Biochemistry of acetylcholine

1. ACETYLCHOLINE
Prof. Kwena

2. Nerve cell

3. • Acetyle choline is neurotransmitter at many
synapses and at nerve-muscle junctions.
• The presynaptic membrane of cholinergic
synapse (one that uses acetylcholine as
neurotransmitter) is separated from
postsynaptic membrane by a gap (500 A)
called a synaptic cleft.

4. • The end of of the presynaptic axon is filled
with synaptic vesicles containing
acetylcholine.
• The arrival of a nerve impulse leads to release
of acetylcholine into the cleft.
• The acetylcholine then diffuses to the
postsynaptic membrane where they combine
with specific receptors.

5. • This produces a depolarization of post
synaptic membrane
• This is propagated along the electrical
excitable membrane of the second nerve cell.
• Acetylcholine is then hydrolized by
acetylcholinestrase enzyme.
• Polarization of the postsynaptic membrane is
restored.

6. • Acetylcholine is released in packets
• Acetylcholine also opens cation gates in the
postsynaptic membrane.
• Acetylcholinestrase inhibitors are used as
drugs and poisons- neostigimine and
physostigimine

7. Acetylcholine Precursors:
• Choline and acetyl-CoA
• Synthesizing enzymes: Choline acetyl
transferase
• Metabolizing enzymes: Acetylcholinesterase
• Metabolites: Choline and acetate

8. • The chemical compound acetylcholine (often
abbreviated ACh) is a neurotransmitter in
both the peripheral nervous system (PNS) and
central nervous system (CNS) in many
organisms including humans.

9. • Acetylcholine is one of many
neurotransmitters in the autonomic nervous
system (ANS) and the only neurotransmitter
used in the motor division of the somatic
nervous system (sensory neurons use
glutamate and various peptides at their
synapses).

10. • Acetylcholine is also the principal
neurotransmitter in all autonomic ganglia.
• Acetylcholine slows the heart rate when
functioning as an inhibitory neurotransmitter.
• However, acetylcholine also behaves as an
excitatory neurotransmitter at neuromuscular
junctions.

11. Structure

12. • Acetylcholine is an ester of acetic acid and
choline with chemical formula
CH3COOCH2CH2N+(CH3)3.
• This structure is reflected in the systematic
name, 2-acetoxy-N,N,N-
trimethylethanaminium.
• Its receptors have very high binding constants.

13. Function
• Acetylcholine has functions both in the
peripheral nervous system (PNS) and in the
central nervous system (CNS) as a
neuromodulator.
• In the peripheral nervous system,
acetylcholine activates muscles, and is a major
neurotransmitter in the autonomic nervous
system.

14. • In the central nervous system, acetylcholine
and the associated neurons form a
neurotransmitter system, the cholinergic
system, which tends to cause anti-excitatory
actions.

15. In the peripheral nervous system
• In the peripheral nervous system,
acetylcholine activates muscles, and is a major
neurotransmitter in the autonomic nervous
system.
• When acetylcholine binds to acetylcholine
receptors on skeletal muscle fibers, it opens
ligand-gated sodium channels in the cell
membrane.

16. • Sodium ions then enter the muscle cell,
initiating a sequence of steps that finally
produce muscle contraction.
• Although acetylcholine induces contraction of
skeletal muscle, it acts via a different type of
receptor (muscarinic) to inhibit contraction of
cardiac muscle fibers.

17. autonomic nervous system
• In the autonomic nervous system,
acetylcholine is released in the following sites:
• all pre- and post-ganglionic parasympathetic
neurons

18. • all preganglionic sympathetic neurons
– preganglionic sympathetic fibers to suprarenal
medulla, the modified sympathetic ganglion; on
stimulation by acetylcholine, the suprarenal
medulla releases epinephrine and norepinephrine
• some postganglionic sympathetic fibers
– pseudomotor neurons to sweat glands.

19. Plasticity
• ACh is involved with synaptic plasticity,
specifically in learning and short-term
memory.
• Acetylcholine has been shown to enhance the
amplitude of synaptic potentials following
long-term potentiation in many regions,
including the dentate gyrus, CA1, piriform
cortex, and neocortex.

20. • This effect most likely occurs either through
enhancing currents through NMDA receptors
or indirectly by suppressing adaptation.

21. • The suppression of adaptation has been
shown in brain slices of regions CA1, cingulate
cortex, and piriform cortex, as well as in vivo
in cat somatosensory and motor cortex by
decreasing the conductance of voltage-
dependent M currents and Ca2+-dependent K+
currents.

22. Excitability and inhibition
• Acetylcholine also has other effects on
neurons.
• One effect is to cause a slow depolarization by
blocking a tonically-active K+ current, which
increases neuronal excitability.
• Alternatively, acetylcholine can activate non-
specific cation conductances to directly excite
neurons

23. • An effect upon postsynaptic M4-muscarinic
ACh receptors is to open inward-rectifier
potassium ion channel (Kir) and cause
inhibition.

24. • The influence of acetylcholine on specific
neuron types can be dependent upon the
duration of cholinergic stimulation.
• For instance, transient exposure to
acetylcholine (up to several seconds) can
inhibit cortical pyramidal neurons via M1 type
muscarinic receptors that are linked to Gq-
type G-protein alpha subunits.

25. • M1 receptor activation can induce calcium-
release from intracellular stores, which then
activate a calcium-activated potassium
conductance which inhibits pyramidal neuron
firing.

26. • On the other hand, tonic M1 receptor
activation is strongly excitatory.
• Thus, ACh acting at one type of receptor can
have multiple effects on the same
postsynaptic neuron, depending on the
duration of receptor activation.

27. • Recent experiments in behaving animals have
demonstrated that cortical neurons indeed
experience both transient and persistent
changes in local acetylcholine levels during
cue-detection behaviors.

28. • In the cerebral cortex, tonic ACh inhibits layer
4 medium spiny neurons, the main targets of
thalamocortical inputs while exciting
pyramidal cells in layers 2/3 and layer 5.
• This filters out weak sensory inputs in layer 4
and amplifies inputs that reach the layers 2/3
and layer L5 excitatory microcircuits.

29. • As a result, these layer-specific effects of ACh
might function to improve the signal noise
ratio of cortical processing.
• At the same time, acetylcholine acts through
nicotinic receptors to excite certain groups of
inhibitory interneurons in the cortex, which
further dampen down cortical activity.

30. • Another theory interprets acetylcholine
neuromodulation in the neocortex as
modulating the estimate of expected
uncertainty, acting counter to norepinephrine
(NE) signals for unexpected uncertainty.

31. • Both modulations would then decrease
synaptic transition strength, but ACh would
then be needed to counter the effects of NE in
learning, a signal understood to be 'noisy'.

32. Synthesis and degradation
• Acetylcholine is synthesized in certain neurons
by the enzyme choline acetyltransferase from
the compounds choline and acetyl-CoA.
• The enzyme acetylcholinesterase converts
acetylcholine into the inactive metabolites
choline and acetate.

33. • This enzyme is abundant in the synaptic cleft,
and its role in rapidly clearing free
acetylcholine from the synapse is essential for
proper muscle function.

34. • Certain neurotoxins work by inhibiting
acetylcholinesterase, thus leading to excess
acetylcholine at the neuromuscular junction,
thus causing paralysis of the muscles needed
for breathing and stopping the beating of the
heart.

35. Synthesis cont’d
• Synthesis of acetylcholine is facilitated by the
enzyme, choline acetyltransferase (CAT).
• This enzyme combines choline with acetate
derived from acetyl coenzyme A (CoA).
• Choline is taken up into cholinergic nerves by
a high affinity transport process (sodium-
choline cotransport) that is indirectly coupled
to the energy stored by the Na/K pump
ATPase

36. • This transporter process is inhibited by
hemicholinium-3 (HC-3).
• HC-3 has no immediate effect on
neurotransmission, but can cause cholinergic
nerve fibers eventually to run out of
transmitter.
• In the presence of HC-3, the more rapidly
cholinergic fibers are stimulated, the more
rapidly they run out of ACh.

37. Acetyl coA + Choline = Acetylcholine

38. Receptors
• There are two main classes of acetylcholine
receptor (AChR), nicotinic acetylcholine
receptors (nAChR) and muscarinic
acetylcholine receptors (mAChR).
• They are named for the ligands used to
activate the receptors.

40. • Acetylcholine (ACh) has diverse actions on a
number of cell types mediated by two major
classes of receptors.
• Nicotinic receptors are ligand-gated ion
channels.
• Muscarinic receptors are part of the
transmembrane, G protein coupled receptor
family.

41. Nicotinic
• Nicotinic AChRs are ionotropic receptors
permeable to sodium, potassium, and chloride
ions.
• They are stimulated by nicotine and
acetylcholine.
• They are of two main types, muscle type and
neuronal type.

42. • The former (muscle type)can be selectively
blocked by curare and the latter (neuronal) by
hexamethonium.
• The main location of nicotinic AChRs is on
muscle end plates,
• autonomic ganglia (both sympathetic and
parasympathetic),
• and in the CNS

43. Myasthenia gravis
• The disease myasthenia gravis, characterized
by muscle weakness and fatigue, occurs when
the body inappropriately produces antibodies
against acetylcholine nicotinic receptors, and
thus inhibits proper acetylcholine signal
transmission.
• Over time, the motor end plate is destroyed.

44. • Drugs that competitively inhibit
acetylcholinesterase (e.g., neostigmine,
physostigmine, or primarily pyridostigmine)
are effective in treating this disorder.
• They allow endogenously-released
acetylcholine more time to interact with its
respective receptor before being inactivated
by acetylcholinesterase in the gap junction.

45. Muscarinic
• Muscarinic receptors are metabotropic, (act
through coupling with a G- protein) and affect
neurons over a longer time frame.
• They are stimulated by muscarine and
acetylcholine, and blocked by atropine.
• Muscarinic receptors are found in both the
central nervous system and the peripheral
nervous system, in heart, lungs, upper GI tract
and sweat glands.

46. • Extracts from the plant Deadly nightshade
included this compound (atropine), and the
blocking of the muscarinic AChRs increases
pupil size as used for attractiveness in many
European cultures in the past.

47. • Now, ACh is sometimes used during cataract
surgery to produce rapid constriction of the
pupil.
• It must be administered intraocularly because
corneal cholinesterase metabolizes topically-
administered ACh before it can diffuse into the
eye.

48. • It is sold by the trade name Miochol-E (CIBA
Vision).
• Similar drugs are used to induce mydriasis
(dilation of the pupil), in cardiopulmonary
resuscitation and many other situations.

49. Sub-Types of Nicotinic receptors
• There are two major subtypes of nicotinic
receptors;
• those found in the neuromuscular junction of
skeletal muscle (nicotinic muscle, Nm) and
• those found in autonomic ganglia and other
parts of the nervous system (nicotinic
neuronal, Nn).

50. • When ACh or other agonists occupy the
receptor site on the external surface of the
cell membrane, there is a conformational
change in the ion channel and an increase in
conductance to the ion(s) for which that
channel is selective.

51. • Thus, when Nm receptors are activated, there
is an influx of cations through the ion channel
and depolarization of the motor end plate.
• In short, nicotinic receptors rather directly
transduce the ACh external messenger into an
action on the cell.

53. Acetylcholine receptors: Muscarinic
Receptor
subtypes
Agonists Antagonists Second messenger
M1
Methach
oline
MT-7 toxin
Telenzepine
Pirenzepine
PI; G-protein
M2
Methach
oline
Triptramine
Himbacine
Methoctramine
AFDX116
G-protein:
Modulates K+
channel
cAMP: Inhibition

54. M3
Methacholi
ne
Darifenacin
HHSiD
pFHHSiD
PI; G-protein
M4
Methacholi
ne
MT-3 toxin
Tropicamide
PD102807
G-protein:
Modulates K+
channel
cAMP: Inhibition
M5
Methacholi
ne
. PI; G-protein

55. Transduction of Ach message
• Transduction of the ACh message is more
complex in the muscarinic family of receptors.
• And the family of muscarinic receptors is more
complex than the nicotinic family.
• There are at least 5 muscarinic receptor
subtypes expressed in humans.

56. • For most purposes it is sufficient to
concentrate on M1, M2 and M3 receptors.
• M1 receptors are located in autonomic ganglia
and the central nervous system.
• M2 receptors are located mainly in the
supraventricular parts of the heart.
• M3 receptors are located in smooth muscles
and glands, and on endothelial cells in the
vasculature.

57. • M1 and M3 receptors are coupled to the
enzyme phospholipase C (PLC) through a G
protein.
• When the receptor is activated the enzyme
increases splitting of phosphatidylinositol
polyphosphates of the cell membrane into
(mainly) inositol-1,4,5-trisphosphate (IP3) and
diacylglycerol (DAG).

58. • IP3 contains many charged phosphate groups
and is water soluble.
• It is thus released into the interior of the cell
and acts on IP3 receptors on the surface of the
endoplasmic (or sarcoplasmic) reticulum.
• IP3 receptors increase the release of Ca from
the ER and increased cytosolic Ca is thus part
of the intracellular message from ACh at the
surface membrane.

59. Message transduction

60. • In the example illustrated here, an M3
receptor on a smooth muscle cell promotes
smooth muscle contration by promoting
increased cytosolic free Ca ion.
• Another part of the transduced message from
ACh at the cell surface is diacylglycerol- DAG.

61. • Because DAG is lipid soluble it remains in the
cell membrane.
• Its presence in the membrane, along with
increased intracellular Ca activates a protein
kinase, protein kinase C (PKC).
• PKC is involved in turn in regulating a number
of other enzyme activities.

62. • The bottom line is that M1 and M3 receptors
generally mediate excitatory responses in
effector cells.
• Thus, M1 receptors promote depolarization of
postganglionic autonomic nerves,
• and M3 receptors mediate contraction of all
smooth muscles (an apparent exception to be
noted below) and increased secretion in
glands

63. • It is useful to remember that excess ACh levels
in the body (for example caused by inhibition
of AChE) are associated with GI cramping,
salivation, lacrimation, urination, etc.

64. • An apparent, but important, exception to the
general rule that ACh stimulates all smooth
muscle is the effect of ACh on blood vessels.
• When injected intravenously (see Virtual Lab
pathway for examples), ACh causes
vasodilation and decreased blood pressure.

65. • This is mediated by an effect of ACh on the
endothelial cells of the vasculature.
• In response to activation of M3 receptors on
the surface of the endothelial cells there is
increased intracellular Ca and activation of the
enzyme nitric oxide synthase (NOS).

66. • This results in increased synthesis of the highly
diffusable free radical, nitric oxide (NO).
• NO diffuses from endothelial cells into the
adjacent smooth muscle cells of the
vasculature.
• In those cells, NO activates the cytoplasmic
enzyme, guanylate cyclase.

67. • This increases intracellular cyclic-3',5'-
guanosine monophosphate (cyclic GMP or
cGMP), which promotes relaxation of the
vascular smooth muscle cells.

68. • Note that relaxation of vascular smooth
muscle by ACh is an indirect effect that is
utterly dependent on the presence of intact
endothelial cells.
• If the endothelium is removed, ACh exerts a
stimulatory effect on vascular smooth muscle
cells, as it does on other smooth muscle cells.

69. • The interaction between endothelial and
vascular smooth muscle cells was
demonstrated by Robert Furchgott who was
awarded the Nobel Prize for his work in this
area.

71. • M2 muscarinic receptors, in contrast to M1
and M3 receptors, tend to mediate inhibition
of cellular activity.
• They do so through G proteins that inhibit
adenylyl cyclase (opposite of the activation of
adenylyl by beta adrenergic receptors) and by
activation of K channels in the plasma
membrane.

72. • Clinically important examples of K channel
activation by ACh are especially prominent in
the supraventricular parts of the heart.
• Thus, it is important to be familiar with the
effects of ACh on the cellular
electrophysiology of the heart.

74. • This diagram shows the respective SA node,
atrial and AV nodal action potentials as they
might occur in a control situation.
• The SA node action potentials appear first in
time, because this is the site of the normal
pacemaker.
• The diastolic depolariztion that leads
smoothly into a relatively slow upstroke action
potential.

75. • This is characteristic of a pacemaker cell and a
cell with a relatively low membrane voltage.
• The slow upstroke action potential happens
because Na channels are inactivated to a
significant extent, leaving the Ca channels to
carry the 'spikes' in these nodal cells.

76. • The second tracing shows that the wave of
excitation has passed a cell in the atrium.
• The upstroke is fast showing that Na channels
were 'ready to go' in the atrial cell.
• The action potential repolarizes after some
delay, showing that K channels activate
somewhat slowly in a voltage and time
dependent manner.

77. • The bottom tracing shows that the wave of
excitation has been conducted to a cell in the
AV node.
• The electrophysiological properties of the AV
node are similar to those of the SA node.

78. • Thus AV nodal cells also show spontaneous
diastolic depolarization and slow upstroke
action potentials.
• It should not be surprising that AV nodal
rhythms are among the most common
arrhythmias.

80. • These tracings demonstrate the effects of ACh
on supraventricular cells of the heart, most
easily accomplished by stimulating the vagus
nerve.
• Activation of K channels by muscarinic
receptors brings about important changes in
the electrophysiology of supraventricular cells.

81. • This diagram shows decreased heart rate,
characteristic of vagal or ACh effects.
• The top tracing shows that K channel
activation inhibits spontaneous activity of the
SA node cells (slower spontaneous diastolic
depolarization, a greater maximal diastolic
potential [more negative and further from
threshold]).
• These effects combine to decrease heart rate.

82. • In the second tracing, atrial cells display
decreased action potential duration because
there is no 'delay' for increasing K permeability of
the membrane when the membrane becomes
depolarized (K permeability is already increased
by ACh action).
• The effect on action potential duration shortens
the atrial refractory period.
• This is critical in patients who may have atrial
flutter or fibrillation.

83. • As shown in the bottom tracing, the AV node
cells respond to ACh much as SA node cells.
• They show a decreased rate of spontaneous
diastolic depolarization and a greater maximal
diastolic potential (closer to the K equilibrium
potential).

84. • Although it is not apparent in this diagram,
the AV nodal cells also conduct more slowly
(would be seen as an increased P-R interval on
EKG) and, when appropriately tested, display
increased refractory period of the AV node.

85. • This latter phenomenon is extremely
important for limiting ventricular rate in
patients with supraventricular
tachyarrhythmias.
• Note that all of these seemingly diverse
effects of ACh are mediated by an increase in
membrane permeability to K.

86. Inactivation of acetylcholine

87. • Acetylcholine (ACh) is terminated by
hydrolysis, which is greatly accelerated by one
or more of the cholinesterase enzymes.
• Acetylcholinesterase (AChE) is present in high
concentration in cholinergic synapses.
• Butyrylcholinesterase, also known as
pseudocholinesterase is important for
hydrolyzing ACh in the circulation.

88. • It is important to recognize that the
neurotransmitter actions of acetylcholine are
terminated by a chemical reaction that forms
two products (choline and acetate) which are
essentially inactive.

89. • Diffusion of ACh from the synaptic region
plays a minor role because AChE is so active.
• By contrast, the neurotransmitter actions of
catecholamines are terminated mainly by
diffusion away from the postsynaptic
receptors; a process greatly facilitated by the
active re-uptake of the catecholamine into the
presynaptic nerve terminal.

90. AChE inhibitors
• AChE inhibitors, also designated AChEIs,
include echothiophate, edrophonium,
neostigmine, physostigmine.
• Other AChEIs include various so-called nerve
gas agents such as sarin and soman.

91. Drugs acting on the acetylcholine
system
• Blocking, hindering or mimicking the action of
acetylcholine has many uses in medicine.
• Drugs acting on the acetylcholine system are
either agonists to the receptors, stimulating
the system, or antagonists, inhibiting it.

92. ACh receptor agonists/antagonists
• Acetylcholine receptor agonists and
antagonists can either have an effect directly
on the receptors or exert their effects
indirectly, e.g., by affecting the enzyme
acetylcholinesterase, which degrades the
receptor ligand.
• Agonists increase the level of receptor
activation, antagonists reduce it.

93. Associated disorders
• ACh Receptor Agonists are used to treat
myasthenia gravis and Alzheimer's disease.

94. Direct acting-stimulants
• These are drugs that mimic acetylcholine on
the receptor. In low doses, they stimulate the
receptors, in high they numb them due to
depolarisation block.
• Acetyl l-carnitine
• Acetylcholine itself
• Bethanechol
• Carbachol

95. Stimulants- cont’d
• Cevimeline
• Muscarine
• Nicotine
• Pilocarpine
• Suberylcholine
• Suxamethonium

96. Cholinesterase inhibitors
• Most indirect acting ACh receptor agonists
work by inhibiting the enzyme
acetylcholinesterase.
• The resulting accumulation of acetylcholine
causes continuous stimulation of the muscles,
glands, and central nervous system.

97. • They are examples of enzyme inhibitors, and
increase the action of acetylcholine by
delaying its degradation;
• some have been used as nerve agents (Sarin
and VX nerve gas) or pesticides
(organophosphates and the carbamates).

98. clinical use
• They are administered to reverse the action
of muscle relaxants,
• to treat myasthenia gravis,
• and to treat symptoms of Alzheimer's disease
(rivastigmine, which increases cholinergic
activity in the brain).

99. Reversible
• The following substances reversibly inhibit the
enzyme acetylcholinesterase (which breaks
down acetylcholine), thereby increasing
acetylcholine levels.
• Many medications in Alzheimer's disease
– Donepezil
– Galantamine
– Rivastigmine
– Tacrine

100. • Edrophonium (differs myasthenic and
cholinergic crisis)
• Neostigmine (commonly used to reverse the
effect of neuromuscular blockers used in
anaesthesia, or less often in myasthenia
gravis)
• Physostigmine (in glaucoma and
anticholinergic drug overdoses)

101. • Pyridostigmine (in myasthenia gravis
• Carbamate insecticides (e.g., Aldicarb)
Huperzine A

102. Irreversible
• Semi-permanently inhibit the enzyme
acetylcholinesterase.
• Echothiophate
• Isofluorophate
• Organophosphate Insecticides (Malathion,
Parathion, Azinphos methyl, Chlorpyrifos,
among others)

103. • Organophosphate-containing nerve agents
(e.g., Sarin, VX)
• Victims of organophosphate-containing nerve
agents commonly die of suffocation as they
cannot relax their diaphragm.

104. Reactivation of acetylcholine esterase
• Pralidoxime

105. ACh receptor antagonists
• Antimuscarinic agents
• Atropine
• Ipratropium
• Scopolamine
• Tiotropium

106. • Ganglionic blockers
• Mecamylamine
• Hexamethonium
• Nicotine (in high doses)
• Trimethaphan

107. • Neuromuscular blockers
• Atracurium
• Cisatracurium
• Doxacurium
• Metocurine
• Mivacurium

108. • Pancuronium
• Rocuronium
• Succinylcholine
• Tubocurarine
• Vecuronium
• Hemicholine

109. Synthesis inhibitors
• Organic mercurial compounds, such as
methylmercury, have a high affinity for
sulfhydryl groups, which causes dysfunction of
the enzyme choline acetyltransferase.
• This inhibition may lead to acetylcholine
deficiency, and can have consequences on
motor function.

110. Release inhibitors
• Botulin acts by suppressing the release of
acetylcholine;
• where the venom from a black widow spider
(alpha-latrotoxin) has the reverse effect.

111. Other/uncategorized/unknown
• Surugatoxin