Chapter06-Acetylcholone & Serotonin

8/4/2019 Chapter06-Acetylcholone & Serotonin

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Chapter 6: Acetylcholine and Serotonin

How comes ACh is linked to nerve gases?

Knowing about how the neurotransmitters acetylcholine (ACh) and serotonin (5-HT) aresynthesized, stored, and inactivated;

Knowing about how the metabolizing process for ACh breakdown is interrupted by nervegases and the resulting toxic effect.

Knowing about organization and function of cholinergic and serotonergic systems.

Learning issues from this chapter:

Purposes of learning:

Structural features of ACh

Synthesis of ACh is done bycholine acetyltransferase

ACh is formed from two precursors: choline and acetyl coenzyme A (acetyl CoA)

To understand the roles of these transmitters in regulating neuronal signaling andmediating several brain functions;

To understand how ACh- and 5-HT-like drugs act the receptors in tissues or organs thatthey target and resulting physiological effects;

To understand pathological changes due to the dysfunction of the ACh synthesis andloss of cholinergic and serotoneric neurons.

How is serotoninergic system linked clinically to mood change and psychiatric disorders?

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ACh SYNTHESIS and STORAGE

Choline and acetyl coenzyme A (acetyl CoA) are precursors for making ACh;

The choline comes mainly from fat in our diet (choline-containing lipids), and is also producedin the liver. Acetyl CoA is generated within all cells by the metabolism of sugars and fats;

ACh is synthesized biologically by a catalyzing enzyme choline acetyltransferase (ChAT),which transfers the acetyl group (-COCH3) from acetyl CoA to choline to form ACh;

Choline acetyltransferase (ChAT) is present in the cytoplasm of the cell, and this enzyme isfound only in neurons that use ACh as their transmitter. This specificity allows us to identifycholinergic neurons by staining for ChAT;

Control of the ACh synthesis rate:The

availability of its precursors inside the cellcholinergic neurons make more ACh when morecholine and/or acetyl CoA is available;The rateof cell firing (when the neurons are stimulatedto fire at a higher rate, they make more ACh).

ACh is packaged into synaptic vesicles by thevesicular acetylcholine transporter (VAChT);

ACh is synthesized in a single step from two precursors;

VAChT can be blocked by a drug, vesamicol;

Synthesized ACh molecules that wouldnormally have been transported into the vesiclesby VAChT now remain in the cytoplasm of theterminal, what is happening?

Thus, ACh release is reduced in the presence

of vesamicol that blocks VAChT. 2


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ACh RELEASE and BREAKDOWN

Upon neuron stimulation, ACh-containing vesiclesfuse with the plasma membrane in response toelevated cytoplasmic Ca2+(see Fig. 3.5 for Ca2+ influx);

The release of ACh is dramatically affected byvarious animal and bacterial toxins;

A toxin found in the venom of the black widowspider, Latrodectus mactans, leads to a massiverelease of ACh at synapses in the PNS;

Overactivity of the cholinergic system causesnumerous symptoms, including muscle pain in theabdomen or chest, tremors, nausea and vomiting,

salivation, and copious sweating;Botulinum toxin blocks ACh release at theneuromuscular junction by preventing fusion ofsynaptic vesicles with the nerve terminal membrane;

This inhibition of cholinergic activitycan deadly lead to muscular paralysis.

What is the Neuromuscular junction?The synapse where the axon of a motornerve meets the muscle fiber plasmamembrane, thus transmitting messagesfrom the brain which cause the muscle tocontract. In vertebrates, the signal passes

through the neuromuscular junction viathe neurotransmitter ACh.

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ACh RELEASE and BREAKDOWN (continued)

Acetycholinesterase (AChE) is responsible for ACh break down

AChE breaks down the transmitter into choline and acetic acid;

Within the cell, AChE is located in several strategic locations:1) inside the presynaptic cell, where it can metabolize excess ACh that have been synthesized;

3) at neuromuscular junctions, which are specialized synapses between neurons and musclecells where ACh is released by motor neurons to stimulate muscular contraction.

2) present on the membrane of the postsynaptic cell to break down molecules of ACh after theirrelease into the synaptic cleft;

AChE is secreted by muscle cells into the space betweenmuscle cell and cholinergic nerve endings. Immediately aftera squirt of ACh causes a particular muscle to contract, thetransmitter is metabolized extremely rapidly so that themuscle can relax until the next command arrives to squirt

out some more ACh and contract that muscle once again.

Once ACh has been broken down within the synapticcleft, the liberated choline is then taken back up into thecholinergic nerve terminal by a choline transporter in themembrane of the terminal.

The choline transporter is subject to the blockade by the

drug hemicholine-3 (HC-3). As a result, the rate of AChproduction declines (why ACh is not accumulated?)

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AChE inhibitors and their clinical significance

Physostigmine (Eserine), neostigmine (Prostigmin) and pyridostigmine (Mestinon) are drugsselectively inhibit activity of AChE. The result is the reduction or blockade of the inactivation(breakdown) of ACh.

Physostigmineis able to cross the blood-brain barrier and exerts on the CNS. Poisoning byaccidentally taking (it is a compound isolated from Calabar beans, the seeds of a woody plant

found in the Calabar region of Nigeria) leads to an overactivity of central cholinergic synapsesdue to accumulation of ACh. The symptoms include slurred speech, mental confusion,hallucinations, loss of reflexes, convulsions, and even coma and death.

Therapeutic significance of neostigmine and pyridostigmine (do NOT cross the BBB) intreatment of an autoimmune neuromuscular disorder, myasthenia gravis.

Patients with myasthenia gravis develop antibodies against their own muscle cholinergicreceptors. This is an autoimmune disorder in which a part of the human body is attacked by

ones own immune system.

Normally, ACh released at neuromuscular junctions, and the muscle cholinergic receptors can

respond to the neurotransmitter to trigger contraction;

In this case, the antibodies block the muscleacetylcholine receptor, and the loss of receptorfunction causes the patients muscles to be less

sensitive to ACh, which in turn leads to severeweakness and fatigue;

By inhibitingAChE,neostigmine orpyridostigmine prolongs the action of ACh at theneuromuscular junction, which causes increasedstimulation of the remaining undamagedcholinergic receptors.

Physostigmine, neostigmine & pyridostigmineare reversible AChE inhibitors.

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AChE inhibitors and their clinical significance (continued)

Some other AChE inhibitors are irreversible (therefore it is permanent), which have beendeveloped as nerve gases to be used as chemical weapon, such as Sarin and Soman;

Symptoms of nerve gas poisoning due to rapidACh accumulation and overstimulation ofcholinergic synapses throughout both the CNSand PNS include slurred speech, mentalconfusion, hallucinations, loss of reflexes,convulsions, and even coma and death.

A reversible AChE inhibitor, pyridostigmine,is used as an antidote against Sarin or Soman.Its mechanism is said that the temporaryinteraction of pyridostigmine with the enzymeprotects AChE from permanent inactivation bythe nerve gas.

These agents are designed to be dispersed as a vapor cloud or spray, which allows their entryinto the body through skin contact or by inhalation. In either case, the drug quickly penetrates in

to the bloodstream and is distributed to all organs, including the brain.

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Organization and Function of the Cholinergic System

Cholinergic synapses are in the neuromuscular junctionsresponsible for initiating and controlling muscle contract.

Cholinergic synapses in the parasympathetic andsympathetic branches of the autonomic nervous system

Preganglionic neurons are located within the CNS and send theiraxons to the autonomic ganglia that in turn innervate target organs;

The preganglionic neurons of both branches and postganglionic

neurons of parasympathetic branches are cholinergic;The postganglionic neurons of sympathetic branches are adrenergic.

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Organization and Function of the Cholinergic System (continued)

In the brain, the cell bodies of cholinergic neurons are located primarily in the striatum,nucleus basalis, substantia innominata, medial septum, diagonal band nuclei (Basal forebraincholinergic system, BFCS), pedunculopontine tegmental nucleus and dorsolateral tegmentalnucleus.

A diffuse collection of cholinergic nerve cells called BFCS comprises the major source of adense cholinergic innevation of the cerebral cortex, hippocampus and limbic system structures.

Basal forebrain cholinergic system (BFCS)

There is evidence that the BFCS plays an important role in cognitive functioning, and damageto this system may contribute to the dementia observed in Alzheimer's disease.

The cholinergic interneurons in the striatum (basal ganglia) should play a role of balance withthe dopaminergic neurons. A neurotransmitter imbalance happened in Parkinsons disease due toneurodegeration of dopaminergic neurons.

Anatomy of cholinergicpathways in the brain

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S b f h li i Ni i i


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Subtypes of cholinergic receptors: Nicotinic

There are two major acetylcholinergic receptor subtypes

Nicotinic receptors: Named because they respond selectively to the agonist nicotine, an alkaloid found in the leavesof the tobacco plant;

It is classified to be ionotropic receptors that possess an ion channel as an integral part of thereceptor complex;

Comprise 5 subunits that come together in the cellmembrane forming the ion channel in the center. Two -subunits, each of which helps form an ACh binding siteon the receptor. Both binding sites must be occupied byACh molecules to open the nicotinic receptor channel;

When ACh binds to a nicotinic receptor, the channelopens very rapidly and Na+ and Ca2+ ions enter the neuronor muscle cell. This flow of ions causes a depolarization

of the cell membrane, thereby increasing the cell'sexcitability.

The exact proteins (subunits) that make up neuronaland muscle receptors are different. This structuraldifference leads to significant pharmacologicaldifferences between neuronal and muscle receptors.

In fact, muscle nicotinic receptors are not as sensitiveto nicotine as are the nicotinic receptors located in the

brain and autonomic nervous system. This is whysmokers only obtain the psychological effects of nicotinewithout experiencing muscle contractions or spasms.

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S b f h li i Ni i i ( i d)


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Subtypes of cholinergic receptors: Nicotinic (continued)

Nicotinic receptors mediate fast excitatory responses in boththe CNS and PNS. In the CNS, the nicotinic receptors can belocated 1)presynapticallyright on the terminals (axoaxonicsynapse). In this case, ACh binding to nicotinic receptorsenhances the release of other type transmitter from this nerve

terminal. 2) postsynapticllyon the dendrites or cell bodies ofreceiving neurons (axondendritic or axonsomatic synapse). In thiscase, activation of nicotinic receptors by ACh can stimulate cellfiring.

Nicotinic receptor desensitization

When nicotinic receptors are subject to continnuous agonist exposure, they may become desensitize,but this status will spontaneously switch toresensitization to be able to respond again toa nicotinic agonist;

Desensitization means that an altered state of the receptor in which the channel remains closedregardless of whether molecules of an agonist such as ACh or nicotine are bound to the receptor.

Depolarization block:Nicotinic stimulation maycause a persistent depolarization of the cellmembrane, in which the resting potential of the

membrane is lost and the cell cannot be exciteduntil the agonist is removed and the membranere-polarized. A chemical relative of ACh calledsuccinylcholine is AChE resistant, so it continuouslystimulates the nicotinic receptors and induces adepolarization block of the muscle cells.

Drugs producing depolarization block are good

used in certain surgical procedures where anesthesiaalone may not provide sufficient relaxation.

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S bt f h li i t M i i


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Subtypes of cholinergic receptors: Muscarinic

Muscarinic receptors are all metabotropic;

There are five different types of muscarinic receptors (designated M1 to M5), each with specificpharmacological characteristics and coded for by a different gene;

Muscarinic receptors operate through several different second-messenger systems. Some

activate the phosphoinositide second-messenger system, while others inhibit the formation ofcyclic adenosine monophosphate (cAMP).

Another important mechanism of muscarinic receptor action is the stimulation of K+ channelopening. This leads to a hyperpolarization of the cell membrane and a reduction in cell firing.

Muscarinic receptors are widely distributed in the

brain. Some areas containing high level of muscarinicreceptors are the neocortex, hippocampus thalamus,striatum, and basal forebrain. the receptors in theneocortex and hippocampus play an important role inthe cognitive effects of ACh, whereas those in thestriatum are involved in motor function.

Muscarinic receptors in the brain:

Muscarinic receptors in the PNS:

High densities in the cardiac muscle of the heartand in the smooth muscle of many organs, such asthe bronchioles, stomach, intestines, bladder, andurogenital organs. These peripheral muscarinicreceptors are activated by ACh released frompostganglionic fibers of the parasympatheticnervous system; 11

S bt f h li i t M i i ( ti d)


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Subtypes of cholinergic receptors: Muscarinic (continued)

Activation of the muscarinic receptors in the cardiac muscle cells: a slowing of heart rate anda decrease in the strength of contractiona decrease in the cardiac output;

Activation of the muscarinic receptors in the smooth muscle cells: activation of smooth musclecells, thus causing contraction of the muscle;

Activation of the muscarinic receptors in the secretory glands: produces salivation, sweating,and lacrimation (tearing).

A blockade of muscarinic receptors seen in the side effect of many drugs used for psychiatric disorders results in a

symptom called dry-mouth.

Muscarinic receptor agonists in nature and their clinical significance

Muscarinic receptor agonist-like substances are from many plants leaves or seeds. Becausetheir Ingestion mimics many of the effects of parasympathetic activation, they are called

parasympathomimetic agents.

Poisoning due to accidental ingestion of these agonist like-substances leads to exaggeratedparasympathetic responses including lacrimation, salivation, sweating, pinpoint pupils related toconstriction of the iris, severe abdominal pain and painful diarrhea due to strong contractions ofthe smooth muscles of the viscera. High doses can even cause cardiovascular collapse,convulsions, coma, and death;

The antagonist like-substances are also from nature. Since they would inhibit the actions of theparasympathetic system, they are called parasympatholytic agents;

Muscarinic antagonists have several current medical applications: pupil dilation for MiddleAges women and eye examination; secretion reduction in the patient's airways for anesthesiaprocedure during surgery; counter effect on poisoning with a cholinergic agonist, et al.

Toxic effects of muscarinic antagonists: when high doses, the CNS effects include restlessness,

irritability, disorientation, hallucinations and delirium. The worst case would be CNS depression,coma and eventually death by respiratory paralysis. 12


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S t i SYNTHESIS


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Serotonin SYNTHESIS

Serotonin is another monoamine neurotransmitter, by looking at the structure of

catecholaminesthey have a same monoamine NH2. So called 5-hydroxytryptamine (5-HT);

Structural features of serotonin:

Biochemically derived from tryptophan, that is primarily found in the gastrointestinal (GI) tract(from protein in our diet),platelets, and CNS of humans and animals;

Synthesis:

There are two steps in the biochemical pathway. The firststep is catalyzed by the enzyme tryptophan hydroxylase (TH),which converts tryptophan to 5-hydroxytryptophan (5-HTP);

5-HTP is then acted upon by aromatic amino acid decarborylase

(AADC) to form 5-HT; The conversion of tryptophan to 5-HTP is rate-limiting inthe 5-HT pathway, which means that tryptophanhydroxylaseis the rate-limiting enzyme for 5-HT synthesis;

Tryptophan hydroxylase is a specific marker for neuronsthat make 5-HT (these are called serotonergic neurons). This

because that the 2

nd

enzyme AADC is located both in thepathways of catecholamines and 5-HT syntheses;

Serotonin synthesis in the brain can be stimulated bygiving animals a large dose of tryptophan, but 5-HTPadministration is even more effective because it isconverted so rapidly and efficiently to 5-HT.

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5-HT is a well-known contributor to feelings of well-being;

so it is known as a "happiness hormone" despite not being ahormone.

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Serotonin SYNTHESIS (continued)

Pharmacological depletion of 5-HT: A way to experimentally assess the role of thisneurotransmitter in various behavioral functions;

The drug para-chlorophenylalanine (PCPA), which selectively inhibits 5-HT synthesis byirreversibly inhibiting tryptophan hydroxylase (TH). One or two high doses of PCPA can reducebrain 5-HT levels in rats 80 to 90% for as long as 2 weeks, until the serotonergic neurons makenew molecules of tryptophan hydroxylase that haven't been exposed to the inhibitor;

Cocktail amino acids: animals are given a cocktail containing a large quantity of aminoacids except for tryptophan. Because the large neutral amino acids in the cocktail inhibit entryof the remaining tryptophan into the brain, it leads to a temporary depletion of brain 5-HT.

Clinically, cocktail method is used to

demonstrate the critical role of 5-HT in moodregulation and further suggest that in patientssuccessfully treated with antidepressantmedications, symptom improvement maydepend on continued activity of theserotonergic system.

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Serotonin STORAGE RELEASE and INACTIVATION


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Serotonin STORAGE, RELEASE and INACTIVATION

The processes that regulate storage, release, and inactivation are similar for serotonin andthe catecholamines: (Release skipped)

Serotonin is transported into synaptic vesicles using the same vesicular transporter, VMAT2(vesicular monoamine transporter), found in dopaminergic and noradrenergic neurons;

Consequently, the VMAT blocker reserpine depletes serotonergic neurons of 5-HT, just as itdepletes catecholamines in dopaminergic and noradrenergic cells;

Serotonergic autoreceptors control 5-HT release in the same way as the DA and NEautoreceptors. Terminal autoreceptors directly inhibit 5-HT release, whereas other autoreceptorson the cell body and dendrites of theserotonergic neurons (somatodendriticautoreceptors) indirectly inhibit release by

slowing the rate of firing of the neurons;

Somatodendritic autoreceptors are of the5-HT1A subtype, whereas the terminalautoreceptors are either of the 5-HT1B or5-HT1D subtype, depending on the species;

In addition to being triggered by nerve cell

firing, the release of 5-HT can be stimulateddirectly by a family of psychostimulants,a series of compounds, amphetamine. Thisrelease mechanism is independent of nervecell firing (to be discussed in Chapter 11).

The inactivation process is done by thesame mechanism as NE and DA do, i.e.

Reuptake.5-HT is removed by its transporterfrom the synaptic cleft after use.

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Serotonin STORAGE RELEASE and INACTIVATION (continued)


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Serotonin STORAGE, RELEASE and INACTIVATION (continued)

5-HT transporter is a key target of some types of drug action. Inhibition of 5-HT transporterresults in an inhibition of 5-HT reuptake. The drugs to be able to inhibit this transporter iscalled selective serotonin reuptake inhibitors (SSRIs);

SSRIs has become a new class of antidepressant drugs based on the idea of inhibiting 5-HT

reuptake since late 1980s; Certain abused drugs such as cocaine and MDMA likewise interact with the 5-HT transporter,but they are not selective in their effects because they also influence the DA transporter;

The enzymaticbreakdown of 5-HT iscatalyzed by monoamine oxidase (MAO,that metabolizes DA and NE) to yield themetabolite 5-hydroxyindoleacetic acid (5-HIAA). The level of 5-HIAA in the brains ofanimals or in the cerebrospinal fluid ofhumans or animals is often used as ameasure of the activity of serotonergicneurons;

This is because when serotonergic

neurons fire more rapidly, they make more5-HT and there is a corresponding increasein the formation of 5-HIAA.

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Organization and Function of the Serotonergic System


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Organization and Function of the Serotonergic System

The serotonergic system originates from cell bodies in the brain stem where the 5-HT-containing

cells groups (serotonergic nuclei) are named with B and projects to all forebrain areas;

Serotonergic cell body groups inraphe nuclei of human brainstem andtheir axon projections

(Median raphe nucleus)

Almost all of the serotonergic neurons in the CNS arefound along the midline of the brainstem(medulla,pons, and midbrain), these groups are called raphe

nuclei.

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Organization and Function of the Serotonergic System (continued)


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Organization and Function of the Serotonergic System (continued)

The firing rate of serotonergic neurons inthe cat dorsal raphe

The raphe nuclei at different areas participate in different modulatory effects includingpsychiatric behaviors, motor and sensory processing, as well as pain sensation.

Dorsal raphe and median raphe: studies by aresearch group suggest that serotonergicneurons in these nuclei modulate motor and

sensory coordination by facilitating the outputof motor systems while simultaneouslyinhibiting sensory processing;

Nuclei of raphe magnus and pallidus (B1/B2):5-HT-containing nerve cell bodies in thesetwo nuclei send their axon projections to the

spinal cord dorsal horn synapsing with thesensory neurons for mediating the painsensory input from the periphery.

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Serotonergic Receptors


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Serotonergic Receptors

There is a large family of serotonin receptors, most of which are Metabotropic;

At least 15 5-HT receptor subtypes have been identified. Among these is a large family of 5-HT1 receptors (that is, 5-HT1A, 5-HT1B, and so forth), a smaller family of 5-HT2 receptors, andadditional receptors designated 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7;

AII of the above receptors are metabotropic, except for the 5-HT3

receptor, which is anexcitatoryionotropic receptor;

we will focus on the 5-HT1A and 5-HT2A receptors, which are the best-known serotonergicreceptors in terms of their cellular and behavioral effects.

5-HT1A receptors

Found in many brain areas, but they are particularly concentrated in the hippocampus, theseptum, parts of the amygdala, and the dorsal raphe nucleus;

These receptors are located 1) postsynaptically to 5-HT-containing nerve terminals in theforebrain; 2) at presynaptically to somatodendritic synapses to function as autoreceptors in thedorsal and median raphe nuclei;

5-HT1A receptors work through two majormechanisms.

1) the receptors reduce the synthesis ofcAMP by inhibiting adenylyl cyclase;

Increase opening of K+ channels andmembrane hyperpolarization leading to adecrease in firing of either the postsynapticcell (in the case of 5-HT1A receptors locatedpostsynaptically) or the serotonergic neuron

itself (in the case of the 5-HT1A autoreceptors). 20

Serotonergic Receptors (continued)


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Serotonergic Receptors (continued)

A number of drugs act as 5-HT1A receptor agonists are listed. Administration of a 5-HT1Areceptor agonist produces several behavioral and physiological effects in animals.

Hyperphagia (overeating): This effect is thought to be due to stimulation of the 5-HT1Aautoreceptors,thereby inhibiting the release of 5-HT to reduce activity of serotonergic neurons and in the forebrain.

5-HT1A receptor activation produces anxiolytic, both in humans and in animal models of anxiety;

Serotonin acting postsynaptically tends to reduce appetite and food intake in both animals andhumans, but stimulation of serotonergic autoreceptors by a 5-HT1A agonist would lead to increasedappetite and overeating because it inhibits the release of 5-HT (using a specific 5-HT1A agonist);

Administration of a 5-HT1A agonist inhibits alcohol consumption, the possibility that suchcompounds might provide some therapeutic benefit in the treatment of alcoholism?

Activation of 5-HT1A receptors located in spinal dorsal horn sensory neurons produces analgesia

reducing pain sensation).5-HT2A receptors

Large numbers of 5-HT2A receptors are present in the cerebral cortex. This receptor subtype isalso found in the striatum, nucleus accumbens and a varietyof other brain areas.

5-HT2A receptors function mainly by activating thephosphoinositide second messenger system (thatactivates PKC), and elevates Ca2+ levels within thepostsynaptic cell.

5-HT2A agonists are hallucinogenic (hallucinationproducing) in humans;

Blockade of 5-HT2A receptors has become a major topic ofdiscussion and research with respect to the treatment of

schizophrenia. New class of anti-schizophrenia drug --- 5-HT2Aantagonist. 21


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