What are the 6 sequential steps in neurotransmission of cholinergic neurons?

1. Synthesis
2. Store
3. Release
4. Bind of ACh
5. degradation of neurotransmitter in synaptic cleft
6. recycle choline and acetate

*Synaptic cleft is the space between the nerve endings and adjacent receptors located on nerves or effector organs*

1. Synthesis of Acetylcholine

Choline transported from extracellular fluid into cytoplasm of cholinergic neuron by energy dependent carrier system that cotransport sodium

inhibited by drug hemicholinium

choline is permanently positive charge (do to quaternary nitrogen) thus cannot diffuse through membrane. only negative charges can

1. Synthesis of Acetylcholine

uptake = rate limiting step in ACh synthesis.

Formed by reaction of Acetyl CoA and choline (catalyzed by choline acetyltransferase)

transported into cytoplasm via sodium cotransporter

2. Storage of acetylcholine in vesicles

ACh packaged and stored into presynpatic vesicles by (via) active transport process coupled to efflux protons

contain ACh & adenosine triphosphate (ATP) & proteoglycan

2. Storage of acetylcholine in vesicles

most synaptic vesicles contain the primary neurotransmitter as well as cotransmitter that will increase or decrease the effect of primary neurotransmitter

protected from degregation in vesicles

3. Release of Acetylcholine

Action potential propagated by voltage-sensitive NA channels arrive at nerve endings,

voltage-sensitive CA channels on presynaptic membrane open, causing increase in concentration of intracellular calcium

elevated calcium levels promote fusion of synaptic vesicles with cell membrane and release in synaptic space

3. Release of Acetylcholine

blocked by botulinum toxins

ex. toxin in black widow spider venom causes all the ACh stored in synaptic vesicle to empty all contents into the synaptic gap

4. Binding to the receptor

ACh released from synaptic vesicle diffuses across synaptic space & bind to either:

2 post synaptic receptors on target cell
presynaptic receptors in the membrane of neuron that released ACh

other target presynaptic receptors

4. Binding to the receptor

Postsynaptic cholinergic receptors on surgae of the effector organs are divided into 2 classes
muscarinic & nicotinic

binding to a receptors leads to biologic response within cell
ex. initiation of a nerve impulse in postganglionic fibers
activation of specific enzyme in effector cells; mediated by second mesengers

5. Degradation of acetylcholine

Acetylcholine is rapidly hydrolyzed by acetylcholinesterase in synaptic cleft.

Acetylcholine broken down into acetate and choline via acetylcholinesterase

6. Recycling choline

recaptured by sodium-coupled, high affinity upttake system that transports the molecule back into the neuron

acetylated into ACh that stored until released by action potential

Transported back into cell where it is transformed into acetylcholine and stored

Review of the 6 steps of Cholinergic
Neurotransmission

1. Acetylcholine synthesis
• Formed by reaction of Acetyl CoA and choline (catalyzed by choline acetyltransferase) and transported into cytoplasm via sodium cotransporter

2. Storage
• Packaged into vesicles via active transport. Matured vesicles also contain proteoglycan and ATP

3. Release
• Action potential propagation triggers opening of calcium voltage-gated channels, which causes calcium influx. Increased intracellular calcium causes vesicles to fuse to cell membrane and release its contents (blocked by botulinum toxin/
black widow spider stimulates release of all contents)

4. Binding to a receptor
• Binds to a specific postsynaptic receptor, which triggers a specific biological effect

5. Degradation
• Acetylcholine broken down into acetate and choline via acetylcholinesterase

6. Recycling of choline
• Transported back into cell where it is transformed into acetylcholine and stored

2 families of cholinoreceptors. What are they

Muscarinic & Nicotinic

What are Muscarinic Receptors

Are found in autonomic effector organs such as
heart, smooth muscle and exocrine glands.
M1-M5: Neurons

M1, M2, and M3 receptors are the only ones that have been functionally characterized

M1: Gastric parietal cells (antagonist Pirenzepine)
(produce slow excitation of ganglia)

M2: Cardiac cells and smooth muscle
(decrease cardiac rate, contractile force)

M3: Exocrine glands and smooth muscle
(cause secretion, vascular relaxation and contraction of visceral smooth muscle.)

What are Muscarinic Receptors

belong to the class of G protein-coupled receptors. In addition to binding ACh recognizes muscarine

Where are the muscarinic receptors located?

found on ganglia of the peripheral nervous system

on the autonomic effector organs (heart, smooth muscle, brain, and excrine glands)

Blocked by Atropine

Mechanism of acetylcholine signal transduction

When M1 or M3 receptors are activated, receptors undergoes conformation change and interacts with G protein that inturn activates phospholipase C.

This leads to hydrolysis of phosphatidulinositol- (4,5)-bisophosphate to yield diacylglycerol and inositol (1,4,5)-trisphosphate.

inositol & and diacylglycerol are secondary messengers

inositol causes increase in intracellular CA. The cation can then interact to stimulate or inhibit enzymes or cause hyperpolarization, secretion, or contraction

Diaxylglycerol activates protein kinase C. which phosphorylates proteins within the cell

Mechanism of acetylcholine signal transduction

M2 subtype on cardiac muscle stimulate G protein which inhibits adenylyl cylcase and increase K (potassium) conductance

The heart responds with a decrease rate and force of contraction

Muscarinic agonist and antagonist

attemps are underway to develop muscarinic agonist and antagonists that are directed against specific receptor types

Pirenzepine, tricycle anticholinergic has greater selectivity for inhibiting (prevent) M1 muscarinic receptors (gastric)

Pirenzepine produced a reflex tacychardia on rapid infusion d/t blockage of M2 receptors.

Pirenzepine then is an alternative proton pump inhibitor in gastric or duodenal ulcers is questionable

Darifenacin greater affinity for M3 and used for overactive bladder.

What are Nicotinic receptors?

weak affinity for muscarine. composed of 5 subunits and its functions as a ligand-gated ion channel.

binding of 2 ACh molecules elicit a conformational change that allows entry of sodium ions, resulting in depolarization of effector cell

Nicotine at low concentration = stimulates receptor
Nicotine at high concentration - blocks the receptor

Blocked by hexamethonium.

What are Nicotinic receptors?

located in CNS, adrenal medulla, autonomic ganglia, and neuromuscular junction (NMJ)

Adverse effects observed with cholinergic drug

Diarrhea
Diaphoresis (sweating)
Miosis (contraction of pupils)
Nausea
Urinary urgency

Direct Acting Cholinergic Agonist (also known as parasympathomimetics)- mimics the effects of ACh by binding directly to cholinoceptors

These agents are classified into 2 groups called

choline esters = ACh and synthetic esters of choline (carbachol and bethanechol)

naturally occurring alkaloids = pilocarpine

Direct-Acting Cholinergic Agonist
Acetylcholine

quaternary ammonium compound that cannot penetrate membranes

lacks therapeutic importance because of its multiplicity of action (diffuse effects) and rapid inactivation by cholinesterase

No therapuetic effect

Neurotransmitter for: Parasympatetic, somatic nerve, and autonomic ganglia

ACh both muscarinic and nicotinic activities

Direct-Acting Cholinergic Agonist

What are Acetylcholines actions

1. Decrease in heart rate and cardiac output
2. decrease in BP
3. Other actions (GI)

What are Acetylcholines actions in decrease heart rate and cardiac output

mimics vagal stimulation

ACh produces decrease in cardiac rate (negative chronotrpy) and stroke volume = reduction in rate of firing of SA node

(vagal activity regulates heart by releasing ACh at SA node)

What are Acetylcholines actions in BP

causes vasodilation and lowering BP by indirect mechanism of action

activates M3 receptors found on endothelial cells lining smooth muscles of blood vessels which results in production of nitric oxide from arginine

Nitric oxide then diffuses to vascular smooth muscle cells to stimulate protein kinase G leading to hyperpolarization and smooth muscle relaxation

Atropine blocks these muscarinic receptors and prevents ACh from producing vasodilation

In all, Cholinergic receptors on the blood vessels respond by causing vasodilation.

What are Acetylcholine other actiosn

GI: increases salivary secretion and stimulates intestinal secretion and motility (enhances bronchiolar secretion)

Genitourinary tract: increases ton of detrusor urinae muscle, causing expulsion of urine

Eye: stimulating cilary muscle contraction for near vision and in the constriction of the pupillae sphincter muscle, causing miosis (constricting pupil)

Direct- Acting Cholinergic Agonist
Bethanechol

unsubstituted carbamoyl ester which acetate is replaced by carbamate, and choline is methylated (not hydrolized by AChE

lacks nicotinic and is inactivated through hydrolysis by esterases(pseudocholinesterases)

major actions are on smooth muscle musculature, the bladder, and GI tract

1 hour duration of action

Direct- Acting Cholinergic Agonist
Bethanechol actions include

stimulates muscarinic receptors = increase intestinal motility and tone

stimulates dettrusor muscle of bladder

trigone and sphincters are relaxed which increase voiding pressure and decreases bladder capacity to cause expulsion of urine (basically stimulates urination)

used for atonic bladder (to treat postoperative
urinary retention).

Direct- Acting Cholinergic Agonist
Bethanechol adverse reactions include

causes effects of generalized cholinergic stimulations:

sweating
salivation
flushing
decrease BP
nausea
abdominal pain
diarrhea
bronchospasmm

Atropine sulfate may be administered to overcome severe cardiovascular or bronchoconstrictor responses to these agents

Direct- Acting Cholinergic Agonist
Carbachol (carbamylcholine)

has muscarinic and nicotinic actions

lacks methyl group present in bethanechol

is an ester of carbamic acid and poor substrate of AChE. biotransformed but at a much slower rate

Direct- Acting Cholinergic Agonist
Carbachol (carbamylcholine)actions

effects cardiovascular and GI system, because of ganglion stimulating activity; depress these systems

release epinephrine from adrenal medulla by nicotinic actions

causes miosis and spasm of accomodation in which ciliary muscles of the eyes remain in a constant state of contraction

Direct- Acting Cholinergic Agonist
Carbachol therapeutic use

high potency, receptor nonselectivity, and long duration of action; rarely used therpauetically
except
eyes as miotic agent to treat glaucoma causing pupillary contraction and decrease intraocular pressure

action for miosis = 10 to 2o minutes.
intraocular pressure reduced fro 4-8 hours

little or no side effects due to lack of penetration (used opthalmologically)

Direct- Acting Cholinergic Agonist
Pilocarpine

tertiary amine and hydrolysis by AChE
far less potent than ACh and penetrate the CNS at therpauetic doses

exhibits muscarinic activity; used in opthamology

Direct- Acting Cholinergic Agonist
Pilocarpine action

applied in cornea; produced rapid miosis and contraction of ciliary muscle

under miosis, it experiences spasm. vision becomes fixed and distant; making it impossible to focus

most potent stimulators of secretion (secretagogue); sweat, tears, and saliva

promoting salivation in patients with zerostomia resulting from irradiation of the head and neck

Skogren syndrome, which characterizes dry mouth and lack of tears is treated with oral pilocarpine tablets and cevimeline

If you have Miosis (contraction of the the pupil) you treat with?

Pilocarpine

If you have Mydriasis dilation of the pupils) eyes are treated with?

Atropine

Direct- Acting Cholinergic Agonist
Pilocarpine therapuetic use for???

Glaucoma. treat glaucoma and is the drug of choice in emergency lowering of intraoccular pressure of both narrow angle (closed angle) and wide angle glaucoma

opens trabecular meshwork around schlemms canal; droping intraocular pressure as a result of increased drainage of aqueous humor

actions within minutes; last 4-8 hours

Wide-angle Glaucoma

Symptoms
– Gradual loss of peripheral vision, usually in both eyes
– Tunnel vision in the advanced stages

Narrow-angle Glaucoma

Symptoms
– Blurred vision
– Severe eye pain
– Redness of the eye
– Nausea and vomiting
– Sudden Sudden onset of visual onset of visual
disturbance, often in low light
– Halos around lights

Adverse effect:

enter brain and can cause CNS disturbance.
poisoning - parasympathetic effects= sweating (diaphoresis) and salivation; simular effects of consuming mushrooms

parenteral atropine cross blood brain barrier counteracts toxicity of pilocarpine

Indirect Acting Cholinergic Agonist: Acetylcholinesterase inhibitors (reversible)

Stimulate both M & N Receptors

Water Soluble
AChE is an enzyme that specifically cleaves ACh to acetate and choline which terminates its action

located both pre- and postsynaptically in nerve terminal where it is membrane bound

inhibors of AChE indirectly provides a cholinergic action by prolonging the lifetime of ACh produced endogenously at the cholinergic nerve endings; resulting in accumulation of ACh in synaptic space.

These drugs can provoke a response at all cholinoreceptors in the body; both muscarinic and nicotinic of ANS as well as NMJ

Reverse AChe vs. irreverse

Reverse: Examples
Physostigmine
Edrophonium
Pyridostigmine
Neostigmine

Short acting and intermediate acting agents

Irreversible Examples
Echothiophate
Organophosphates
Nerve gasses
Irreversible ihibitors (organophosphates) are lipid soluble.

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Edophonium

short acting inhibitor
binds reversibly to active center AChE preventing hydrolysis of ACh

rapid absorbed and has a short duration; 10-20 minutes due to rapid renal elimation

quaternary amine; limit to peripery

Edrophonium is used to diagnose what?

myasthenia gravis; autoimmune disease caused by the antibodies to the nicotinic receptors at NMJ

degradation, making fewer receptors at NMJ (neuromuscular junction)

intravenous injection causes rapid increase in muscle strength. excess drug may provoke a cholinergic crisis (atropine is the antidote)

used to assess cholinesterate inhibitor therapy by reversing the effects of nondepolarizing neuromuscular blockiers after surgery

myasthenia gravis

In myasthenia gravis the nicotinic receptors (NM) are blocked by antibodies,preventing interaction between Ach and the receptor (Ach: acetylcholine)

Caused by antibodies that block acetylcholine receptors at the post-synaptic junction, which inhibits effect of acetycholine.

causing muscle weakness
Leads to
– Facial muscle weakness
– Double vision
– Difficulty in breathing, talking,
• chewing or swallowing
– Muscle weakness in arms or legs
– Fatigue brought on by repetitive motions

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Physostigmine action

nitrogenous carbamic acid ester found naturally in plans and is a tertiary amine

substrate for AChE, forms carbamoylated intermediate with enzyme which becomes its reversibly inactive

It can enter the CNS. Increases Ach level leading to stimulation of both muscarinic and nicotinic receptors.

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Physostigmine Therapeutic use

Used to increase intestinal and bladder motility,

Produce miosis and spasm of accomodation (lower intraocular pressure)

Reduce intraocular pressure in Glaucoma. but pilocarpine is more effective

overdose of atropine,henothiazine and tricyclic
antidepressants.

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Physostigmine adverse effect

may lead to convulsion when high doses are used

Bradycardia and fall in cardiac output
inhibition of AChE at skeletal NMJ causing accumulation of ACh = paralysis of skeletal muscle

contraction of visceral smooth muscle

Reverse CNS effects of atropine and uncharged tertiary amine can penetrate CNS

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Neostigmine action

synthetic compound, a carbamic acid ester, and it reversibly inhibits AChE (similar to physostigmine)

has a quaternary nitrogen (unlike physostigmine). more polar, absorbed POORLY from GI tract and does not enter CNS

greater skeletal muscle effecct than physostigmine

stimulate contractility before paralyze

intermediate duration of actual; 30 min-2 hours

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Neostigmine Therpaeutic uses

stimulate bladder and GI tract

antidote for tubocurarine (neuromuscular blocking agent)

symptomatically treat myasthenia gravis
preserve endogenous ACh, which stimulate greater number of ACh receptors at muscle endplate

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Neostigmine Therpaeutic Adverse effects

similar to cholinergic stimulation; salivation, flushing, decreased BP, nausea, abdominal pain, diarrhea, and cronchospasm.

does not cause CNS side effects and not used to overcome toxicity of central acting antimuscarinic agents such as atropine

contraindicated when intestinal or urinary bladder obstruction

not for patients with peritonitis or inflammatory bowel disease

Indirect Acting, Inhibit AchE
Isoflurophate
Echothiophate

(Long duration of action – 1 week)
Treatment of open-angle glaucoma

Indirect Acting Cholinergic Agonist:
Acetylcholinesterase Inhibitor Reverse

Pyridostigmine and ambenonium

Pyridostigmine and ambenonium are cholinesterase inhibitors that are used in chronic management of myasthenia gravis

durations of action are intermediate (3-6 hours and 4-8 hours), longer than neostigmine

adverse effect similar to neostigmine

Tacrine, donepezil, rivastigmine, and glantamine

patients with alzheimer disease have deficiency in cholinergic neurons in CNS. This lead to the development of anticholinesterases as a remidies for loss of cognitive function

Tacrine, first available but now replaced by others because of its hepatotoxicity

donepezil and galantamine to delay the progression of alzheimers diseases, those cannot stop its progression

fist line treatment for alzheimer disease (Rivastigmine, galantamine, and donepezil)

Indirect Acting cholinergic Agonist: Anticholinesterase (irreversible):

long lasting icnrease in ACh at all sites where it is released. extremely toxic and developed by the military as nerve agents.

parathion are used as insecticides

Phosphorylate AchE. Nerve gases extremely toxic, generalized cholinergic stimulation, parlaysis of motor function, respiratory failure,
convulsions

Indirect Acting cholinergic Agonist: Anticholinesterase (irreversible):

Echothiophate mechanism of action

organophosphate that covalently binds via phosphate group to the serine-OH group at active AChE. Once this occurs, it permanently inactivates, and restoration of AChE requires synthesis of new enzyme molecules

following these modifications of AChE, the phosphorylated enzyme slowly releases one ehthyl group. Loss of alkyl group (called aging) makes it impossible for chemical reactivator such as pralidoxime to break the bond between the remaining drug and the enzyme.

pralidoxime (PAM) can remove the inhinitor

Indirect Acting cholinergic Agonist: Anticholinesterase (irreversible):

Echothiophate action

cholinergic stimulation, paralysis of motor function (causing breathing difficulties)
convulsions
produce intense miosis (contract)- therpauetic use
intraocular pressure falls from the facilitation of outflow of aqueous humor
atropine in high dosages can reverse echothiophate

Indirect Acting cholinergic Agonist: Anticholinesterase (irreversible):

Echothiophate Therapeutic use

opthalmic solution applied topically to the eye for chronic treatment of open angle claucoma.

NOT the first line agent in treatment of glaucoma.

causing cataracts

Isoflurophate (Diisopropylfluorophosphate, DFP)

AchE is permanently inactivated.

Restoration of of enzyme activity requires the synthesis synthesis of new enzyme molecules.

Used for glaucoma, effect lasts for a week.

Reactivator of Acetylcholinesterase
inhibited by organophosphates: Pralidoxime

Toxicology of Acetylcholinesterase Inhibitors

used as agricultural insecticides in US, which has led to numerous cases of accidental intoxication.

frequently used for suicidal and homicidal purposes

toxicity manifested as nicotinic and muscarinic signs and symptoms

can be peripheral or affect whole body

Toxicology of Acetylcholinesterase Inhibitors
Reactivation of acetylcholinesterase

Pralidoxime can reactivate inhibited AChE though unable to penetrate in CNS

if given before aging of alkylated enzyme occurs, it can reverse effects of echothiophate, except for those in CNS

pralidoxime is less effective, is a weak AChE inhibitor and high doses cause side effects similar to other AChE ingibitors

Toxicology of Acetylcholinesterase Inhibitors:
Other treatments

Atropine is administered to prevent muscarinic side effects:
bronchial secretion & salivation
bronchoconstriction and bradycardia

Diazepam reduces persistent convulsion caused by these agents and should measure maintenance of patients airway, oxygen supply, and artificial respiration

Excessive use of anticholinesterases produce a

cholinergic crisis
salivation
gastrointestinal cramps
lacrimation
poor vision

Use edrophonium to differentiate it from the
myasthenia gravis.