Download experiments with enzymes involved in neurotransmission

Department of Biochemistry and Molecular Biology
Faculty of Medicine, University of Debrecen
BIOCHEMISTRY PRACTICE
EXPERIMENTS ON ENZYMES
INVOLVED IN
NEUROTRANSMISSION
theoretical background
Krisztina Köröskényi, PhD
2016
TABLE OF CONTENTS
ABREVIATIONS
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THEORETICAL BACKGROUND
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1. Neuronal communication, neuronal synapse
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2. Neurotransmission and chemical synapse
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3. Neurotransmitters
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3.1. Acetylcholine
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3.1.1. Cholinesterases
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3.1.2. Determination of pseudocholinesterase (PChE) activity
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3.1.3.Drugs acting on cholinergic system
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3.1.4. Clinical relevance of measuring cholinesterase activity
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3.2. Monoamine neurotransmitters
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3.2.1. Monoamine oxidase enzymes
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3.2.2. Determination of monoamine oxidase activity
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3.2.3. Clinical relevance of monoamine oxidases
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ABREVIATIONS
4-AA
4-aminoantipyrine
acetyl-CoA
acetyl coenzyme A
ACh
acetylcholine
AChE
acetylcholinesterase
CNS
central nervous system
DTNB
5, 5’-dithio-bis-2-nitrobenzoate
FAD
flavin adenine dinucleotide
GABA
gamma-Aminobutyric acid
mAChR
muscarinic acetylcholine receptor
MAO
monoamine oxidase
nAChR
nicotinic acetylcholine receptor
PChE
pseudocholinesterase (butyrylcholinesterase)
TNB
5-thio-2-nitrobenzoic acid
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ENZYMES INVOLVED IN NEUROTRANSMISSION
THEORETICAL BACKGROUND
1. Neuronal communication, neuronal synapse
Neurons communicate with each other via synapses, where the axon terminal of one cell
impinges upon another neuron's dendrite, soma or, less commonly, axon. The human brain
has a huge number of synapses. Each of the 1011 (one hundred billion) neurons has on average
7,000 synaptic connections to other neurons. It has been estimated that the brain of a threeyear-old child has about 1015 synapses (1 quadrillion). This number declines with age,
stabilizing by adulthood. Synapses can be excitatory or inhibitory and either increase or
decrease activity in the target neuron, respectively. There are two fundamentally different
types of synapses: electrical and chemical synapses.
ELECTRICAL SYNAPSE
CHEMICAL SYNAPSE
 the pre- and and postsynaptic cell membranes
 electrical activity in the presynaptic neuron is
are connected by gap junctions that are
converted into the release of a
capable of passing electric current, causing
neurotransmitter that binds to receptors
voltage changes in the presynaptic cell to
located in the plasma membrane of the
induce voltage changes in the postsynaptic
postsynaptic cell.
cell.
 Because of the complexity of receptor signal
 The main advantage of an electrical synapse is
transduction, chemical synapses can have
the rapid transfer of signals from one cell to
complex effects on the postsynaptic cell.
the next.
Table 1. Types of neuronal synapses
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2. Neurotransmission and
chemical synapse
Neurotransmission is the process by which
neurotransmitters
are
released
by
a
presynaptic neuron, and bind to and
activate the receptors of the postsynaptic
Figure 1. The structure of chemical
synapse (from Wikipedia)
neuron.
Stages in neurotransmission at the synapse
1. Synthesis of the neurotransmitter: can take place in the cell body, in the axon, or in
the axon terminal.
2. Storage of the neurotransmitter: in granules or vesicles in the axon terminal.
3. Release of the neurotransmitter: in response to a threshold action potential or graded
electrical potential voltage-gated Ca2+ channels open, allowing Ca2+ ions to enter into
the axon terminal. Ca2+ enters the axon terminal, during an action potential, causing
release of the neurotransmitter into the synaptic cleft.
4. Receptor binding: after its release, the neurotransmitter diffuse across the synaptic
cleft, bind and activate receptors on the postsynaptic neuron. Binding of
neurotransmitters may influence the postsynaptic neuron in either an inhibitory or
excitatory way. The binding of neurotransmitters to receptors in the postsynaptic
neuron can trigger either short term changes, like changes in the membrane potential
called postsynaptic potentials, or longer term changes by the activation of signaling
cascades.
5. Termination: after a neurotransmitter molecule binds to a receptor molecule, it must
be removed to allow for the postsynaptic membrane to continue to relay subsequent
postsynaptic activity. This removal can happen through one or more processes:
 The neurotransmitter may diffuse away
 Enzymes bound to the subsynaptic membrane may inactivate/metabolize the
neurotransmitter.
 Reuptake by pumps which actively transport the neurotransmitter back into the
presynaptic axon terminal.
Chemical synapses can be classified according to the neurotransmitter released: glutamatergic
(often excitatory), GABAergic (often inhibitory), cholinergic (e.g. vertebrate neuromuscular
junction), and adrenergic (releasing norepinephrine).
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3. Neurotransmitters
Neurotransmitters are endogenous chemicals that transmit signals across a synapse or junction
from one neuron (nerve cell) to another "target" neuron, muscle cell or gland cell. More than
100 chemical messengers have been identified. Their common classification and the major
neurotransmitters: are listed in Table 2.
GROUP
NEUROTRANSMITTERS
AMINO ACIDS
glutamate, aspartate, GABA (γ-aminobutyric acid), glycine
AMINES
MONOAMINES dopamine, serotonin (5-HT), norepinephrine, epinephrine, histamine
TRACE AMINES
phenethylamine, tryptamine, tyramine, 3-iodothyronamine, etc.
PEPTIDES
somatostatin, substance P, opioids,
CART (cocaine and amphetamine regulated transcript)
PURINES
ATP (adenosine triphosphate), adenosine
GASES
CO (carbon monoxide), H2S (hydrogen sulfide), NO (nitric oxide)
IONS
zinc
OTHERS
acetylcholine (ACh), anandamide, etc.
Table 2. Main chemical classes of neurotransmitters
The most prevalent transmitter is glutamate, which is excitatory at well over 90% of the
synapses in the human brain. The next most prevalent is GABA, which is inhibitory at more
than 90% of the synapses that do not use glutamate.
3.1. Acetylcholine
Acetylcholine (ACh) was the first neurotransmitter discovered in the nervous systems. It is a
major neurotransmitter in the autonomic nervous system which also acts in the peripheral
nervous system and central nervous system (CNS); cholinergic system, which tends to cause
inhibitory actions. ACh is the only neurotransmitter used in the neuromuscular junction
connecting motor nerves to muscles and the inhibition of it’s effect causes paralysis of the
muscles needed for breathing and stopping the beating of the heart. The arrow-poison curare
acts by blocking transmission of cholinergic synapses (by the inhibition of nicotinic
acetylcholine receptors). Botulin acts by suppressing the release of ACh, whereas the venom
from a black widow spider has the reverse effect (the wastage of ACh supplies and the
muscles begin to contract).
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ACh is synthesized in certain neurons by the enzyme choline acetyltransferase from the
compounds choline and acetyl-CoA. Cholinergic neurons are capable of producing
Cholinesterase enzymes convert ACh into the inactive metabolites choline and acetate. ACh
uses different types of receptors, including nicotinic and muscarinic receptors (Table 3).
NICOTINIC ACh RECEPTOR
(nAChR)
MUSCARINIC ACh RECEPTOR
(mAChR)
 metabotropic receptor
 stimulated by nicotine and ACh
 ionotropic receptors permeable to Na+, K+ and
Ca2+ ions
 two main types, muscle-type and neuronal-type
 main location: muscle end plates, autonomic
ganglia, CNS
 affect neurons over a longer time frame
 stimulated by muscarine and ACh
 location: CNS, peripheral nervous system
of the heart, lungs, upper gastrointestinal
tract, and sweat glands
Table 3. The main features of muscarinic and nicotinic acetylcholine receptor
3.1.1. Cholinesterases
In biochemistry, cholinesterase is a family of enzymes that catalyze the hydrolysis of the
neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a
cholinergic neuron to return to its resting state after activation.
Figure 2. Cholinesterase reaction
There are two separate cholinesterase enzymes in the body: (1) acetylcholinesterase, found in
red blood cells as well as in the lungs, spleen, nerve endings, and the gray matter of the brain,
and (2) pseudocholinesterase (butyrylcholinesterase), found in the serum as well as the liver,
muscle, pancreas, heart, and white matter of the brain. The difference between the two types
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of cholinesterases: the former hydrolyses acetylcholine more quickly; the latter hydrolyses
butyrylcholine more quickly.
Acetylcholinesterase (AChE) found primarily in the blood, on red blood cell membranes, in
neuromuscular junctions, and in neural synapses. AChE, located on the post-synaptic
membrane, terminates the signal transmission by hydrolyzing ACh. The liberated choline is
taken up again by the pre-synaptic nerve and ACh is resynthetized by combining with acetylCoA through the action of choline acetyltransferase. Loss of AChE activity (genetic
abnormalities or enzyme inhibition) leads to accumulation of ACh in the synaptic cleft and
results in impeded neurotransmission, muscle paralysis, seizures and death.
Pseudocholinesterase (also known as plasma cholinesterase, butyrylcholinesterase, PChE)
is a non-specific cholinesterase enzyme that catalyzes the hydrolysis of many different
choline esters. It’s physiological role is unclear, PChE-deficient individuals have no
physiological abnormalities. In contrast to the physiological processes the enzyme plays an
important role in pharmacology and toxicology: it is involved in the catabolism of ester-based
local anaesthetics (e.g. succinylcholine, procaine), heroin, and cocaine. PChE deficiency
lowers the margin of safety and increases the risk of systemic effects of this type of drugs.
3.1.2. Determination of pseudocholinesterase (PChE) activity
Since the neural form of acetyl choline esterase is not easily accessible, in the lab serum
activity of PChE is measured. The substrate used in the assay system is a synthetic compound
butyrylthiocholine iodide. It is used as a tool to distinguish between AChE and PChE.
PChE
activity
is
measured
kinetically, based on the in vitro
reaction summarized in Figure
3. The substrate is hydrolyzed
into thiocholine and butyrate by
PChE.
Thiocholine
forms
mercaptan, which reacts with
oxidizing agent 5,5’-dithio-bis2-nitrobenzoate
(DTNB)
to
form yellow product (TNB, 5Figure 3. Colorimetric PChE activity measurement
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thio-2-nitrobenzoate), which
has a maximum absorption arround 410 nm. In the practice, the activity of PChE is measured
by following an increase in absorbance at 405 nm.
3.1.3. Drugs acting on cholinergic system
Blocking, hindering or mimicking the action of acetylcholine has many uses in medicine.
Drugs acting on the ACh system are either agonists (e.g.Carbachol, Muscarine, Nicotine), to
the receptors, stimulating the system, or antagonists (e.g. Atropine, Scopolamine,
Hexamethonium, Pancuronium, Rocuronium), inhibiting it. These compounds can either have
an effect directly on the receptors or exert their effects indirectly, e.g., by affecting the
enzyme cholinesterase.
Promethazine is a neuroleptic medication and firstgeneration antihistamine of the phenothiazine family.
The drug has strong sedative and weak antipsychotic
effects. It also reduces motion sickness and has
antiemetic and anticholinergic properties. The main
pharmacological targets of promethazine are H1
Figure 4. Medicines containing
promethazine
histamine receptors (antihistaminic activity) and D2
dopamine receptors (sedative and antiemetic actions).
In addition, antiadrenergic, antiserotonine and anticholinergic effects are also known. The
latter one is mediated by strong inhibition of M1 muscarinic AChR. Promethazine - similarly
other phenothiazine derivatives -has ability to inhibit human cholinesterases (both AChE and
PChE), especially at high substrate concentartion range.
AChE inhibitors are present in various fields of life: they are components of venoms and
poisons, used as chemical weapons and insecticides and are common tools of medicine.
In clinical use, they are administered to reverse the action of muscle relaxants, to treat
myasthenia gravis, glaucoma, and to treat cognitive (memory and learning deficits mostly)
symptoms of CNS diseases like Alzheimer's disease, schizophrenia, autism and dementia.
Reversible AChE inhibitors (e.g. carbamates, Neostigmine, Physostigmine) – which are
degraded within a few hours - have been used for medical purposes.. The following
substances are reversible AChE inhibitors: Neostigmine (commonly used to reverse the effect
of neuromuscular blockers used in anaesthesia, or less often in myasthenia gravis),
Physostigmine (in the treatment of glaucoma and anticholinergic drug overdoses)., Caffeine
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(noncompetitive inhibitor). It has also been shown that the main active ingredient in
cannabis, tetrahydrocannabinol, is a competitive inhibitor of AChE.
Irreversible inhibitors semi-permanently inhibit AChE. The usage of them may lead to
muscular paralysis, convulsions, bronchial constriction, and death by asphyxiation. These –
mainly organophosphate-containing - compounds have been used in insecticides (e.g.
Malathion, Parathion) and nerve gases for chemical warfare (e.g., Sarin, Soman, Tabun, VX
gas). Victims of organophosphate-containing nerve agents commonly die of suffocation, as
they cannot relax their diaphragm.
3.1.4. Clinical relevance of measuring cholinesterase activity
The normal range of PChE activity in humans: 3500-8500 U/L.
There are two most common reasons for testing activity levels in the blood:
1. In testing for acute pesticide exposure/poisoning: testing AChE and PChE may be
done to detect acute poisoning or to monitor those with occupational exposure to
these chemicals, such a farm workers or those who work with industrial chemicals.
Following exposure to organophosphate compounds, AChE and PChE activity can
fall to about 80% of normal before any symptoms occur and drop to 40% of normal
before the symptoms become severe. In addition, PChE administration is currently
the only therapeutic tool effective in providing complete protection against the entire
spectrum of organophosphate nerve agents.
2. In testing for succinylcholine sensitivity: About 3% of people have low activity
levels of PChE due to an inherited deficiency and will have prolonged effects from
the muscle relaxant succinylcholine. Total quantitative PChE levels will be evaluated
prior to surgery for patients with a history or family history of prolonged apnea after
use of this drug. Low activity levels of PChE levels indicate that these people may be
at increased risk of experiencing prolonged effects of the muscle relaxant.
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3.2. Monoamine neurotransmitters
Monoamine neurotransmitters are neurotransmitters and neuromodulators that contain one
amino group that is connected to an aromatic ring by a two-carbon chain (-CH2-CH2-). All
monoamines are derived from aromatic amino acids like phenylalanine, tyrosine, tryptophan,
and the thyroid hormones by the action of aromatic amino acid decarboxylase enzymes.
Monoaminergic systems (neuronal networks utilizing monoamine neurotransmitters), are
involved in the regulation of cognitive processes such as emotion, arousal, and certain types
of memory. Drugs used to increase (or reduce) the effect of monoamine may be used to treat
patients with psychiatric disorders, including depression, anxiety, and schizophrenia.
Classical
monoamines
are:
histamine,
catecholamines
(adrenaline/epinephrine,
noradrenaline/norepinephrine, dopamine) and tryptamines (serotonin, melatonin).
Specific transporter proteins called monoamine transporters that transport monoamines in or
out of a cell exist.
After release into the synaptic cleft,
monoamine neurotransmitter action
is ended by reuptake into the
presynaptic
terminal
(repackaged
into synaptic vesicles) or degraded
by the enzyme monoamine oxidase
(MAO).
Figure 4. Monoamine oxidase reaction
3.2.1. Monoamine oxidases
L-monoamine oxidases belonging to the protein family of amine oxidoreductases are found
bound to the outer membrane of mitochondria in most cell types in the body. They catalyze
the oxidative deamination of monoamines (Figure 4.). Oxygen is used to remove an amine
group, resulting in the corresponding aldehyde and ammonia. Monoamine oxidases contain
the covalently bound cofactor FAD and are, thus, classified as flavoproteins.
In humans there are two types of MAO: MAO-A and MAO-B. MAO-A generally
metabolizes tyramine, norepinephrine, serotonin, and dopamine. In contrast, MAO-B mainly
metabolizes dopamine.
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MONOAMINE OXIDASE A
(MAO-A)
 neurons, astroglia
TISSUE
DISTRIBUTION
FUNCTION
SUBSTRATE
SPECIFICITY
 liver, pulmonary vascular
endothelium, gastrointestinal
tract, placenta
MONOAMINE OXIDASE B
(MAO-B)
 neurons, astroglia
 platelets
 catabolism of monoamines
ingested in food
 inactivation of monoaminergic
neurotransmitters
 inactivation of monoaminergic
neurotransmitters
 serotonin, melatonin, adrenaline
and noradrenaline
 Phenethylamine and benzylamine
 both forms break down dopamine, tyramine, and tryptamine equally
Table 4. The main features of monoamine oxidases
3.2.2. Determination of monoamine oxidase activity
There are different methods for the measurement of MAO activity. The quantification of the
aldehyde compound formed (using e.g. isotope techniques) or the determination of the
hydrogen peroxide release are widely used methods. During the practice we use the latter one.
Figure 5. Colorimetric MAO activity measurement
Benzylamine substrate is metabolized into benzaldehyde by MAO. Hydrogen peroxide, which
is released in the course of the reaction in stoichiometric amount, is used to oxidize 4aminoantipyrine (4-AA) with a peroxidase helper enzyme. The oxidized product forms a red
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compound with phenol (Trinder-reaction) and the absorbance of which is then measured
spectrophotometrically. The breakdown of hydrogen peroxide by catalase is blocked by the
use of sodium azide.
3.2.3. Clinical relevance of monoamine oxidases
While people lacking the gene for MAO-A display mental retardation and behavioral
abnormalities, people lacking the gene for MAO-B display no abnormalities except elevated
phenethylamine levels in urine, raising the question of whether MAO-B is actually a
necessary enzyme.
Genetic studies focusing on MAO-A polymorphism revealed that high-activity MAO-A
variants are associated to major depressive disorders and violance, while low activitiy of
MAO-A is linked to autism.
Alzheimer's disease and Parkinson's disease are both associated with elevated levels of MAOB in the brain. MAO-B levels have been found to increase with age, suggesting a role in
natural age related cognitive decline and the increased likelihood of developing neurological
diseases later in life. The prophylactic use of MAO-B inhibitors to slow natural human aging
in otherwise healthy individuals has been proposed, but remains a highly controversial topic.
Polymorphisms of the MAO-B gene have been linked to negative emotionality, and suspected
as an underlying factor in depression.
The differences between the substrate selectivity of the two enzymes are utilized clinically
when treating specific disorders: MAO-A inhibitors have been typically used in the
treatment of depression, and MAO-B inhibitors are typically used in the treatment of
Parkinson's disease.
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