Pharm 1 Test 2 Ch 8-10
I. Differences between Somatic/Voluntary & Autonomic/Involuntary
II. Receptor & Neuron Organization in the Peripheral ANS
A. The anatomy of a synapse (review)
1. Both excitatory and Inhibitory neurons carrying Na mediated AP respond to Ca2+ influx to
affect vesicular exocytotic release. They are Ca2+ obligates.
a) excitatory vesicular release enables Na channels on the post synaptic target
b) inhibitory vesicular release
(1) enables Cl- channels, hyperpolarizing the post synaptic target
(2) or causes the efflux of K form the post synaptic target (creating same hyper
polarization
2. Ca2+ can also influx to post synaptic target causing excitation (raising membrane potential
with it's positive charge
B. Sympathetic
1. Gross anatomy (CNS to PNS)
2. Neuron characteristics
a) sympathetic neurons can have many types of receptors depending on where they are in
the body, so different chemicals or xenobiotics can affect these receptors that may be
G coupled, which would then cause action or inhibition of the neurotransmitter that's
within that neuron (typically norepi, since this is the principle adrenergic
neurotransmitter), in addition to having their own specific effects.
(1) this is part of what makes up a side effect profile
(2) one example of this are adenosine receptors on presynaptic dopaminergic neurons
(a) Giving adenosine that could bind to purinergic receptors on dopaminergic
neurons causes exocytosis
(b) NOT just of NE... because those same vesicles also contain ATP and
neuropeptide Y
(c) which means that drug therapy has all these layers of cause and effect.
(3) Part of the receptor diversity are those receptors that would be targets for the other
vesicular compounds... just as there are α2 and ß2 pre and post synaptic receptors
responding to the primary adrenergic neurotransmitter, norepinephrine, there are pre
and post receptors for the other components in those vesicles (neuropeptide Y and
ATP). Exactly what receptors are there depends on where the neuron is in the body.
b) typically release NE to target receptors
(1) adrenal medulla exception
(2) post gang Ach release to sweat glands exception
3. Synapse Physiology... Making & regulating catecholamines
a) Synthesis is more complicated & yields our 2 major catecholamines Epinephrine
(released systemically as a hormone) and Norepinephrine (released locally)
(1) starts with Tyrosine hydroxylation into L-Dopa by the enzyme tyrosine-3monooxygenase (tyrosine hydroxylase) at the 3 Carbon of the benzene ring.
Tyrosine and L Dopa are both "cathechols."
(a) requires tetrahydrobiopterin as a cofactor
(2) Dopamine is made when L Dopa becomes decarboxylated by the enzyme aromatic
L amino acid decarboxylase.
(a) the carboxylic acid group is removed as CO2 and the H stays behind, stays
bound to the α Carbon
Generalized enzymatic
reaction in any
dopaminergic neuron
Enzymatic action
specific to sympathetic
neurons
Enzymatic action
specific to Adrenal
Medulla
(b) this is a general enzyme is expressed in nearly all cells
(c) it will decarboxylate any L amino aromatic amino acid
(d) this synthesis process for Dopamine is the same in any dopaminergic neuron like in the Substantia Nigra of the basal nuclei
(e) requires pyridoxal phosphate as a cofactor
(3) The enzyme dopamine ß hydroxylase converts Dopamine into Norepinephrine
replacing a Hydrogen on the ß carbon with a hydroxyl group
(a) this enzyme is specifically expressed in sympathetic nerves because of the
genes in this neuron
(b) requires the antioxidant ascorbate as a cofactor, to capture the Oxygen radical
that forms when the hydroxyl group replaces the plain old Hydrogen
(4) Finally, Epinephrine is made when one of the Hydrogen's on the Nitrogen is
replaced with a methyl group by the enzyme phenylethanolamine N methyl
transferase
(a) this enzyme is specific to the adrenal medulla
(b) requires the cofactor S-adenosylmethionine
b) Whether or not all synaptic components are involved depends on where int he body
which catecholamine is being produced
(1) 2ndary active transport via "Na+ driven transporter" in axon membrane transports
Tyrosine into the axon terminus against it's concentration gradient
(2) Tyrosine is converted to L Dopa and then to Dopamine in the cytoplasm of the
neuron by their enzymes
(3) Dopamine is taken up into the vesicle by a V Type transporter & inside the vesicle,
the DßH enzyme converts it to Norepinephrine
(a) DßH is in that vesicle because way back when it was transcribed in the rough
ER, it was tagged as "that kind of a protein," and was packaged thusly.
(b) If this process gets fucked up, the vesicles will all have Dopamine, and not
NorEpi...
(c) Reserpine is a drug that inhibits vesicular reuptake
(4) Vesicle awaits Ca2+ influx from arriving AP and then moves to the membrane and
spills NE via exocytosis.
(5) NE moves across the synaptic cleft and interacts with α or ß receptors, depending
on the tissue
c) Regulation
(1) there are no post synaptic membrane enzymes (like AChE)
(2) but there are multiple ways that the affects of NE are modulated (be able to list
these in a
(a) presynaptic reuptake transporters that take the whole compound back into the
presynaptic neuron to 2 different fates:
i) NE can be reuptook into the vesicle
ii) NE can be metabolized by MonoAmineOxidase (MAO), which works by
aliphatic deamination, which inactivates it.
(b) BLOCKING PRESYNAPTIC REUPTAKE OF NE by Cocaine at this transporter
causes euphoria due to increase of NorEpi. Locally it causes profound
vasoconstriction.
(c) NE can also engage an α2 receptor presynaptically
i) binds with presynaptic α2 receptor
ii) α2 receptors are coupled to Gi, just like the muscarinic receptors that are
affected by ACh... so they cause the same presynaptic negative feedback
inhibition with an agonist, which we found out because...
iii) phentolamine (nonspecific α1 α2 receptor antagonist) administration showed
no contraction but a five fold increase in synaptic NE - because the only α1
receptors were post synaptic. This is how Precedex works. It's a selective α
2 agonist, which inhibits the presynaptic exocytotic release.
iv) this also means that a "α2 antagonist will increase NE levels." This sounds a
little fishy to me when worded this way. Abstracts I read posit that α2
blockers partially increase NE because they "inhibit the negative feedback or
Norepinephrine release" but also because they are antagonizing the
postsynaptic α2 receptors as well. The latter makes more sense to me
because - first of all antagonists shouldn't do the opposite (like an inverse
agonist), second an antagonist at an α2 Gi coupled protein... well, it would
just nullify the Gi activity right? so the presynaptic neuron would simply be
at the mercy of regular Ca2+ influx... it wouldn't have INCREASED Ca2+
influx... This abstract was from 1997 - have we found the answer?
(d) NE can engage in ß2 receptor presynaptic positive feedback
i)
NE binds with presynaptic ß2 receptors, which are coupled to Gq, which do
cause an increase in intracellular Ca2+, either from the ER, SR or plasma
influx. Comparing this "up" modulation to the α2 antagonist "up"
modulation listed above, this seems far more likely and more accurate when
assessing the drawing.
(e) for all these neurotransmitters, there is also simple diffusion away from the
synaptic cleft...
d) Adrenergic metabolism happens in 2 places - one of which is by starting with MAO
breaking apart the amine from the NorEpi in the presynaptic neuron
(1) the cytoplasmic side of the outer membrane surface of the mitochondria is
specifically where the MAO is
(2) MAO will metabolize any monoamine
4. Metabolism also occurs in the liver as released NorEpi diffuses away
5. Receptors
a) α receptors
b) ß receptors
C. Parasympathetic
1. CNS to PNS
2. neuron characteristics
3. Synapse Physiology... Making & regulating Acetylcholine
a) cholinergic Ach synthesis and regulation is pretty simple (compared to sympathetic)
b) 2ndary active transport via "Na+ driven transporter" in axon membrane transports
Choline into the axon terminus against it's concentration gradient
(1) Na K ATPase exchanges 3 Na out for 2K in to correct Na symport above
c) Choline combines with AcetylCoA under the influence of Choline Acetyl Transferase
Enzyme
(1) AcetylCoA is an activated carrier of acetate groups (just as ATP is an activated
carrier of phosphate), Acetyl CoA can transfer the acetate portion with the help of
an enzyme
(2) to make Acetylcholine
d) Formed Acetylcholine is transported (against it's concentration gradient) into a vesicle
using a V type ATPase
e) vesicle awaits signal of increased Ca2+ which creates the impetus for the exocytotic
release of Ach into the synaptic cleft
(1) P type ATPase will pump Ca2+ back out against it's gradient
f) Once Ach is released...what happens to it
(1) The electronic AP has now been converted to a chemical mediator - and there's a
bunch of it (the Ach) in the synaptic cleft that needs to be regulated
(2) Different mechanisms:
(a) Ach in the synapse is metabolized by Acetylcholinesterase (most significant
mechanism)
i) AChE is embedded on the post synaptic membrane and is a serine protease
ii) it has a high Km, metabolizes very quickly via 2 phases (like any serine
protease):
(1) breaks the covalent bond between Choline and acetate, releasing the
choline
(2) then water comes in and breaks the bond between the serine and the
acetate, releasing acetate and regenerating the serine proteases ability to
work
(b) Presynaptic Negative Feedback Inhibition - Released ACh also activates a
presynaptic muscarinic receptor which creates a decrease in exocytotic release
of ACh.
i) M receptor is activated because a concentration gradient exists once all that
ACh is released.
ii) Once ACh binds to the muscarinic receptor, it's G protein to which it's
coupled (which is a Gi protein) then does it's G protein thing - in this case,
because it's Gi, decrease cAMP, shut down Ca2+ influx and cause K efflux
iii) this is how it causes a "decrease in exocytotic release"
(c) there is also evidence of an Nn receptor in some places.
i) when ACh works on this receptor, it cause an influx of Na to the presynaptic
neuron, which is excitatory (this is the origin of the AP in the first place)...
ii) this creates the increased exocytosis of Ach
(3) Which means that the amount of ACh at any given synapse is the sum of ACh
release, metabolism, and effects of both negative and positive feedback
g) Other modifications of this ENTIRE process include:
(1) hemicholinium inhibits choline uptake into the presynaptic cell
(2) vesamicol blocks the vesicular uptake transporter (empty vesicles don't create a
response upon exocytosis)
(a) creating of a reverse gradient due to build up in the vesicle does happen, but it's
not that extreme because acetylcholine is a quaternary with a charge
(3) botulinum toxin blocks exocytosis
4. Remember that Ach is released at both pre ganglionic and post ganglionic parasympathetic
ganglion.
a) At the preganglionic terminus, it is released to bind with cholinergic Nn receptors on the
post ganglionic neuron
b) At the post ganglionic terminus, it is released to bind with cholinergic muscarinic
receptors on target organs.
(1) M1, 3, 5 receptors are linked to Gq (PLC, IP3, DAG, increased Ca and PKC)
(2) M2, 4 receptors are linked to Gi (decreased cAMP, K efflux and Ca channel shut
down hyper polarizes cell)
c) Also at the post ganglionic terminus, it is released to Nm receptors on skeletal muscle.
This is the target for Non depolarizing antagonists and agonists like Succinylcholine.
5. Muscarinic receptors, their placement, cellular/functional responses and disease relevance
(Table 8.3)
III. Intro to Muscarinics
A. ACh is the naturally occurring neurotransmitter for cholinergic
receptors
1. virtually no therapeutic effects of ACh
a) too rapidly metabolizing by AChE or
butyrylcholinesterase...but...
2. cholinergic agonists mimic the effect of ACh
3. muscarine is the naturally occurring plant form
B. Receptors found primarily on the autonomic effector cells
innervated by the parasympathetic postganglionic neurons...
1. endothelial cells of blood vessels
2. autonomic ganglia
3. adrenal medulla
C. receptors
1. M1 - M5
a) M1, 3, 5 coupled to Gq
b) M2,4 coupled to Gi
2. named for compound that it is selective for, muscarine, produced by poisonous mushroom
a) general agonist responses
(1) GI
(a) increased tone and motility, anabolism!
(2) URINARY
(a) go pee! contract detrusor, increase voiding pressure, decrease capacity,
increase ureteral peristalsis, relax trigone
(3) EXOCRINE
(a) stimulates glands receiving sympathetic or parasympathetic cholinergic
innervation... diaphoresis
(4) RESPIRATORY
(a) bronchial smooth muscle stimulation (constriction)
(5) CV
(a) vasodilation of certain vasculature mediated by M3 receptors on endothelium?
penis - erection, vasculature to some glands
(b) otherwise, negative "homeostatic effects via M2, M3 - negative ionotrope,
negative chronotrope, negative dromotrope (reduces conduction velocity) as
stated below
D. Analogs of Acetylcholine have therapeutic value because they are not metabolized as
fast
1. ACh is the same as Muscarine but in a non rigid structure
2. conjoiners top row and plant analogs bottom row
3. By adding small groups like methyl, amine or both to synthetic analogs, these compounds
become more resistant, if not completely resistant, to serine protease hydrolysis by AChE.
They become competitive agonists, and only respond to the concentration of ACh versus
themselves... meaning that the only way to "reverse" their binding is to saturate the
environment with something that has preferential affinity. So, if you give an AChE
INHIBITOR, you basically slow ACh metabolism, giving it a chance to build in numbers and
bind (and give the synthetic a chance to diffuse away).
4. 2 types of Analogs:
a) ACh Synthetic Congeners: Methacholine, Carcachol, Bethanechol
(1) Carbachol is almost completely resistant to hydrolysis
(a) carbechol has strong nicotinic agonist activity
(b) induces miosis for ophthalmic surgery, and is used to treat glaucoma
(c) Gaddis slides again cite some, more minimal CV effects, presumably because
carbechol has significant nicotinic activity, but does also seem to prefer M3
binding binding to M2, which may be why it's helpful in the eyes and less
effective in the heart
(2) Methacholine is the only synthetic that undergoes mild metabolism by AChE.
Drugs
Therapies
Pharmacology
(a) methacholine used to diagnose bronchial hyperactivity in the clinical absence of
asthma...it can cause severe constriction in asthmatics but otherwise shows
constriction in everyone. rescue equipment and ß 2 inhalation agonists should
be available during testing
(b) Gaddis slides say the most predominant effects of Methacholine are on the CV
system... which is true and why rescue equipment should be available.
i) M2/M3 Cardiac system effects:
(1) decreased HR (SA node)
(2) moderate to severe decrease in atrial contractility with slight decrease in
ventricular contractility
(3) decreased conduction velocity
(4) AV blocks
ii) can be exaggerated if patients are taking ß2 receptor antagonists (ß
blockers) for cardiovascular disease
(3) Bethanechol
(a) no nicotinic activity
b) Naturally occurring Alkaloids
(1) Muscarine
(a) resistent to AChE hydrolysis
(b) no nicotinic activity
(c) significant M2M3 CV effects
(2) Pilocarpine
(a) predominant agonist affect on M2, M3 generalized sweat glands
(3) Arecholine
(a) nicotinic activity
(b) resistent to ACh hydrolysis
E. Muscarinic Receptor Antagonists
1. prevent the effects of ACh binding, by binding in it's place, to muscarinic receptors in:
a) smooth muscle
b) cardiac muscle
c) glands
d) sympathetically innervated sweat glands
e) peripheral ganglia (I don't know of any muscarinic receptors here, maybe referring to
this muscarinic antagonists that have nicotinic activity?)
f) and the CNS
2. little nicotinic receptor affinity but the ones that are quaternary ammonium compounds do
have MORE nicotinic activity, which has significance for NMB and blocking ganglionic
activity (including sympathetic cholinergic)
3. 3 classes:
a) Naturally occurring alkaloids
(1) Atropine (the prototype - and most other antagonists are not significant different)
(2) Scopolamine
b) semisynthetic Atropine
(1) homatropine and quaternary derivatives
c) Synthetic congeners
(1) pirenzepine
(2) tolterodine
(3) oxybutynin
4. Functional groups important for antimuscarinic activity
a) essential for activity
(1) Tropine
(2) Tropic Acid
b) important
(1) presence of a free hydroxyl group int he acyl portion of the ester is important for
activity
5. Effects of muscarinic antagonists are dose dependent - because different organs are
variably affected by cholinergic sensitivity.
a) sweat glands, salivary glands, the heart have a lot of muscarinic receptors... so they are
sensitive to small doses
b) GI tract is not that innervated by the parasympathetic branch, so it is only affected at
high doses
c) there also have to be interneurons to carry on messaging
d) Atropine overdose - red as a beet, dry as a bone, blind as a bat, hot as a firestone, mad
as a hatter
e) because atropine, nor any muscarinic receptor antagonist is receptor specific,
increased efficacy of some compounds may be because they hit multiple M receptors
f) hitting multiple receptors, especially presynaptic ones can also effect exocytosis
leading to paradoxical responses
6. Pharmacological effects & therapies
a) CV
(1) increased heart rate after transient small decrease (likely from a presynaptic
receptor antagonism)... used for bradycardia
(a) effects can depend on how significant vagal tone is for that person since it is
blocking M2 receptors on the SA node - healthy young people with good vagal
tone will get a good 35-40 beat response with 2mg - an older person, infant or
CHFer will not.
(2) prevent or abolishes cardiac arrest occurring as a result of electrical vagal
stimulation during a code by reducing stimulation of vagal tone. Again, affects can
vary.
(3) reverses bradycardia associated with sympathomimetics like AChE inhibitors... so
given with AChE during NMBA reversal to inhibit bradycardia cholinergic activity
(4) minimal affect on BP since most vascular beds don't have much cholinergic
innervation
(5) dilation of cutaneous vessels (red as a beet) likely occurs as a compensatory
mechanism for decreased sweating (dry as a bone)
b) CNS
(1) atropine has minimal CNS effects at therapeutic doses but toxic doses can cause
CNS excitation (irritability --> hallucinations), followed by depression, then death
(2) scopolamine crosses the BBB easier and has many CNS effects
(a) therapeutic doses cause CNS depression: drowsiness, amnesia, fatigue, less
REM sleep, euphoria
(b) with severe pain, scopolamine can mimic excitatory effects - delirium,
restlessness, hallucinations
(c) scopolamine effectively used as a depressant in a localized way for motion
sickness when placed behind the ear
c) RESPIRATORY
(1) block parasympathetic vagal tone and dry secretions (ipratropium)
d) EYES
(1) local or systemic administration will cause mydriasis and cycloplegia allowing for
ophthalmology examination
(2) results in photophobia and far vision
(3) contraindicated in closed angle glaucoma d/t intraocular pressures
(4) effects can be reversed by Pilocarpine agonist
e) URINARY
(1) antimuscarinics used in overactive bladder reduce parasympathetic tone
7. Containdications and ADR all make sense if you just think about the consequences of
receptor blockade
a) ADR: constipation, blurred vision cognitive impairment, xerostomia, dyspepsia
b) contra: urinary tract obstruction, GI obstruction, closed angle glaucoma, BPH
c) though, even though there is a lack of receptor specificity, if you can deliver locally,
some ADRs can be avoided and meds can be used safely
IV.Anticholinesterase Agents
A. Don't get it twisted - the above name means "Things that build up the ACh levels by
inhibiting the enzyme that breaks ACh down
1. ACh - Acetylcholine, a tetrahedral compound. "Muscarine is the plant version"
2. Acetylcholinesterase - the enzyme that breaks it down
3. Acetylcholinesterase Inhibitor - really, an analog of ACh... these are compounds that inhibit
the enzyme by occupying it's active site, as ACh WOULD so that ACh is free to build up in
number and saturate a synaptic cleft, thereby displacing cholinergic receptor site agonists
(succ) or antagonists (NDMAs)
4. 2 classes produced by a single gene:
a) simple homomeric
(1) soluble, for export
b) heteromeric
(1) in synapses on outer membrane surface and tin basal lamina of skeletal muscle
junctions
5. A SEPARATE gene encodes for butyrylcholinesterase
a) synthesized int he liver
b) primarily found in plasma
B. Review the 3 sites of the AChE enzyme and the basic principle of how different functional
groups inhibit it
1. enzyme functional areas:
a) acyl group - the active site - where the electrophilic Serine Oxygen is - when ACh binds
here, that O is acylated because CH3 C double O is an acetate group.
(1) When the O is acylated, the choline group is liberated and goes away. THEN the
water comes in, and because carbonyl acetate groups are so prone to hydrolysis,
the second step of serine protease metabolism happens licit split and the enzyme is
regenerated.
b) choline site - where the choline group on ACh lines up to... because the quaternary N
with the positive charge is weakly attracted to the...
c) peripheral anionic site (not to be confused with the "oxyanion hole," which is where that
electrophilic oxygen was)
2. MOA of Different inhibitors (carbamate endings, phosphoric endings) all just slow down the
rate of serine regeneration by stabilizing the serine modification for longer than ACh's
acetate group does.
a) Carbamoyl lasts longer than carbonyl and phosphoric lasts LOOOONG time. These are
the organophosphates and some of their serine bonds are irreversible and toxic (nerve
gases)
C. Classes
1. Acetyl Inhibitors - this IS ACh
2. Carbamoyl Inhibitors - Stigmines... inhibition lasts 3-4 hours (not 150 µsec)
a) neostigmine, physostigmine, pyridostigmine, rivastigmine
b) all have a phenyl ring/choline group - this is what bonds to AChE's anionic region
c) all have an ester bond and a nitrogen - this is a carbamate, which forms a more stable
bond with the electrophilic oxygen on the serine unit in the acetylcholinesterase (a
serine protease) than does ACh's carbonyl group
(1) the serine becomes carbamolated instead of acylated
(2) so it stays bonded longer... so we say it is more resistant to hydrolysis
(3) (When ACh undergoes the initial electrophilic attach from the enzyme, it's left
behind acetyl form is VERY labile to hydrolysis... which is what frees up the acetate
group into circulation and the enzyme turnover)
(4) ACh turnover is incredible - just 150 µsec
d) so ACh builds up and preferentially displaces whatever NMB agent is blocking the Nm
cholinergic site at the NMJ (for example)
3. SANS ESTER edrophonium alcohol, tacrine, donepezil & galantamine... REVERSIBLE
a) has the phenyl but doesn't have an ester -acetate or -carbamate or anything to bond
with the serine - it doesn't modify the serine
b) so it doesn't undergo the 1st step if serine protease metabolism, but it's N+ of the
quaternary does still work at the anionic region of the choline site
c) the choline group just disassociates after stopping infusion...time/saturation
d) (donepezil technically has an ester doubled bonded oxygen, but it's not where the
serine of the serine protease could line up with it.) It does stay bonded for longer
though.
4. Phosphoryl inhibitors - organophosphates - can be irriversible
a) DFP (diisopropylflourophosphate), tabun, soman, parathion, diazinon, dursban and
malathion
b) forms extremely stable phosphorylated serine conjugates
c) structural analogs contributing to stability
(1) alkyl groups in phosphorylated enzyme are ethyl or methyl groups = pontanseuous
regeneration after HOURS
(2) alkyl groups in phosphorylated enzyme are secondary or tertiary covalent bonds,
they are irreversible and new enzyme must be synthesized - takes DAYS, so you'll
probably be dead.
5. Enzyme re-activators (what soldiers carry around with them incase they're exposed to
nerve gases
a) Pralidoxime (2-PAM chloride)
D. so, AN ANCHORING POINT HERE...
1. pharmacological properties of antiAChE agents are similar to the effects of locally
stimulated release of ACh... it's causing the rebuilding of a critical mass
2. which means that this whole chapter was really the first part of the last chapter in different
words.
3. Muscarinic stimulation of cholinergic receptors (Nn, Nm, and M) happens with the release
of ACh or the administration of an AChE inhibitor (AKA, the antiAChE agent) after a ACh
antagonist.
4. Which means that the parasympathetic effects we see from muscarinics = ACh = what's
seen with a AChE inhibitor after cholinergic receptor antagonism
a) stimulation of muscarinic receptor responses at autonomic effector organs (sweat
glands)
b) stimulation, followed by depression and paralysis, of all autonomic ganglia (including
the adrenal gland) and skeletal muscle (nicotinic actions)
c) stimulation, with occasional subsequent depression, of cholinergic receptor sites in the
CNS
E. Useful Pharm bits
1. quaternary compounds don't cross barriers a) poorly absorption via GI, skin or BBB
b) but they DO have nicotinic activity - Neostigmine is the stand out here
2. other sites of important AChE inhibitor therapy are the CNS, EYES, Intestines & NMJs
V. Matching Symptom profiles to Pharmacodynamic profiles
A. Symptoms... what system is causing this
B. What needs to happen - activation or inhibition of which system (what ligand/receptor/G
protein)
C. what MOA is needed to achieve the goal without causing undue ADR
D. What class of drugs can accomplish this goal