhesion type GPCR) –> formation of a Ca2+ permeable pore within the presynaptic nerve membrane which allows Ca2+ influx to trigger NT release (so debated if Ca2+ independent)
Beta-bungarotoxin
INHIBITS ACh RELEASE:
By binding to and blocking shaker-type potassium channels (so localises to cholinergic neurons), then Phospholipase A2 activity degrades membrane lipids in the active zone
Die as a result of respiratory failure
Alpha-methyltyrosine
Competitive inhibitor of tyrosine hydroxylase
Reduces amount of NA produced
In the past, used for preoperative treatment of phaeochromocytoma (adrenal tumour that secretes high levels of Adr and NA)
Atenolol (metoprolol)
Beta 1 adrenoceptor selective antagonist
Treatment of hypertension, angina, cardiac dysrhythmias
Atracurium
Non depolarising blocker at NMJ
Unstable at physiological pH and so spontaneously breaks down in the plasma.
Induces histamine release from mast cells so associated with transient hypotension.
Atropine + benzilylcholine
Reversible muscarinic antagonist (inhibits PNS so causes a large increase in HR) - ‘antimuscarinic agent’
Naturally occurring alkaloid
Can be used to produce dilation of pupils (mydriasis)
Very long duration of action
Used to treat effects of AChE inhibitor poisioning
Can cross BBB - causes marked restlessness and increase in temperature accentuated by an inhibition of sweating. Can be used to treat central and peripheral effects of irreversible anticholinesterase poisoning
Botulinum toxin A-G (BoNT)
Cleaves various SNARE proteins
BDFG synaptobrevin
AE snap-25
Preferentially on cholinergic neurons
C-terminus of heavy chain binds to ganglioside receptor (i.e. GT1b) and complex is endocytosed
Antitoxin only works within 30 mins
N-terminus translocates the light chain from the endosomal lumen into the cell cytoplasm by making a channel in the endosomal membrane
Light chain is a zinc dependent protease and once in cytoplasm cleaves the target snare
Injected muscle can no longer contract - wrinkles relax and soften. BoTX-A = BOTOX
Poisoning: somatic muscle weakness, autonomic signs associated with loss of cholinergic activity (i.e. PNS loss constipation/blurred vision/dry skin/urinary retention) but HR may be slowed rather than increased due to action on NA nerves
Caffeine (a methylxanthine)
Competitive antagonist for A1 receptor (type of P1 - for adenosine)
Carbidopa (+ benserazide)
Inhibitor of DOPA Decarboxylase (DDC) in the periphery (can’t cross BBB)
Reduces side effects of L-DOPA treatment (along with encaptone)
Cevimeline
Muscarinic M3 receptor agonist
Used to increase salivation and lacrimation in Sjögren’s syndrome (autoimmune disease of glands that secrete fluid)
Alphamethyl-Noradrenaline
False transmitter
More selective than NA on alpha2 but less on alpha1 (alpha2 selective agonist)
Consequently less vasoconstriction in the blood vessels following sympathetic nerve stim (decreased alpha1 mediated smooth muscle contraction and increased negative feedback on NA release)
antihypertensive - reduce BP. Used during pregnancy.
Cocaine
Inhibitor of uptake 1 (NET/SERT/DAT)
Darifenacin (+solifenaxin, oxybutynin, tolterodine)
Selective muscarinic M3 antagonist so used in treatment of urinary incontinence (M3 mediates detrusor muscle of bladder)
Decamethonium
Depolarising antagonist of nicotinic receptors at NMJs
Tyramine
Indirectly sympathomimetic monoamine
Poor agonist at adrenoceptors but excellent substrates for NET/uptake 1. Then transported by VMAT into vesicles, displacing NA, which is subsequently expelled into synaptic space by reverse transport via NET
NET inhibitors like imipramine interfere
Tyramine: normally hydrolysed by MAOs. If a large amount of tyramine-rich food is ingested, the release of NA can be sufficient to cause widespread vasoconstriction and a fatal rise in blood pressure: cheese effect. People taking MAO inhibitors should avoid tyramine-rich foods.
Dipyridamole
Inhibits nucleoside transporter (NsT) so indirectly increases external concentration of adenosine
A vasodilator and antiplatelet agent
Disulfiram
Inhibits dopamine beta hydroxylase (DBH)/aldehyde dehydrogenase
May act by chelating the Cu2+ ion which is an essential cofactor of the enzyme, or may attack the sulfur-handling system for the methyl donor S-adenosyl methionine.
Can be used in treatment of alcohol abuse as it inhibits aldehyde dehydrogenase, which causes acetaldehyde to build up when alcohol is imbibed
Dobutamine (+xanolerol)
Beta1-adrenoceptor selective agonist. Used in cases of cardiogenic shock to increase CO but can cause cardiac dysrhythmias
Donepezil
Short acting anticholinesterase, also reaches the CNS
Spans the aromatic gorge to interact with both PAS and active site. Has inhibitory effect on protein aggregation (beta-amyloid protein) and also inhibits AChE.
Selective for AChE>BuChE so fewer side effects
Doxazosin
Alpha1- adrenoceptor antagonist
Dyflos
Long acting irreversible anticholinesterase
PO3 + labile F - releases labile group then serine hydroxyl group is phosphorylated. V stable. Undergoes aging - makes even more stable
Can be used in glaucoma
Ecothiophate
Irreversible long acting anticholinesterase
PO3 + labile organic group. Labile group splits off, serine hydrolase is phosphorylated. Undergoes aging.
Can be used in glaucoma
Edrophonium
Reversible, non-covalent, short acting anticholinesterase
Quaternary ammonium
Action limited to periphery
Binds to CAS through cation-pi with W84
2-10 min
used for tensilon test of myasthenia gravis (+ individual feels temporary improvement in the facial weakness and ptosis within 5-10mins of edrophonium injection)
cannot cross BBB as charged
Ephedrine
Indirectly acting sympathomimetic amine, promotes catecholamine release
Used as a nasal decongestant because of NA-mediated vasoconstriction it produces in the vasculature of the nose. May have some direct action on beta2adrenoceptors int he bronchi
Poor agonist at adrenoceptors but excellent substrates for NET/uptake 1. Then transported by VMAT into vesicles, displacing NA, which is subsequently expelled into synaptic space by reverse transport via NET
Imipramine and other NET inhibitors interfere with effect
Encaptone
COMT inhibitor
Used in Parkinson’s disease with L-DOPA and carbidopa: also reduces metabolism of L-DOPA in the periphery
Formoterol
Beta2-adrenoceptor selective agonist (long-acting)
Guanethidine (+bretylium +guanadrel)
Directly blocks NA release by a nerve impulse
Agents can transiently stimulate release of NA because of capacity to displace amine from stores
Selectively accumulate into neurons by Uptake 1/NET (compete with NA so can potentiate exogenously applied NA)
Stored into vesicles, replace NA, can transiently serve as indirectly-acting sympathomimetic agents, but after block the release of NA evoked by APs
Guanethidine not an agonist at postsynaptic adrenoceptors so doesn’t elicit a response
In past was used to treat uncontrolled hypertension
Hemicholinium
Inhibits choline transporter (ChT) which normally cotransports choline with Na+ from the extracellular fluid into the cytoplasm of the cholinergic neuron
Supply of choline is the rate limiting step of ACh synthesis
Hexamethonium
Use-dependent blocker of ganglionic nicotinic receptor
decamethonium more specific
Imipramine (+amitryptyline)
Tricyclic antidepressant, inhibits the norepinephrine transporter (NET). Interferes with indirectly sympathomimetic amine action
Indacaterol
Beta2 adrenoceptor agonist (ultra long acting)
Used in treating COPD
Ipratropium, Tiotropium(+homatropine +methsoopolamine)
Muscarinic antagonist - semi-synthetic derivative
Produces bronchodilation
Used to treat COPD and asthma
L-DOPA
DOPA decarboxylase (DDC) substrate. Can cross BBB so used to boost DA synthesis in the Parkinsonian brain Used in conjunction with carbidopa and entacaptone to reduce peripheral decarboxylation and so reduce side effects
Labetalol/Carvedilol
Mixed alpha1/beta adrenoceptor antagonist
Alpha1 blockade –> vasodilation
Beta1 blockade –> reflex sympathetic increase in HR
Together –> decrease BP
Can be used in treating hypertension associated with pregnancy
Malathion
Irreversible long acting anticholinesterase
Used as an insecticide to kill lice
Methotrexate
Indirectly increases the extracellular concentration of adenosine (unclear mechanisms)
An antifolate type anti cancer and immunosuppressive drug
Alpha-bungarotoxin
Irreversibly antagonises adult Nm nAChR (alpha1)2beta1deltaepsilon + brain (alpha7)5. Ganglionic nAChR insensitive
Benzilylcholine mustard
Irreversible non selective muscarinic antagonist
Nicotine (+lobeline)
Stimulates then depolarising blocking agent of nAChR ganglionic
Tertiary amine
Butoxamine
Selective beta2 antagonist
Methyldopa
Results in alpha-methylnoradrenaline production. False transmitter. PNS= antihypertensive. CNS= alpha2 agonist
Action similar to clonidine. Hypotensive drug during pregnancy
Mirabegron
Beta3 adrenoceptor selective agonist
Relaxes detrusor smooth muscle and increases bladder capacity: used for patients with an overactive bladder.
Potential for treatment of obesity as promotes lipolysis?
Moclobemide
Selective MAO-A inhibitor (preferentially degrades serotonin, NA, DA)
Nebivolol
Beta1 adrenoceptor antagonist
Secondary action makes it chosen antihypertensive: one of its metabolites is a beta3 adrenoceptor agonist, which mediates increased NO production and thus vasodilation
Neostigmine
Medium acting anticholinesterase, also activates nAChR directly at NMJ
Basic group interacts with CAS and transfers carbamyl (i.e. instead of acetyl) group to Ser203. Carbamylated AChE is more stable than acetylated AChE and takes minutes to hydrolyze so medium acting
Charged so only acts peripherally
Used IV to reduce neuromuscular blockade after surgery (and give orally to limit parasympomimetic effects) and orally to treat myasthenia gravis
Pancuronium
Non depolarising blocker at NMJ. Inhibits cardiac muscarinic receptor so induces mild tachycardia
Metabolised by liver or excreted unchanged in the urine
Phenoxybenzamine
Irreversible alpha adrenoceptor antagonist and NET/ENT inhibitor.
Blocks ENT at 10 times lower conc than needed to block uptake 1 NET.
Antihypertensive drug (obsolete) as they reduce BP so much that it triggers reflex tachycardia. Covalently binds to the receptor and causes long lasting inhibition. So retains clinical use in preparing patients with phaeochromocytoma for surgery (since surgical manipulation tens to cause a massive release of catecholamine into the circulation)
Phentolamine
Non selective alpha adrenoceptor antagonist Antihypertensive drug (obsolete) as they reduce BP so much that it triggers reflex tachycardia
Phenylephrine and oxymetazoline
Alpha1 adrenoceptor selective agonist
Nasal decongestant
Phenylephrine also used to treat acute hypotension e.g. from septic shock
Pilocarpine
Non selective muscarinic agonist
Naturally occurring cholinomimetic alkaloid: tertiary amine
Stable to hydrolysis by AChE
Used to treat glaucoma + emergency lowering of intraocular pressure of both open-angle and angle-closure glaucoma. It contracts the ciliary muscle –> traction on trabecular network around Schlemm canal –> immediate drop in intraocular pressure due to increased drainage of aqueous humour.
Action within a few minutes, lasts 4-8h, can be repeated
Also rapid miosis, accommodation, sweat, tears, saliva (low selectivity)
Pirenzepine
M1 selective antagonist
Decreases gastric acid secretion so can treat ulcer (but now H2 receptor agonists and PPIs better)
Synthetic
Pralidoxime
AChE reactivator,
Used as an antidote for organophosphate poisoning
Cationic group interacts with the anionic site of AChE, which brings the oxime group into close proximity with the phosphorylated serine. Oxime = v strong nucleophile, and takes the phosphate, which frees the serine hydroxyl group. Only works before aging. Can’t reach CNS
Prazosin (+terazosin +doxazosin)
Alpha1 adrenoceptor selective antagonist
Decrease peripheral vascular resistance and lower BP by causing relaxation of both arterial and venous smooth muscle. Thus useful to treat hypertension without same reflex tachycardia as non selective alpha adrenoceptor antagonists
Terazosin and doxazosin have longer half lives than prazosin
Propranolol
Non selective beta adrenoceptor antagonist
Treatment of hypertension:
1. decreases CO 2. Inhibits renin release 3. Decrease in TPR with long term use 4. Inhibits SNS so causes mild decrease in HR
Reserpine
Inhibits vesicular monoamine transporter (VMAT) by binding very tightly to the amine binding site. Leads not only to a block of uptake but also to a long-lasting depletion of stored NA/5-HT since the vesicles allow leakage of the stored amine into cytoplasm where it is metabolised by MAO. Acts in periphery and brain.
Used as an antihypertensive, but discontinued as causes profound psychological depression (perhaps due to 5-HT depletion).
Rivastigmine
Medium acting reversible anticholinesterase, can reach the CNS as uncharged and lipophilic
Useful in mild to moderate dementia assocated with Alzheimer’s disease and Parkinson’s
Salbutamol (+terbutaline +cleributerol)
Beta2 adrenoceptor selextive agonist (short acting)
Used mainly for bronchodilator action in ashthma. Taken by inhalation as needed.
Salmetarol (+formoterol)
Beta2 adrenoceptor selective agonist (long lasting)
Used prophylactically in chronic asthma.
Solifenacin
M3 antagonist
Suramin
Agonist for all P2X and some P2Y receptors (ATP antagonist)
Suxamethonium (succinylcholine)
Depolarising blocker at the NMJ. Short duration of action and thus much faster recovery compared to other NM blockers. Short duration as hydrolysed by plasma cholinesterase (BuChE)
Tamsulosin (+silodosin)
Alpha1A-adrenoceptor selective antagonist
Allow better bladder emptying (alpha1 adrenoceptors mediate contraction of smooth muscle of the bladder, this inhibits) and thus reduce urinary retention associated iwth benign prostatic hypertrophy.
Alpha1B mediates vasoconstriction: these thus produce much less postural hypotension
Tetanus neurotoxin (TeNT)
Cleaves synaptobrevin in specific inhibitory interneurons in the CNS
Enters body through puncture wounds in skin. Spreads through tissue spaces into the lymphatic and vascular systems
Enters nervous system at NMJ: C-terminal heavy chain binds to some ganglioside that are enriched on the peripheral terminals of motor neurons. This enables endocytosis.
Moves towards cell body in endoscope by retrograde axonal transport. Discharged into intersynaptic space. Binds to presynaptic membrane of inhibitory interneurons, is endocytosed. Acidic nature of synaptic vesicles causes insertion of the N-terminal light chain into the cytoplasm where it cleaves synaptobrevin. Inhibitory interneurons thus unable to release GABA/glycine thus disinhibition of motor neurone which is then more excitable which leads to tetanic contractions of skeletal muscle.
Tranylcypromine (+isocarboxazid +phenelzine)
Non specific MAO inhibitor
Triethylcholine
Competitive substrate for choline acetyltransferase (CAT)
Converted to acetyltriethylcholine which is then released in place of ACh. It is far less potent at both n+mAChR, so is a ‘false transmitter’.
Tropicamide
Reversible muscarinic antagonist (short acting)
Synthetic
Varenicline
Agonist for nicotinic receptors in CNS - full agonist for (alpha7)5, partial agonist for (alpha4)2(beta2)3 (this partial effect, unlike complete nicotine withdrawal, allows it to stimulate some dopamine release in the mesolimbic dopaminergic pathway that helps in relieving some withdrawal symptoms, but some serious mental/mood problems)
Vesamicol
Inhibits vesicular ACh transporter (VAChT). Non-competitive and reversible blocker.
Xylazine
Selective alpha2-adrenoceptor agonist
Vet use
Used as sedative, often in combination with ketamine
No imidazoline receptor effect
Xylometazoline and methoxamine
Alpha adrenoceptor-selective agonist
Topically given to relieve nasal congestion by causing vasoconstriction in nasal mucosa
Yohimbine (+idazoxan)
Alpha2 adrenoceptor selective antagonist
Amphetamine
Release stored NT (ie displaces NA from storage vesicles)
Trimetaphan
Competitive antagonist of ganglionic nAChR
Epibatidine
Highly potent nicotinic agonist selective for ganglionic and CNS receptors
Many autonomic side effects so not used clinically
TMA, DMPP
NAChR agonist on autonomic ganglion, no block but three times more potent than nicotine and slightly more ganglion-sensitive
Selegyline
Selective MAO-B inhibitor
Ipratropium
Reversible non-specific muscarinic cholinergic antagonist
Scopolamine
Muscarinic antagonist, and block exceptional sympathetic neurons that are cholinergic to salivary and sweat glands
Naturally occurring alkaloid
Cyclopentolate
Muscarinic antagonist, short-acting
Synthetic
Tripitramine
M2 selective muscarinic antagonist
Physostigmine
Reversible covalent anticholinesterase inhibitor. Intermediate acting.
Charged so only acts peripherally
Basic group interacts with CAS and transfers carbamyl (i.e. instead of acetyl) group to Ser203. Carbamylated AChE is more stable than acetylated AChE and takes minutes to hydrolyze so medium acting
Physostigmine can be used to treat glaucoma by topical application to the eye (same mechanism as pilocarpine?)
Paramion
Irreversible anticholinesterase inhibitor
Tacrine
Anti-AChE
Uncharged, so can cross BBB so useful in treating Alzheimer’s disease
(NB loss of cholinergic neurons in basal forebrain and resultant decrease in cholinergic NT thought to underlie, at least in part, the memory loss/intellectual degeneration/personality changes of AD)
Recently withdrawn due to severe hepatotoxicity
Normetanephrine
Uptake 2 inhibitor (ENT)
Selegyline
Selective MAO-B inhibitor
Clorgyline
Selective MAO-A inhibitor
Mivacurium
Non-depolarising blocking agent at NMJ. Hydrolysed by BuChE
Gallamine
Gallamine is 1st synthetic successor to curare
nAChR antagonist at NMJ
- at higher concentrations is a M2 selective antagonist so causes changes in HR. Now replaced by compounds with fewer side effects
Carbachol/Carbamylcholine (+methacholine)
Directly-acting muscarinic agonist/cholinomimetic agent
Synthetic esters of choline
For methacholine - presence of beta methyl group improves selectivity for mAChR and reduces hydrolysis by cholinesterases
Bethanechol
Directly-acting muscarinic agonist/cholinomimetic agent
Chemically a hybrid of methacholine and carbachol
Lacks nicotinic actions but non-selectively activates mAChR subtypes
Stimulates detrusor of bladder via M3 receptor, whereas trigone and sphincter muscles are relaxed. –> urination –> bethanechol increases bladder function
Procaine
Local anaesthetic
Hydrolysed by BuChE > AChE
Clonidine
Selective alpha2 adrenoceptor agonist
Antihypertensive - partly via inhibition of NA release, partly via CNS alpha2 stimulation reducing SNS outflow (presynaptic modulation): Clonidine binds to imidazoline receptors
Adjunct in treating drug addiction: helps ameliorate some adverse sympathetic actions during drug withdrawal in addicts
Dextroamfetamine (Ritalin, MDMA)
Indirectly acting sympathomimetic amine.
Poor agonist at adrenoceptors but excellent substrates for NET/uptake 1.
Has an alpha-methyl group that makes it a poor substrate for MAO, so acts as a weak inhibitor of MAO. Is a weak base so once taken up by VMAT reduces transvesicular pH gradient and thus vesicular packaging of amines. Some displaced NA leaves nerve ending to stimulate the receptors. Used in narcolepsy and ADHD and abused recreationally.
Ritalin (methylphenidate) and MDMA have similar effects though MDMA also stimulates 5HT2 receptors (so is hallucinogenic).
Corticosteroids: cortisone and hydrocortisone
Uptake 2 inhibitor (ENT)
Adrenaline
Given in acute cardiac failure, acute severe asthma, or anaphylactic shock (where it coutneracts the systemic vasodilation and reduction in tissue perfusion that is caused by massive histamine release)
Acts on bronchial smooth muscle to relieve bronchospasm
Given with local anaesthetics to produce vasoconstriciton at the site of injection, prolongs their action
Isoprenaline
Beta-selective adrenoceptor agonist. Beta2 = bronchodilator so used to be used in asthma treatment but stopped due to beta1= cardiac effects (problem)
Nadolol and Timolol
Similar but more potent than propranolol:
i.e.
Non selective beta adrenoceptor antagonist
Treatment of hypertension:
1. decreases CO 2. Inhibits renin release 3. Decrease in TPR with long term use 4. Inhibits SNS so causes mild decrease in HR
Timolol reduces production of aqueous humor in the eye. Used topically in treatment of chronic open-angle glaucoma.
Adenosine
Given as a bolus injection to terminate supraventricular tachycaria (slows rate and force of contraction of the heart via A1 receptors)
Nitrodilators
Donate NO in vivo rapidly –> vasodilation –> antianginal therapy
Nicotinic acid
• Inhibits liver triglyceride production and VLDL secretion when used in very large doses. Increases levels of tissue plasminogen activator.
Define gap junction
Small protein tubular structure that allow free movements of ions from the interior of one cell to the interior of the next. Electrical synapses
When are electrical synapses needed in the CNS? Give examples
Synchronous firing
Thalamus, also circadian rhythm in SCN nucleus
How wide is the synaptic cleft?
20-50nm
List the effects of sympathetic stimulation
Adrenal hormone release
HR up
BP up
Bronchioles dilate
Intestinal motility and secretion inhibited
Glucose metabolism increases
Pupils dilate
Hairs become erect due to piloerector muscles
Cutaneous and splanchnic blood vessels constrict
Blood vessels in skeletal muscle dilate
Where do the two neurons in series involved in efferent autonomic innervation originate from and end? What type of fibres are they? Which neurotransmitters are involved??
1: spinal cord to autonomic ganglion. Myelinated B fibres. ACh
2. autonomic ganglion to effector organ. Unmyelinated C fibres. ACh/NA
Give an exception to the two-neuron in series model
Adrenal medulla - directly innervated by a nerve from the spinal cord.
Where do parasympathetic nerves originate from? Where do they supply?
Craniosacral
Cranial: III (ciliary ganglion), VII, IX, X. Head and neck, vagus does thoracic cavity and first 2/3 colon
Sacral S2-4: Pelvic splanchnic nerves. Supplies lower GI tract, bladder, genitalia
Where are parasympathetic ganglia located?
Close to target organ or within wall of target organ
Where do sympathetic nerves originate from?
Thoracolumbar
Cell bodies in lateral horns
T1-L2/L3
What are the neurotransmitters involved in splanchnic nerve transmission?
ACh
NANC: VIP, substance P, CCK, NO
Where are the sympathetic ganglia located?
Paravertebral on each side of the vertebral column. Also to some unpaired prevertebral ganglia (coeliac, superior and inferior mesenteric) on ventral surface of aorta, aorticorenal and pelvic ganglia
What are the neurotransmitters released by the second neuron in sympathetic transmission?
NA and ATP
ATP faster effect, NA more long-lasting effect
Ones supplying sweat glands ACh
Describe the postsynaptic arrangement involved in peripheral sympathetic neurotransmission
Bulbous varicosities distributed along axons within their target organ
At each varicosity, autonomic axons form an en passant synapse with their end organ target
Increases number of targets that a single axonal branch can influence
Describe the postsynaptic arrangement involved in peripheral parasympathetic neurotransmission
Probably varicosities like SNS but less well characterised
Give two situations of dual SNS and PNS innervation, one where they have the same effect, and the other where they have opposing effects
Same: Salivary glands. Both promote salivary secretion
Different: HR and force of myocardial contraction both enhanced by SNS and reduced by PNS
When an organ is innervated by both the SNS and PNS, which provides the dominant tone? Give evidence
PNS for humans (other animals not always)
Giving atropine to patient markedly increases HR (abolishes PNS input) but giving propranolol only slightly decreases HR (abolishes SNS input)
How can the SNS impact tissues that are only PNS innervated?
Circulating catecholamines can bind to receptors on those tissues, i.e. bronchial smooth muscle relaxes with circulating adrenaline
What are the proportions of Adr and NA released by the adrenal medulla?
80% A 20% NA
What are the primary SNS and PNS receptors acting on the heart and what are the effects?
Beta1 SAN increase HR, atrial muscle increase force, AVN increase automaticity, ventricular muscle increase automaticity and force
M2 SAN decrease HR, atrial muscle decrease force, AVN decrease conduction velocity/AVN block, no effect on ventricular muscle
What are the primary SNS and PNS receptors acting on blood vessels and what are the effects?
Beta2 dilation: muscle, veins
Alpha constriction: coronary, viscera, skin, brain, erectile tissue, salivary glands, veins
Para: only effects via M3 on erectile tissue and salivary glands, not on other blood vessels
What are the primary SNS and PNS receptors acting on the bronchi and what is the effect?
Beta2 smooth muscle circulating adrenaline dilated
Constriction of smooth muscle and secretion of glands via M3
What are the primary SNS and PNS receptors acting on the GI tract and what is the effect?
Alpha1/2/beta2: smooth muscle decreased motility, sphincters constrict
M3:increase motility, dilation, secretion
M1: increased gastric acid secretion
What are the primary SNS and PNS receptors acting on the bladder and what is the effect?
Beta2: relaxation
Alpha1: sphincter contraction
M3: contraction and sphincter relaxation
What are the primary SNS and PNS receptors acting on the uterus and what is the effect?
Pregnant: alpha contraction
Non pregnant: beta2 relaxation
What are the primary SNS and PNS receptors acting on the male sex organs and what is the effect?
Alpha ejaculation, M3 erection
What are the primary SNS and PNS receptors acting on the eye and what is the effect?
Alpha pupil dilation, beta ciliary muscle relaxation
M3 pupil constriction, ciliary muscle contraction
What are the primary SNS and PNS receptors acting on the skin?
Sweat gland secretion via M3
Piloerection via alpha
Salivary gland secretion alpha, beta, M3
Lacrimal gland secretion M3
What are the primary SNS and PNS receptors acting on the kidney?
Beta1 renin secretion
What are the primary SNS and PNS receptors acting on the liver?
alpha beta2 glycogenolysis, gluconeogenesis
What are the primary SNS and PNS receptors acting on the adipose tissue?
Beta3 lipolysis, thermogenesis
What are the primary SNS and PNS receptors acting on the pancreatic islets?
alpha2 insulin sec down
How many neurons in the ENS?
100 million
How is the ENS subdivided?
Myenteric/Auerbach’s plexus between longitudinal and circular muscle layers of the gut. Control GI motility.
Submucosal/Meissner’s plexus in gut submucosa. Regulate body fluid homeostasis
Define conduction
Passage of an impulse along an axon or muscle fibre
Define transmission
Passage of an impulse across a synaptic or neuroeffector junction
Which drugs influence conduction?
Local anaesthetics
Describe the process of synthesising small molecule NTs
In presynaptic terminals
Enzymes to do this synthesis produced in the soma and transported to the nerve terminal cytoplasm by slow axonal transport
Normally take up precursors for synthesis by transporters found in the plasma membrane of the terminal
Loaded into vesicles
Final steps of synthesis occur inside the vesicles
Describe slow axonal transport
0.5-5mm/day
Anterograde transport
From soma to nerve terminal
Used to take enzymes for small molecule NTs to the periphery
What are small molecule NTs stored in?
Small clear-cored vesicles
40-60nm diameter
Centre clear in EM
Describe the synthesis of peptide neurotransmitters
Precursor peptides and enzymes for synthesis produced in the cell body of a neuron
Packaged into vesicles
Shuttled to nerve terminal via fast axonal transport
What are peptide NTs stored in?
Large dense-core vesicles
90-250nm
Electron-dense in electron micrographs
Describe fast axonal transport
400mm/day
Used to transport large dense-core vesicles containing neuropeptides from soma to peripheral nerve terminal. Move the vesicles down microtubule tracts
What is the microtubule-dependent motor protein used in fast axonal transport?
Kinesin
What is the microtubule-dependent motor protein used in retrograde axonal transport?
Dynein
What is retrograde transport used for?
Target-derived growth factors such as nerve growth factor can reach the nucleus of a neuron where it can influence survival
Viral proteins and neurotoxins hijack this mechanism
Also carries things to further degrade or salvage e.g. used vesicles, degradable products
What is the purpose of storing NTs in synaptic vesicles?
Enable exocytosis, protect from protease and hydrolases, stop passive leaking from pre-synaptic membrane
Describe what Katz showed
When an intracellular microelectrode is used to record the membrane potential of a muscle cell, an AP in the presynaptic motor neuron can be seen to elicit a transient depolarisation of the postsynaptic muscle fibre - an EPP
- Spontaneous changes in muscle cell membrane potential occurs even in the absence of stimulation of the presynaptic motor neuron (MEPP). Both these and EPPs are sensitive to blockade by curare.
- If stim the nerve to the junction under conditions of low extracellular Ca2+ (low transmitter release) the EPPS that occur were multiples of the MEPP. So ACh released in a quantal manner
How big is an EPP and how big is a MEPP?
EPP 40-50mV
MEPP 0.5mV
What is a MEPP?
Miniature end plate potential
Synchronous release of 5000 molecules ACh
Maintenance of physiological responsiveness of skeletal muscle (lacks inherent tone)
What is an exception to the rule that NT release is quantal?
Release at the synapse between photoreceptors and bipolar neurons in the retina
What is the relation between Ca2+ conc and NT release?
NT release proportional to Ca2+ conc to the 3rd or 4th power
What is the name for vesicles:
- Already docked to the release sites
- Associated to the cytoskeleton nearby
- Releasable pool (within 200microseconds)
2. Reserve pool
How are vesicles recycled?
- Uptake NT into synaptic vesicles by ATP-driven transport. Proton pump acidifies vesicle interior to give an electrochemical gradient across the synaptic vesicle membrane. This provides the energy for the uptake of NT through specific transport proteins that are specialised for the various NTs.
- Full synaptic vesicles move to active zone of presynaptic plasma membrane
- Dock
- ATP-dependent prefusion reaction that primes them for Ca2+ dep release
- Ca2+ triggers completion of fusion
- Synaptic vesicles rapidly retrieved by clathrin-mediated endocytosis/ ‘kiss and run’
- Coated vesicles shed the coat and recycle to the interior of the synaptic terminal
- Empty vesicles either refill immediately or pass through an endosomal intermediary sorting station. Local recycling of synaptic vesicles enables the terminals to sustain repeated rounds to transmitter release independently of the distal cell body.
What are congenital myasthenic syndromes?
Genetic disorders that cause muscle weakness by affecting neuromuscular transmission:
- Affect AChE
- Autoimmune attack of AChR
- Defects in ACh due to altered synaptic vesicle traffic (impairment in endocytosis or reduced supply of vesicles from soma)–> Inadequate number of synaptic vesicles available for release during sustained presynaptic activity
What is familial infantile myasthenia?
Synaptic vesicles that are smaller in diameter but normal in number. Results in problems initiating voluntary movement
What is Lambert-Eaton myasthenic syndrome?
Occasional complication in patients with certain kinds of cancers.
Evidence: intracellular recordings - number of quanta in EPPs greatly reduced, while amplitude of spontaneous MEPPs is normal. So impairs evoked NT release, but doesn’t affect the size of individual quanta.
Autoimmune destruction of CaV in pre-synaptic membrane. Voltage insufficient for vesicle release, leads to problems initiating voluntary movement.
What are SNAREs?
V associated with vesicle and t associated with target.
Segment in cytosolic domain called a SNARE motif - 60-70 aa and contains heptad repeats that can form coiled-coil structures.
Describe the interactions of the SNAREs
Synaptobrevin is a V SNARE anchored in vesicle membrane by hydrophobic carboxy terminus.
Central 70 aa residue region forms an alpha helix which complexes with SNAP25 and syntaxin (t snares).
This structure is known as the core trans-SNARE complex and consists of 4 alpha helices (one synaptobrevin, one syntaxin, 2 SNAP-25).
Helices lie parallel to each other and form a leucine zipper that brings the vesicle and plasma membranes close together.
What is the Ca2+ sensor for exocytosis?
Synaptotagmin isoforms I, II and IX. Anchored to vesicles via N-terminus. C termini contain C2A and C2B which provide the binding sites for Ca2+.
In the presence of Ca2+, C2 domains bind synaxin and SNAP-25 and to membranes containing acidic phospholipids.
The C2B domain is specifically able to bind to membranes enriched in PIP2.
Which terminus anchors synaptobrevin and synaptotagmin in the vesicle?
Synaptobrevin = C Synaptotagmin = N
What are synapsins?
Proteins on the surface of vesicles that link vesicles to cytoskeleton, regulated by phosphorylation. In non-P state, synapsins bind to vesicles.
When phos by PKA/CaM Kinase II vesicles dissociate.
Means vesicles move to active zone.
Critical for maintaining the reserve pool.
Where are C2 domains found?
Synaptotagmin
PKCs
Low affinity binding site for Ca2+
How do synaptotagmin’s C2’s work when they have a low affinity for Ca2+?
Physically located in the same place as CaV - active zone. Develops a Ca2+ rich microdomain as Ca2+ is poorly diffusible.
What undoes the SNARE complexes once fusion has occurred?
NSFs using ATP
What type of receptor do most non-peptide neurotransmitters bind to?
Ionotropic LGIC
What type of receptor do most peptide neurotransmitters bind to?
Metabotropic GPCRs
Why are some NTs released faster than others?
Differences in spatial arrangement of vesicles relative to presynaptic Ca2+ channels
What is it called when the NT released from a varicosity inhibits the releasing varicosity?
Autoinhibition
Which synapses use ACh?
Preganglionic fibres of SNS and PNS
Postganglionic PNS
Motor
Preganglionic fibres terminating in the adrenal medulla
What is the rate limiting step in the synthesis of ACh? What inhibits this?
Supply of choline
Choline transported from ECF into cytoplasm by ChT choline transporter. Cotransports Na+. Can be inhibited by hemicholinium.
What else is in ACh vesicles?
Clear cored = with ATP at ration 10:1
Some cholinergic nerves have dense cored vesicles containing VIP
Where is AChE found?
Tethered to post synaptic membrane in synaptic cleft
Also in the presynaptic terminal
What are the two types of cholinesterase and what is their specificity?
AChE specific for ACh
Butyrylcholinesterase/pseudocholinesterase BuChE - broader substrate specificity. Also breaks down local anaesthetic procaine.
Where is BuChE made?
Liver
Found mainly in plasma
Trace quantities in skin, gut, brain
What is the aim of presynaptic AChE?
Guards against accumulation of cytoplasmic ACh outside storage vesicles.
What is the structure of AChE?
3 tetramers of AChE attached by disulphide bonds to a long collagenous tail that binds to a heparin sulfate proteoglycan on the basement membrane post-synaptically
Where are nAChRs expressed?
NMJ, autonomic ganglia, adrenal medulla, CNS, some other non-neuronal cells
What is the structure of nAChR?
LGIC
Cys-Loop family
Pentamer with at least 2alpha subunits present.
Each subunit 4 transmembrane M1-M4 helices
M2 helix from all 4 subunits forms the pore
Normal adult Nm: (alpha1)2beta1deltaepsilon
Normal ganglionic Nn: (alpha3)2(beta4)3 or (alpha3)2(beta2)3
Brain: (alpha4)2(beta2)3 or (alpha7)5
Where does ACh bind to the nAChR?
Binds to interface between alpha and neighbouring gamma or delta subunits. Minimum 2 molecules ACh required for activation of channel.
What is the nAChR permeable to?
Na+ K+
(alpha7)5 Ca2+
What is the mechanism of action of the two bungarotoxins?
Alpha = binds to ACh binding site on Nm irreversibly antagonises adult Nm. Beta = Binds to shaker type K+ channels, so localised to presynaptic membrane. PLA2 activity degrades active zone lipids
What can block the nAChR?
Nm: Alpha bungarotoxin
Decamethonium
Depolarising suxamethonium
Non-depolarising d-tubocurarine/Atracurium/pancuronium
Nn:
Nicotine - analogous to depolarising. First stim then blocks by causing persistent depolarisation
Trimetaphan - analogous to non-dep blocking agents
Hexamethonium - blocks in a use dependent manner so likely to cause an open channel block. Shortens duration of current flow as open channel either becomes occluded or closes.
Brain: (alpha7)5 also alpha bungarotoxin
How do you block the NMJ?
- Depolarising agents - act as agonist to Nm
2. Non-depolarising agents - act as competitive antagonists to Nm
Why would you want to block the NMJ?
Endotracheal intubation - can provide complete muscle relaxation at lower anaesthetic doses so more rapid recovery from anaesthesia and reduce postoperative respiratory depression
Describe depolarising blocking agents of the NMJ
Suxamethonium/Succinylcholine
More resistant to degradation by AChE
Continuous activation of nAChR
Phase I: Membrane depolarises. Brief period of excitation (fasciculations) followed by flaccid paralysis. This is because Nm channels maintain cell membrane in depolarised condition, inactivating NaV, so blocking further APs.
Phase II: Membrane gradually repolarises but Nm channels become desensitised.
Short duration of action so much faster recovery. Due to hydrolysis by plasma cholinesterase.
What are the side effects of suxamethonium?
Bradycardia due to direct muscarinic action
Increased ocular pressure
Increased serum K+ - can trigger ventricular dysrhythmia or cardiac arrest
Malignant hypothermia
Prolonged paralysis if liver disease or genetic deficiency of plasma cholinesterases
How do non-depolarising blocking agents work?
E.g. tubocurarine
Competitive antagonists for Nm. Compete with endogenous ACh without stimulating it. Prevent dep and inhibit muscular contraction leading to flaccid paralysis.
How do you overcome non-depolarising agents?
AChE inhibitors to increase the conc of ACh (to compete!)
Describe ganglionic stimulants. Why might you use them?
Selective agonists for Nn
Nicotine and lobeline
Epibatidine - powerful analgesic but side effects ruled it out from clinical use
Varenicline partial agonist (alpha4)2(beta2)3 and full for (alpha7)5. Parial agonism allows it to stim some dopamine release in the mesolimbic dopaminergic pathway (unlike total nicotine loss) which helps in alleviating some withdrawal symptoms. Serious mental problems have been reported.
Use to ease smoking withdrawal
Which type of muscarinic AChR modulates neurotransmitter release?
M2 activation decreases transmitter release
M1 Signalling pathway Location Agonist Antagonists
Gq/11
Gq –> PLCbeta –> PIP2 hydrolysis –> IP3 and DAG –> Ca2+ activation (IP3 polar so diffuses into cytoplasm and activates ER membrane-bound IP3Rs) and PKC activation (DAG lipophilic so stays in membrane).
DAG can be acted on by DAG lipase to make arachidonic acid, which may trigger a distinct Ca2+ influx pathway in some cell types, but most importantly precursor of prostaglandins and leucotrienes.
PIP2 depletion also reduces K+ efflux via M current (normally PIP2 binding to their C-terminus enables activation). Brings about a late EPSP many seconds after initial fast EPSP.
Peripheral and central neurons, gastric acid secretion
Agonist: non-selective: ACh, carbachol, pilocarpine
Antagonist: non-selective: Atropine, benzilylcholine, tropicamide, ipratropium. Irreversible: benzilylcholine mustard. Selective: pirenzipine
What is the M current?
A current that raises the threshold for firing an AP. PIP2 mediated.
M2 Signalling pathway Location Agonist Antagonists
Gi/o
Inhibition of AC –> inhibit cAMP and PKA production –> decreased phosphorylation of CaV –> decreases their open probability –> decreases Ca2+ influx –> decreased NT release + decreased excitability of heart cells.
Second mechanism in heart:
Beta gamma subunit of Gi/o opens
S (G protein coupled inwardly rectifying K+ channels) in the SAN and AVN. Triggers K+ efflux, hyperpolarisation, negative chronotropic.
Heart, presynaptic nerve terminals
Agonist: non-selective: ACh, carbachol, pilocarpine
Antagonist: non-selective: Atropine, benzilylcholine, tropicamide, ipratropium. Irreversible: benzilylcholine. Selective antagonist = gallamine, tripitramine.
M3 Signalling pathway Location Agonist Antagonists
Gq/11
Gq –> PLCbeta –> PIP2 hydrolysis –> IP3 and DAG –> Ca2+ activation (IP3 polar so diffuses into cytoplasm and activates ER membrane-bound IP3Rs) and PKC activation (DAG lipophilic so stays in membrane).
Smooth muscle contraction due to Ca2+i. Vascular endothelial cells, Ca2+i –> CaM –> eNOS (arginine–> citrulline+) –> NO –> diffuses into underying vascular smooth muscle –> activates soluble GC –> cGMP –> PKG –> relax the vascular smooth muscle.
Agonist: non-selective: ACh, carbachol, pilocarpine. Selective = cevimeline
Antagonist: Non-selective: Atropine, benzilylcholine, tropicamide, ipratropium. Irreversible antagonist: benzilyl choline mustard. Selective antagonist: darifenacin
M4 coupling?
Gi/o
M5 coupling?
Gq/11
What are the two types of muscarinic agonist?
- Choline esters like ACh and carbachol, bethanechol
2. Naturally occurring cholinomimetic alkaloids e.g. pilocarpine
Describe muscarinic antagonists chemically
`Ester and basic group like ACh, but also a bulky aromatic group replacing the acetyl moiety
What are the types of antimuscarinic agent?
- Naturally occurring alkaloids - atropine and scopolamine
- Their semi-synthetic derivatives - ipratropium
- Synthetic agents - pirenzipine, darifenacin
Compete for ACh binding site on mAChR
Most non selective, a few selective (pirenzipine M1, tripitramine M2, darifenacin, solifenacin M3)
Name agents that dilate pupils
Alpha 1 = dil M3 = constrict
So can use M3 antagonist atropine (but duration v long) so normally use short acting agents like tropicamide
Or adrenoceptor agonists - phenylephrine
How do you treat urinary incontinence?
Darifenacin M3 antagonist
What is the turnover of AChE?
10,000 molecules/sec/active site
What is the distribution of BuChE?
Widespread
Liver, skin, brain, GI smooth muscle, soluble in plasma
What does BuChE do?
Physiologically don’t know
Hydrolyses butyrylcholine more rapidly than ACh, ester-type local anaesthetics e.g. procaine, and suxamethonium
Describe the cholinesterases physically
Active site at base of deep and narrow cavity called aromatic gorge
At entrance to gorge there is a PAS peripheral anionic site
Base of gorge catalytic anionic site
What is the role of the PAS?
Peripheral anionic site
Recognising ACh as the substrate - weak pi-cation interaction between heteroaromatic ring of W279 tryptophan on AChE and the charged quaternary nitrogen of ACh
What about the aromatic gorge helps move the captured ACh to the active site?
Gorge lined with 14 conserved aromatic residues which can also form pi-cation interactions with ACh
Also an electrostatic gradient
Which ions in the CAS bind ACh?
Catalytic anionic site
Cation-pi interaction between another tryptophan W84 and quaternary nitrogen atom of ACh critical
Binds in the acyl pocket
What is the constriction in the gorge formed from and what is its consequence?
F330 and Y121 two aromatic sidechains
Smaller now than AChE
Indicates protein must undergo considerable conformational changes locally to widen the gorge and let the substrate through
What is the esteratic site?
Binding in the CAS and acyl pocket
Catalytic triad glu, hist, ser. Residues arranged so hist (+) and glu (-)donate elecrons in a charge-relay system, making the hydroxyl group of the serine highly reactive as a nucleophile.
Serine forms a covalent bond with ACh, forming a tetrahedral intermediate with the acetyl moiety.
What is the oxyanion hole?
Oxyanion (ser bound to acetyl) stabilised by interactions with backbone amide groups in area called oxyanion hole. Here ACh bond is broken, releasing choline and leaving acetyl-serine. Serine-acetate bond spontaneously hydrolysed by water to release acetate and regenerate the enzyme.
What is the difference between AChE and BuChE?
65% homology
3 aromatic residues of AChE PAS missing in BuChE PAS (2 different amino acid residues) - means BuChE has weaker affinity than that of AChE for ACh and other drugs.
6 of 14 conserved aromatic residues in AChE gorge are replaced by aliphatic amino acids in the gorge of BuChE. BuChE thus more flexible and can accommodate a wider range of substrate than AChE.
What are indirectly acting cholinergic agonists?
Anticholinesterases!
Give an example of an anticholinesterase that is also a directly acting cholinergic agonist
Neostigmine
List the categories of AChE by mechanism of inhibition
Mechanism of inhibition:
1. Reversible
a) non-covalent Edrophonium, tacrine
b) covalent Physostigmine, neostigmine, rivastigmine
2. Irreversible
Organophosphates e.g. Dyflos, parathion, ecothiophate
List the categories of AChE by duration of action
Short acting = edrophonium, tacrine
Intermediate acting = physostigmine, neostigmine, rivastigmine
Long acting = organophosphates
What determines duration of action of AChE?
Strength of anionic site interaction
How might AChE contribute to neurodegeneration? How may this be blocked?
PAS may serve as pathological chaperone for amyloid beta peptides, and promoting the formation of insoluble oligomer or amyloid plaque that is neurotoxic.
Donepezil spans the gorge to interact with the PAS and the CAS. Also shown to have inhibitory effect on protein aggregation.
What is the odd one out of the stigmines?
Rivastigmine - rest charged and so only act peripherally. Rivastigmine = lipophilic so useful in mild to moderate dementia.
Pyrimidostigmine
Medium acting covalent reversible anticholinesterase
Used orally to treat myasthenia gravis with a longer action than neostigmine
Describe the long acting anticholinesterases
Organophosphorous compounds - pentavalent phosphorous atom connected to either 3 O or 2 O 1 S along with a labile group like fluoride (dyflos DFP) or organic group (ecothiophate, parathion)
Labile group released then serine hydroxyl group phosphorylated
Extremely stable
What is aging?
In long acting anticholinesterases like dyflos and ecothiophate/parathion, the oxygen-phosphorous bond can undergo spontaneous rearrangement in favour of stronger bonding between enzyme and inhibitor. Once this has occurred, the duration of AChE inhibition is increased even further, so organophosphate inhibition is essentially irreversible.
Praloxime
Interacts with anionic site of AChE
Used to treat poisoning by irreversible anticholinesterases
Brings oxime group into close proximity of phosphorylated serine
Oxime group very strong nucleophile and causes the phosphate to transfer to the oxime, freeing the serine hydroxyl group.
Limited to within a few hours of exposure as phosphorylated AChE undergoes aging that renders the phosphorylated group unsusceptible to nucleophilic attack.
How do you treat poisoning by irreversible anticholinesterases?
Atropine to counteract symptoms (can cross BBB)
Praloxime to reactivate enzyme
What is the structure of catecholamines?
Catechol aromatic ring with two proximal hydroxyl groups and amine flanked by a two carbon linkage that often has an alcoholic -OH at beta position (but not dopamine)
What is the synthesis pathway for adrenaline?
L-tyrosine pumped in cotransported with Na+
Tyrosine hydroxylase (Rate limiting step)
DOPA
DOPA decarboxylase
Dopamine
Stored in vesicles VMAT2 driven by proton gradient
Dopamine beta-hydroxylase
Noradrenaline
In adrenal medulla:
PNMT Phenlyethanolamine N-methyltransferase
Adrenaline
What is isoprenaline?
Synthetic derivative of NA used experimentally
Which synthesis pathways are DOPA decarboxylases involved in?
Dopamine
NA
L-histidine to histamine
L-tryptophan to serotonin
What is dopamine beta hydroxylase concentration used for and why can it be used for it?
Used in vesicles to make NA from dopamine
So released with NA
Not rapidly degraded or susceptible to any uptake mechanism
Conc in plasma and body fluids used as index of overall sympathetic nerve activity
What does DBH deficiency cause?
Failure of NA synthesis
Severe orthostatic hypotension
Which hormone regulates PNMT?
Cortisol produced by surrounding adrenal cortex through the adrenal portal system
Reduction in cortisol levels reduces Adr:NA ratio
Also regulates DBH levels
Which drugs can interfere with catecholamines?
alpha-methyltyrosine: competitive inhibitor TOH
L-DOPA: can cross BBB so used to bypass rate limiting step and boost DA synth in Parkinson’s
Combine with carbidopa/benserazide which inhibits peripheral decarboxylase and so blocks some of the unwanted side effects of L-DOPA.
Disulfiram: inhibit DBH by chelating Cu2+ (its cofactor) or attacking sulphur-handling system of the methyl donor. Inhibition aldehyde dehydrogenase, so used in dealing with alcohol addiction.
Methyldopa: false transmitter. Antihypertensive. Less active than NA at alpha1, more active at alpha2 –> decreased alpha1 mediated constriction, more alpha2 mediated negative feedback. Primarily acts on alpha2 adrenoceptors in CNS where reduces NA outflow.
Clonidine: as alpha-methylNA.
Reserpine: inhibit VMAT by binding to amine binding site.
What is the ratio of storage of NA:ATP?
4:1 (nb ACh was 10:1)
Name the drugs that inhibit ACh and NA loading into vesicles
ACh: Vesamicol
NA: Reserpine
How do you directly block an adrenergic neuron?
Guanethidine and bretylium - displace amine from storage sites so can transiently stimulate the release of NA. Compete with NA for Uptake1/NET so potentiates exogenously applied NA.
Repeated low doses, or in the aftermath of a large dose, block release of NA evoked by APs, but spontaneous release unaffected –> so vesicle release processes unaffected
How can you trigger NA release in absence of depolarisation?
Indirectly acting sympathomimetic amines e.g. tyramine, dextroamphetamine, ephedrine
Not adrenoceptor agonists themselves
NET1
VMAT
Displace NA from vesicles
Pumped into synaptic space by reverse transport of NET (and partly metabolised by MAO.
After this there is no stimulation with vesicle release (as they contain tyramine)
How do the different indirectly acting sympathomimetic amines act on MAO?
Tyramine = substrate Dextroamfetamine = weak inhibitor
Which other drugs act somewhat like indirectly acting sympathomimetic amines?
Ritalin/methylphenidate and MDMA (but MDMA also acts on 5HT2 receptors)
Define tachyphylaxis
Repeated administration produces less and less response
How do you abolish the effects of indirectly acting sympathomimetic amines?
Reserpine - destroy the transmitter stores
How do you modify catecholamine release?
- Modify synthesis
- Modify storage
- Directly bock adrenergic neurons
- Indirectly acting sympathomimetic amines
- Stimulating presynaptic receptors
Why is there greater tachycardia in antihypertensive therapy with non-selective alpha-blockers than with alpha1-adrenoceptor blocking agents?
Blocking alpha 2 –> reduces the inhibitory effect of alpha2 on presynaptic nerve terminals
Which receptors are present on nerve terminals to modify NT release?
Autoreceptors: Alpha2 - reduces
Beta2- enhance
Other:
M2, deltaopioid, prostaglandin EP3 - decrease NA release
P2Y1 G1 sympathetic neuron presynaptically, facilitate NT release by inhibiting M current. A current that raises the threshold for firing an AP. PIP2 mediated.
What mediates the neuronal effect of alpha2?
GIRK opening by betagamma subunit Gi/o –> hyp
How is NA transmission inactivated?
Uptake 1 and 2
Uptake 1 more important
Describe uptake 1 and its pharmacological modulation
Neuronal cells High affinity Low capacity NET transporter Na+ dep Blocked by cocaine, tricyclic antidepressants including imipramine and amitriptyline. Blocked by phenoxybenzamine at 10x higher conc than blocks uptake 2. 75% so primarily responsible for termination of transmitter action NA repackaged into vesicles
Describe uptake 2 and its pharmacological modulation
Non-neuronal cells Low affinity High capacity ENT transporter Not Na+ dep 25% NA Inhibited by normetanephrine (NA metabolite), corticosteroids, and phenoxybenzamine irreversibly
How are catecholamines metabolised? What is the end product
MAO and COMT MAO oxidative deamination amine to aldehyde, then alcohol dehydrogenase metabolises to corresponding carboxylic acid. COMT methylates aromatic hydroxyls. PNS: MAO then COMT --> VMA --> urine CNS: MOPEG
Where is MAO? What does it do in each location?
Outer mitochondrial membrane
Noradrenergic nerve terminals - metabolises NA leaking from vesicles
Liver, intestinal epithelium - inactivates circulating monoamines carried in the portal venous blood from the gut.
What are the two MAO isoforms? How are they different (include drugs)?
MAO-A: degrades serotonin, NA, DA. Moclobemide used in depression (serotonin)
MAO-B: degrades DA more rapidly. Selegyline used in Parkinson’s (DA).
Non-selective MAO inhibitors = tranylcypromine, isocarboxazid, phenelzine.
Where is COMT?
Cytosol in liver and adrenal medulla. NOT NA nerve terminals.
Diagnostic test for pheochromocytoma?
Marked increase in urinary excretion VMA
How do you treat Parkinson’s?
L-DOPA, can cross BBB and reach CNS. Converted to dopamine.
Also need to take carbidopa to inhibit peripheral DDC to stop L-DOPA metabolism in the periphery.
Also take entacaptone to inhibit COMT in the periphery (also less L-DOPA metabolism).
Alpha1 Receptor Second messenger and effector Agonist potency order Selective agonist Selective antagonist Location Physiological role
Gq/11
PLCbeta–> PIP2–> DAG and IP3 –> increase Ca2+ and PKC
Na>A»Iso
Agonist: Phenylephrine, oxymetazoline
Antagonist: Prazosin, doxazosin (tamsulosin silodosin selective alpha-1A)
Postsynaptic effector cells esp smooth muscle
1A: bladder. Contraction of trigone and sphincter muscles of urinary bladder
1B: Vasoconstriction (blood vessels to viscera, brain, skin)
Also:
Mydriasis
Salivary secretion
Decreased GI motility and tone due to relaxation of smooth muscles
Localised secretion of sweat
Piloerection
Ejaculation
Increased glycogenolysis and gluconeogenesis
Alpha2 Receptor Second messenger and effector Agonist potency order Selective agonist Selective antagonist Location Physiological role
Gi/o
Inhibit AC, inhibit cAMP, decrease Ca2+ channels, increase K+ channels.
A>NA»ISO
Agonist: Clonidine partial, methylnoradrenaline
Antagonist: Yohimbine
Presynaptic nerve terminals, platelets, lipocytes, smooth muscle
Inhibit NT release
Platelet aggregation
Vasoconstriction
Inhibit insulin release from pancreatic beta cell
Beta1 Receptor Second messenger and effector Agonist potency order Selective agonist Selective antagonist Location Physiological role
Gs
AC cAMP PKA MLCK phos relax smooth muscles
ISO> NA> A
Agonist: dobutamine, xamotterol
Antagonist: Atenolol, metoprolol, nebivolol
Postynaptic effector cell in heart, presynaptic nerve terminals, juxtaglomerular apparatus of renal tubes, ciliary body
Increase force of myocardial contraction + inotropy, increased HR + chronotropy
Beta2 Receptor Second messenger and effector Agonist potency order Selective agonist Selective antagonist Location Physiological role
Gs
AC CAMP PKA Phos and inhibit MLCK relax smooth muscles
ISO> A> NA
Sabutamol, salmeterol, formoterol, indacaterol
None clinically useful (but butoxamine we do have, not useful)
Postsynaptic effector cells, especially cardiac and smooth muscle
Relax bronchial and tracheal smooth muscle, relax smooth muscle of vessels supplying hear and skeletal muscle, relax uterine smooth muscle (if non-pregnant), hepatic glycogenolysis
Beta3 Receptor Second messenger and effector Agonist potency order Selective agonist Selective antagonist Location Physiological role
Gs
AC cAMP - NO?
ISO > NA=Adr
Agonist: mirabegron
No selective antagonist
On posysynaptic effector cells, esp lipocytes and heart
Lipolysis, thermogenesis, relaxation bladder detrusor muscle.
How can a beta1 antagonist stimulate vasodilation?
Nebivolol
Metabolite is a beta3 agonist
NO production up
Vasodil
Why do you use Adr not NA to treat asthma and anaphylaxis?
Want to relieve bronchospasm
Adr has higher efficacy than NA on beta2 receptors (present on smooth muscle of bronchus and trachea)
Explain the effect of widespread sympathetic stim on the body
- Peripheral excitatory Constrict blood vessles to skin, kidney and mucous membranes alpha1
- Peripheral inhibitory Relax gut alpha1
Relax bronchial tree - breathe more beta2
Relax blood vessels to skeletal muscle beta2 - Cardiac excitatory + ino and chrono beta1
- Metabolic actions. Enhanced glycogenolysis and liberation FFAs from adipose beta2 and 3
- Endocrine actions Endocrine modulation insulin secretion, renin secretion, pituitary hormones alpha2
- CNS actions (resp stim, increase in wakefulness and psychomotor activity, reduction in appetite)
- Prejunctional actions to inhibit mainly release NT alpha2
What is the effect of adrenaline on blood pressure and heart rate?
Beta1: + ino + chrono –> increased HR
Alpha1: Constricts arterioles in skin, mucous membranes and viscera –> increase peripheral resistance
Beta2: Vasodil skeletal muscle vascular bed –> decrease peripheral resistance
So cumulative increase systolic BP slight decrease diastolic BP. slight decrease TPR. Increase HR
What is the effect of NA on blood pressure and heart rate?
Alpha1: strong effect. Rise in TPR due to intense vasoconstriction most vascular beds. Increases both sys and dias BP to an extent high enough to stim baroreceptors –> vagal activity –> reflex bradycardia. If + atropine, stim of beta1 –> tachycardia.
Beta2: doesn’t induce compensatory vasodil
So greater total vasoconstriction than Adr and large increase peripheral resistance. Increase sys and dias BP, decrease pulse rate.
What are the effects of isoprenaline on BP and HR?
Only acts on beta adrenoceptors
Beta1: tachycardia
Beta2: vasodil - decrease diastolic BP -
Large drop BP
What are the non-selective alpha and beta agonists and antagonists?
Alpha: Agonist Xylometazoline, methoxamine, Antagonist: Phentolamine, phenoxybenzamine Beta: Agonist: Isoprenaline Mixed: Propranolol, Agonist: Adr NA Antagonist: Labetalol, carvedilol mixed alpha1/beta
When should you not sue non-selective beta blockers and why?
COPD and asthma
Blockade of beta2 in bronchial smooth muscle can exacerbate condition and lead to a serious crisis.
Diabetic receiving insulin
Blockade of beta2 –> decreased glycogenolysis and decreased glucagon secretion (can lead to hypoglycaemia)
What is the cholinergic transmission to the vas deferens and how do you prove it?
ATP and ACh
Biphasic/twin-peaked
First peak abolished by suramin (ATP antagonist)
Late peak blocked by prazosin alpha1 adrenoceptor antagonist
Completely abolished when both are present
ACh in vas = erection
What is activity dependent tuning?
Need higher conc of Ca2+ to elicit release of peptide transmitters, so they are preferentially released at high rates of neural stim
What are the purinergic receptors?
Purinoceptors
ATP ADM AMP Adenosine receptors
P1/adenosine receptors A1 A2A A2B A3: Adenosine>AMP>ADP>ATP. GPCR
P2: ATP>ADP>AMP>Adenosine
Divided into
P2X1-7: Ligand gated. Homo/heterotrimeric ATP-gated cation channels
P2Y1,2,4,6,11-14: GPCR Gq. some don’t know natural agonists yet.
What stores ATP into vesicles?
VNUT
How is ATP released?
Either from cytosol through large membrane channels e.g. pannexins
Or from vesicles via Ca2+ dep exocytosis
Or via NtT nucleotide transporters
How is ATP metabolised?
Ectonucleotidases to ADP to Adenosine
Adenosine deaminase Adenosine to inosine
How is Adenosine taken up from the extracellular fluid?
NsT nucleoside transporters
Describe the structure and location of P2X receptors
Non selective cation channels (equal Na+ K+ and with significant Ca2+ perm)
ATP opens
Homo or heterotrimer
Each subunit comprises two transmembrane segments TM1 and TM2 separated by an ectodomain containing 10 conserved cysteine residues that form disulphide bonds. Disruption of these can sometimes impair trafficking of these proteins to the plasma membrane.
Projects on an axis of symmetry projecting as a perpendicular from the cell membrane.
ATP binding site formed between 2 adjacent subunits, so each receptor binds 3 ATPs.
Describe the location and roles of P2X receptors. How may these be pharmacologically useful?
P2X1 smooth muscle i.e. sympathetic to vas deferens, blood vessels. Fast response. Example - mice lacking P2X1 receptor gene have drastically reduced fertility because of much reduced sperm count. Maybe target as contraceptive? Also parasympathetic to urinary bladder fast response.
P2X2,4,6 postsynaptic neurons. ATP released from urothelial cells when bladder distended, P2X3 mediate the nerve responses to distension, providing mechanosensory feedback involving both the micturition reflex and pain. Maybe target in pain and detrusor instability?
How do you potentiate adenosine?
Dipyridamole - blocks NsT so indirectly increases the concentration of extracellular adenosine
Methotrexate, unsure how.
When can adenosine concentration rise above the submicromolar range?
Stressful conditions (Hypoxia, ischaemia) –> endothelial cells, neutrophils, cardiomyocytes, smooth muscle cells, glial cells release ATP through pannexins/connexions –> dephos to adenosine by ectonucleotidases
What is the coupling of the different adenosine/P1 receptors? What is the effect?
A1 Gi/o. Slows rate and force of contraction in the heart. So used to terminate supraventricular tachycardia. Presynaptic inhibitory effects on release of excitatory NT in CNS and periphery. A2A Gs A2B Gs - both 2A and 2B relaxation vascular smooth muscle cells. A3 Gi/o Ischaemic preconditioning Potentially anti-inflammatory act via cAMP Some also via PLC and thus Ca2+, MAPK Can alter KV and CaV
What are the antagonists of P1 receptors?
Caffeine competitive antagonist A1
theophylline
What are the three forms of NOS?
- Neuronal NOS: CNS and NANC nerves
- Inducible NOS: induced in macrophages and other cells by bacterial LPS and other inflammatory cytokines ie IFNgamma
- Endothelial NOS: expressed in platelets and endothelial cells
What is the mechanism of action of NO?
Shear stress (force exerted on blood vessel wall by movement of blood past it)
Protein kinase B/Akt (ca2+ independent)
NO
diffuses
activates sGC via interacting with haem moiety of the enzyme
cGMP
PKG
Relaxation smooth muscle
Tonic release NO needed for maintenance of a normal BP
NO also inhibits platelet aggregation
NO also activates some K+ channels, esp KCa, which hyperpolarises the vascular smooth muscle
NO inhibits monocyte adhesion and migration, adhesion and aggregation of platelets, and smooth muscle and fibroblast proliferation
Summary: antiatherosclerotic and antihyeprtensive
What does cGMP do in the eye?
cGMP present in absence of light
Binds to and activates cGMP gated ion channel
Na+ and Ca2+ influx (dark current)
Depolarises outer segment of photoreceptors
Which organs utilise NO?
Upper airways, GI tract, male sexual organs
GI: NO regulates muscle tone of sphincter in the lower oesophagus, pylorus, sphincter of Oddi, anus. Accommodation reflex fundus. Peristaltic reflex of intestine. nNOS disorders may be responsible for motility disorders in the GI tract.
How does diabetes affect gastric emptying?
Diabetes Type I - reduced sec insulin - reduced nNOS protein in enteric neurons - inadequacy in NO-mediated NANC transmission - distended stomach and pyloric hypertrophy
What is achalasia?
Oesophagus cannot move food down
Loss of nitrergic neurons supplied to oesophageal sphincter muscles
What are the main neuropeptides released from noradrenergic and cholinergic neurons?
NPY NA
VIP cholinergic
Why do neuropeptides require more APs to release?
Dense-cored granules do not cluster at the active zone
Requires a more generalised increase in intracellular Ca2+
Why do neuropeptides produce a prolonged response?
Not rapidly removed from ECF - their action terminated by diffusion or by extracellular peptidases.
What are the 3 main types of NANC transmission?
Purinergic
Nitrergic
Neuropeptides
In the autonomic ganglion, which neurotransmitters are responsible for each stage of the response?
N EPSP
M2 IPSP
M1 slow EPSP
Peptides late slow EPSP
Name the brain-gut peptides
Substance P
CCK
VIP
Name the opioid peptides
Leucine encephalin
alpha-endorphin
Dynorphin A
Name the pituitary peptides
Vasopressin
Oxytocin
ACTH
Name the hypothalamic-releasing peptides
TRH
LHRH (LH)
Somatostatin (GHIH)
