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Neuroscience with Pharmacology 2

Neuromuscular Junction 2: Pharmacology

Sir Henry Dale

Acetylcholine

“Quaternary” nitrogen

+

1. Principles and methods for studying ACh pharmacology at the NMJ

2. Targets for drug action at the NMJ

3. Drugs affecting ACh Receptors (activation)

4. Drugs affecting ACh Esterase (inactivation)

5. Drugs affecting ACh release

6. Drugs affecting ACh synthesis and storage.

Neuromuscular Junction - Pharmacology

1. Principles and methods

Drugs acting at the NMJ are useful as:

Experimental tools

Clinical treatments

• Target specificity/Ligand specificity• Agonist/Antagonist• Dose/Response• Efficacy (EC50/IC50)/Affinity (KD)• Competitive/Non-competitive• Clinical/Non-clinical uses• Side-effects

When “doing drugs” a good pharmacologist needs to know:

2

Biological response (->efficacy, EC50)

Ligand binding (->affinity, KD)

Drug assays at the NMJ are based on:

Measuring muscle contractions in response torelease of neurotransmitter by nervestimulation….

Single nerve stimulus : Twitch

Repetitive stimulus : Tetanus

1 sec

Atropine d-Tubocurarine

Specificity and dose-response of antagonists

EC50

Ligand binding visualised…

α-bungarotoxin

Control

10µm α-BTX

But normally measured chemically (eg radioactively-labelled ligand)…

3

Action potential

…add µ-conotoxinX

…add d-tubocurarine

Measuring EPP’s….

Testing drugs on ACh receptors

Iontophoresis

Bath application

Patch clamp

I

V

Ch.2

2 mV

30.00 ms

s

10 µM ACh

2 mV

60 s

2. Targets for drug action at the NMJ

Targets in neuromuscular pharmacology :

Synthesis

Storage

Release

Action

Inactivation

α-bungarotoxind-tubocurarineatracuriumsuxamethonium

edrophoniumneostigminesarin

hemicholinium

vesamicol

botulinium (A-D)

Targets of drug action at the neuromuscular junction

3. Drugs affecting ACh Receptors (activation)

4

ACh + R ACh-R ACh-R*

ACh-R0

General scheme for Agonist-Receptor Interaction (illustrated by ACh binding to its Receptors)

“desensitized”

bound activated

[ACh].[R]

[ACh-R]KD = = ~ 80 µM

8 nm

The nicotinic ACh Receptor at NMJ

/ε

ACh

AChR/AChE

Anionic site Esteric site

αε

Anionic site Esteric site

αε

Stereospecifity of skew (“gauche”) configuration ofACh/receptor binding. (View as “magic eye” stereo pair.)

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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http://en.wikipedia.org/wiki/Quaternary_ammonium_muscle_relaxants

ACh

Decamethonium Pancuronium

ACh

ACh

ACh

Na+

K+

dTC

Reversible competitive antagonism of the ACh receptor. 1

dTC

ACh ACh

ACh ACh

dTCACh dTC

ACh

Reversible competitive antagonism of the ACh receptor. 2Drugs that block ACh Receptors (antagonists)

Example:d-tubocurarine

Example:α-bungarotoxin

ACh

αBTX

ACh

ACh

ACh

AChACh

AChαBTX

Irreversible competitive antagonism of ACh Receptor

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”

2

2

AcetylcholineSuxamethonium

6

dTC dTC neo sux sux sux neo direct

Sux

Na+

+

Na+

K+

+

Dual effect of suxamethonium (succinyl choline)

1. Depolarisation

Sux

+

Na+ Na+

K+

Dual effect of suxamethonium (succinyl choline)

2a. Sodium inactivation (depolarising block)

Sux

Dual effect of suxamethonium (succinyl choline)

2b. Desensitization of AChR (depolarising block)

Phencyclidine (PCP,”angeldust”)

Non-competitive antagonists of the nicotinic ACh receptor

Methyl violet 10B(hexamethylviolet; crystalviolet)

4. Drugs affecting ACh Esterase (inactivation)

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Drugs that block ACh esterase

x Antidote:

5. Drugs affecting release

α-latrotoxin

BEFORE

K channel blockers and some toxins enhance transmitter release

AFTER

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

-0.5

-1.0

-1.5

mV

AC

1

0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200s

During

Drugs that inhibit transmitter release

Botulinum toxins = ‘SNARE’ blockers P/Q Ca2+ channel blockers

Ca2+x

x

ω-agatoxin/ω-conotoxin

0 Ca +Ca +4AP TTXDirectDirect +Mg +4AP

Counteracting the depression of ACh release by low Ca2+ or high Mg2+ using 4-AP

6. Drugs affecting synthesis and storage

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Drugs that inhibit transmitter synthesis/storage

Choline uptake

Vesicle filling

x

x

Choline acetyltransferase Inhibitor

X

1. Every step in the ACh cycle - synthesis, storage release,activation and inactivation - is a potential target for drug actionat the NMJ.

2. Clinical uses of drugs acting at the NMJ:- muscle relaxants as adjunct to anaesthesia in surgery- treatment of neuromuscular disease

3. Drugs affecting ACh Receptors:- carbachol, decamethonium, suxamethonium (agonists)- tubocurarine, α-bungarotoxin, atracurium (antagonists)

4. Drugs that block ACh Esterase:- edrophonium, prostigmine, neostigmine, sarin

5. Drugs affecting release:- TEA, 4-AP, α-latrotoxin (agonists)- botulinum toxin, ω-agatoxin (antagonists)

6. Drugs that block uptake, storage and synthesis :- hemicholinium-3, vesamicol, AF64A

Summary