Lecture 4: Neuromuscular Junction: Pharmacology

Neuroscience with Pharmacology 2
Neuromuscular Junction 2: Pharmacology
Sir Henry Dale
Acetylcholine
+
“Quaternary” nitrogen
1

Terminal
Schwann Cell
Synaptic vesicles
Motor nerve
terminal
Mitochondria
Active
Zone
Kranocyte
Motor
endplate
PSD
Basal
Lamina
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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@RRibchester
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….
X
Action potential
…add µ-conotoxin
…add d-tubocurarine
…Reduce Ca 2+ and/or add Mg 2+
EPP’s
(muscle action potential blocked)
EPP’s in low Ca/high Mg
Mg 2+
Ca 2+
5 mV
10.00 ms
5 mV
10.00 ms
7

Transmitter release (EPP amplitude; Quantal content) depends on [Ca 2+ ] 4
Mg 2+
Slope=4
Dodge & Rahamimoff (1967) JPhysiol
Na +
Ca 2+
K +
8

Ch.2
2 mV
30.00 ms
Inhibiting and Enhancing ACh Release: Ca/K-dependent
0 Ca Direct +Ca +4AP +Mg +4AP Direct TTX
Testing drugs on ACh receptors
Iontophoresis
I
10 µM ACh
2 mV
60 s
Bath application
s
V
Patch clamp
9

Drug assays at the NMJ are based on:
w Biological response (->efficacy, EC50)
w Ligand binding (->affinity, K D )
EC 50
10

Ligand binding visualised…
Control
α-bungarotoxin
10µm α-BTX!
But normally measured chemically (eg radioactively-labelled ligand)…
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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-R 0
“desensitized”
[ACh].[R]
K D = = ~ 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 Ca 2+ channel blockers
Botulinum toxins = ‘SNARE’ blockers
ω-agatoxin/
ω-conotoxin
Ca 2+
x
x
Latrotoxin enhances transmitter release
α-latrotoxin
BEFORE
During
AFTER
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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 . 0 0 m s
5 m V
5 . 0 0 m s
5 m V
5 . 0 0 m s
Neostigmine (5 µM)
15
Half Decay Time
Hepes Buffered Ringer Solution
Neostigmine (5µM)
C h . 0
Time (mS)
10
5
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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@RRibchester
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)
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Atropine
d-Tubocurarine
Drugs that block ACh Receptors (antagonists)
Reversible
Example:
d-tubocurarine
Irreversible
Example:
α-bungarotoxin
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ACh
Na +
ACh
dTC
dTC ACh
K +
19

ACh
ACh
ACh
ACh
dTC ACh
dTC
Na +
ACh
K +
20

ACh
Na +
ACh
αBTX
αBTX ACh
K +
21

ACh
ACh
ACh
ACh
ACh
ACh
ACh
ACh
αBTX
ACh
Suxamethonium
Acetylcholine
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 +
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Targets of drug action at the neuromuscular junction
vesamicol
botulinium (A-D)
hemicholinium
α-bungarotoxin
d-tubocurarine
atracurium
suxamethonium
edrophonium
neostigmine
sarin
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#NeuroMJ
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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,
• Mg 2+ , 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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