Neuroscience with Pharmacology 2
Neuromuscular Junction 2: Pharmacology
Sir Henry Dale
Acetylcholine
+
“Quaternary” nitrogen
1 Terminal Schwann Cell
Synaptic vesiclesMotor nerve Mitochondria terminal Kranocyte Active Zone
PSD Basal Motor Lamina endplate
Junctional Folds
Neurotransmitters are released by exocytosis from synaptic vesicles
2 Excitation synchronises neurotransmitter release
S
End-Plate Current (EPC)
End-Plate Potential (EPP)
20 mV
2 ms
Quantal analysis of EPPs shows a “safety factor” of 2-5
Actual m !
Threshold m !
3 Targets in neuromuscular pharmacology :
® Synthesis
® ® Storage ® ® Release
® ® Action
® ® ® Inactivation
Neuromuscular Junction - Pharmacology
1. Principles and methods of studying ACh pharmacology at the NMJ
2. Targets for drug action (synthesis, storage, release, action, inactivation)
3. Drugs affecting postsynaptic (nicotinic) ACh Receptors
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4 Drugs acting at the NMJ are useful as: w Experimental tools w Clinical treatments
Several techniques can be used to assay the effects of drugs at NMJ’s
1. Muscle contraction 2. Intracellular EPP recording 3. Iontophoresis/patch clamp 4. Ligand binding
5 Measuring muscle contractions in response to release of neurotransmitter by nerve stimulation….
dTC dTC neo sux sux sux neo direct
6 Measuring EPP’s….
Action potential X …add µ-conotoxin …add d-tubocurarine
…Reduce Ca2+ and/or add Mg2+
EPP’s EPP’s in low Ca/high Mg (muscle action potential blocked)
2+ Mg
Ca2+
5 mV 10.00 ms
5 mV 10.00 ms
7 Transmitter release (EPP amplitude; Quantal content) depends on [Ca2+]4
Mg2+
Slope=4
Dodge & Rahamimoff (1967) JPhysiol
Na+ Ca2+ K+
8 Inhibiting and Enhancing ACh Release: Ca/K-dependent
0 Ca Direct+Ca +4AP +Mg +4AP DirectTTX
Testing drugs on ACh receptors
Iontophoresis I 10 µM ACh
2 mV
60 s Bath application s
Ch.2
2 mV 30.00 ms V
Patch clamp
9 Drug assays at the NMJ are based on:
w Biological response (->efficacy, EC50)
w Ligand binding (->affinity, KD)
EC50
10 Ligand binding visualised…
Control
α-bungarotoxin 10µm α-BTX!
But normally measured chemically (eg radioactively-labelled ligand)…
11 General scheme for Agonist-Receptor Interaction (illustrated by ACh binding to its Receptors)
ACh + R D ACh-R D ACh-R* bound activated
D D ACh-R0 “desensitized”
[ACh].[R] KD = = ~ 80 µM [ACh-R]
2. Targets for drug action at the NMJ
- Presynaptic
- Intrasynaptic
- Postsynaptic
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12 Choline acetyltransferase Inhibitor
X
AF64A
Drugs that inhibit transmitter synthesis/storage
Choline uptake
Hemicholinium-3 x x
Vesicle filling
Vesamicol
13 Drugs that inhibit transmitter release
P/Q Ca2+ channel blockers Botulinum toxins = ‘SNARE’ blockers
ω-agatoxin/ ω-conotoxin
2+ Ca x
x
Latrotoxin enhances transmitter release
BEFORE During AFTER
14 Drugs that block ACh esterase
Acetylcholine
Edrophonium Neostigmine Sarin
x Antidote: Pradiloxime
Anticholinesterases prolong EPPs
Control
C h . 0 C h . 0
C h . 0
5 m V 5 m V 5 . 0 0 m s 5 . 0 0 m s
5 m V 5 . 0 0 m s
Half Decay Time Neostigmine (5 µM) 15 Hepes Buffered Ringer Solution
Neostigmine (5µM)
10 (mS)
Time 5 C h . 0
0 T1 T2 T3 T4 EPP Train Number
5 m V 5 . 0 0 m s
15 3. Drugs affecting postsynaptic (nicotinic) ACh Receptors
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The nicotinic ACh Receptor at NMJ 8 nm
/ε
16 nACh Receptor Pharmacology - Neuromuscular Junction
Agonist Antagonist/blocker nicotine d-tubocurarine (curare) acetylcholine α-bungarotoxin carbachol atracurium decamethonium pancuronium suxamethonium (I) suxamethonium(II)
17 Atropine d-Tubocurarine
Drugs that block ACh Receptors (antagonists)
Reversible
Example: d-tubocurarine
Irreversible Example: α-bungarotoxin
18 ACh
Na+
ACh
dTC dTCACh
K+
19 ACh ACh
ACh ACh
dTCACh dTC
Na+
ACh
K+
20 ACh
Na+
ACh
αBTX αAChBTX
K+
21 ACh ACh ACh
ACh ACh ACh ACh ACh
αBTX ACh
SuxamethoniumAcetylcholine
2
2
Not broken down by AChE (nmj) Broken down by BuChE (blood plasma) Short lasting Not counteracted by cholinesterase inhibitor Used clinically for brief muscle relaxation “Depolarising blocker”
22 dTC dTC neo sux sux sux neo direct
Sux Na+
Sux
K+
23 Na+ Na+
Sux + +
K+
Na+ Na+
Sux + +
K+
24 Na+ Na+
Sux +
K+
Sux Na+
Sux
K+
25 Targets of drug action at the neuromuscular junction
hemicholinium
botulinium (A-D)
α-bungarotoxin edrophonium d-tubocurarine neostigmine atracurium sarin suxamethonium
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26 Summary
1. Every step in the cycle of ACh synthesis, storage, release, activation and inactivation is a potential target for drug action at the NMJ.
2. Drugs affecting storage and synthesis : • AF64A, hemicholinium, vesamicol
3. Drugs affecting release • 4-AP, α-latrotoxin, botulinum toxin, ω-agatoxin, • Mg2+, botulinum toxin, ω-agatoxin, 4-AP, α-latrotoxin,
4. Drugs that block ACh Esterase: • edrophonium, neostigmine, sarin
5. Drugs affecting ACh Receptors: • carbachol, decamethonium, suxamethonium • tubocurarine, α-bungarotoxin, atracurium
6. Clinical uses of drugs acting at the NMJ: • muscle relaxants as adjunct to anaesthesia in surgery • treatment of neuromuscular disease
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