1
Q
The nueromuscular junction
A
- all skeletal muscles are innervated by motor neurons whose cell bodies are located in teh ventral horns of the spinal cord
- impulse travels down myelinated axon to nerve terminal
- transmitted to the muscle at the neuromuscular junction
2
Q
Stages during synaptic transmission
A
- A. Depolarization of the nerve terminal by an action potential.
- B. Activation of voltage sensitive Ca++ channels in the terminal causing influx of Ca++ from extracellular medium.
- C. Fusion of synaptic vesicles with the terminal plasma membrane, causing exocytotic release of ACh.
- D. Diffusion of ACh across the synaptic cleft and binding of ACh to nicotinic ACh receptors (nAChR) in the muscle postsynaptic membrane.
- E. Activation of nAChR-dependent ion channels which increase the permeability of the muscle membrane to Na+ and K+ ions, depolarizing the endplate region.
- F. Initiation of a muscle action potential near endplate region, which spreads through the muscle, and eventually leads to muscle contraction.
- G. Termination of synaptic transmission by hydrolysis of ACh to acetate and choline by AChE
3
Q
Spinal cord showing a motor neuron with its axon projecting to skeletal muscle
A
- Somatic system simplest
- skeletal muscle is innervated by myelinated motor neurons
- all have cell bodies in the ventral horn of the spinal cord
- skeletal muscle is innervated by myelinated motor neurons
- Autonomic system
- intervening ganglia and additional synapses within the ganglia which lead to more complicated pharmacology
4
Q
Motor neuron cell bodies in the ventral horn of the spinal cord
A
- polynaptic pathway
- afferent central processes synapse onto interneurons in the spinal cord, which synapse onto motor neurons in the ventral horn of the spinal column
- Monosynaptic pathway
- synapse directly on to motor neurons
- Renshaw cells are inhibitory interneurons which synapse onto and inhibit motor neurons
- Axons from motor neurons synapse onto and activate skeletal muscle
5
Q
Model of the neuromuscular junction
A
- Electrical activity in the form of action potential are propagated down these myelinated motor neurons which ultimately synpase with muscle
- muscle is the postsynaptic cell
- The signal is sent to the postsynaptic muscle cell by synaptic transmission which occurs in a number of distinct steps
- Results in a local depolarization which can stimulate muscle contraction
6
Q
****Synthesis and release of acetylcholine
A
- ACh is synthesized by the enzyme choline acetyltransferase (CAT)
- choline + acetyl CoA→ACh + CoA
- ACh is synthesized in the cytoplasm of the nerve terminals then transported into synaptic vesicles
- CAT activity can be inhibited by several drugs
7
Q
Electron Microscope Image of the Neuromuscular Junction
A
- Nueromuscular junx is a highly specialized structure
- Muscle membrane is very highly folded under the nerve terminal and the receptors for ACh are located on the crests of these folds
- Receptors are present in extremely high density, which essentially no room for other proteins
- Thin wispy stuff is the basal lamina, a basement membrane structure. Important because:
- Surround each muscle fiber. When there is muscle damage, the muscle starts regenerating and the basal lamina serves as a scaffold for the regenerating muscle to insure the proper orientation. Imparts info concerning the specificity of synpase formation. It preserves a “memory” of the structures of specific synapses.
- Acetylcholine esterase, the enzyme that terminates ACh action, is present on the basal lamina. 90% ACh during synaptic activation is hydrolyzed before it gets to the muscle surface. Very active. Inhibitors of acetylcholine esterase have pronounced physiological effects because they cause large changes in teh amount of synaptic acetylcholine available
8
Q
Schematic representation of the synaptic junction
A
- This is a schematic representation of the NM junction.
- Vesicles dock at the presynaptic membrane.
- The vesicles fuse and release acetylcholine into the synaptic cleft.
- ACh diffuses across the synaptic cleft and binds to the nicotinic AChR in the postsynaptic membrane. This causes a transient increase in the permeability of the membrane to Na+ and K+, followed by desensitization of the receptor.
- The depolarization of the endplate is sufficient to trigger a muscle action potential and thus results in contraction of the muscle.
9
Q
Synthesis Storage and Release of ACh
A
- After termination of ACh action by ACh esterase the released choline is transported back into the nerve terminal by a high affinity transport system which depends upon the membrane potential and external sodium.
- The drug hemicholinium-3 (HC-3) inhibits the uptake of choline ultimately causing the depletion of ACh at synapses. HC-3 is a research tool and not currently used clinically.
- At the nerve terminal choline is converted to ACh by the enzyme choline acetyl transferase (CAT) in the cytoplasm. The ACh is then taken up into the synaptic vesicles.
- Several choline derivatives inactivate choline acetyl transferase, including:
- choline mustard aziridinium ion
- ethylcholine mustard aziridinium ion
- These drugs act as active-site alkylating agents. They block the synthesis of ACh.
10
Q
Botulinus toxin is used to treat dystonias
A
- ACh release is inhibited by botulinus toxin
- acts as a protease to hydrolyze SNAP-25, a protein required for vesicle fusion and NT release
- Very potent; active < 0.1 ng/kg
- Treats dystonias (involuntary muscle spasms)
- eye: blepharospasm
- Inject it locally
11
Q
Clinical uses of botulinum toxin
A
- Persistent muscle spasms-dystonias
- Treatment of hyperhidrosis (under arm sweating)-Botox blocks acetylcholine release which normally contributes to sweating
- Healing of anal fissure
- May be useful in tension and migraine headaches
- Elimination of facial wrinkles- Botulinus toxin is also clinically approved for the use in cosmetic surgery to remove wrinkles due to muscle contraction. The effect is temporary but it can last for weeks.
12
Q
Two types of ACh receptors
A
- Nicotinic and muscarinic
- recognize ACh in two distinct conformations
- ACh can interact with either N or M receptors.
- Nicotinic acetylcholine receptors are the ACh receptors at the NM
- Ligand-gated ion channels
- Muscarine play a major role in ANS and are G-protein couple receptors
- Nicotinic acetylcholine receptors are the ACh receptors at the NM
13
Q
ACh binds to and opens ACh receptors
A
- ACh binds to the nicotinic ACh receptor, causes conformation change
- ACH receptor is a transmembrane protein
- When ACh binds it opes a hole in the middle of the protein through which ions can go through
- The increase in permeability for Na+ and K+ ions that causes the electrical response
- One can purify nicotinic ACH receptor from the muscle. Can incorporate this purified receptor into an artificial membrane and apply ACH and it will conduct a current
- No second messanger system
- Ligand gated ion channel that can be very rapidly activated
- Release of ACh causes a local depolarization
- Needs to be large enough to bring the msucle to threshold and generate an action potential that propagates down the muscle fiber
14
Q
Desensitization of Muscle Nicotinic Receptors by Acetylcholine
A
- We have a muscle fiber and an electrode stuck into it, and we record electrical activity in the muscle.
- Squirt out little bursts of ACh and record in response to that ACh.
- After each burst of ACh we get a discrete depolarization.
- The ACh works through nicotinic ACh receptors in the muscle.
- Now we bring a second pipette to the muscle preparation, also containing ACh, and apply ACh in continuous streams, and at the same time have the other pipette applying short bursts.
- This broad deflection shows the big bolus of ACH.
- As you keep giving the little squirts the response starts to decrease. When we turn off the continuous stream of ACh and continue the brief pulses the response to the little bursts of ACh eventually reappears.
- This is desensitization, in the continuous presence of ACh the response decreases; the time response for the desensitization is quick, within a mater of secs. This is an intrinsic property of the receptor.
15
Q
****Model for Receptor Desensitization****
A
- Initially the receptor is closed.
- When ACh binds it opens the pore, current flows.
- If ACh stays bound there is a different kind of conformation that closes down the pore.
- important because some of the drugs that we will be talking about are partial agonists and produce their desired therapeutic effects by causing receptor desensitization.
- nAChRs exist in three states, resting state (R), activated state (O) and desensitization state (D).
- In order for the desensitization to occur, the receptor has to first be in the activated state for a period of time to depolarize the membrane.
- When the membrane is depolarized for an extended period of time and acetylcholine is bound to the nAChR, it undergoes a confirmation change that closes that channel.
- Some drugs produce their pharmacological effects by causing desensitization of the nAChR.
16
Q
Deinnervation Supersensitivity
A
- When a muscle degenerates or it is deinnervated by cutting the motor nerve that synapses on it, it undergoes a process known as deinnervation Supersensitivity.
- Normally the receptors are clustered at the nerve terminal at the synapse.
- There are very few receptors in the non- synaptic regions of the muscle.
- When the muscle is injured, you start getting receptors in non-synaptic regions of the muscle as well.
- This is due to the synthesis of new receptors and insertion into the muscle membrane.
- This results in an overall large increases (20 x) in new receptors.
- Consequently, de- innervated skeletal muscle becomes supersensitive to pharmacologically applied cholinergic agonists.
- If the nerve grows back and the muscle becomes re-innervated, you lose these additional non-synaptic receptors.
17
Q
Uses of Neuromuscular Blockers
A
They have a number of therapeutic uses:
- Endotracheal intubation-tube placed down the trachea
- Muscle relaxants during surgery
- Resetting of fractures
- During electroshock therapy, to prevent physical trauma.
- During intensive care to prevent high airway resistance and to abolish muscle rigidity.
- These drugs are also used in the cocktail of drugs used for state executions.
18
Q
Competitive Non-Depolarizing Blockers
A
- There are two classes of neuromuscular blockers, competitive non-depolarizing blockers and depolarizing blockers. These are some of the competitive non-depolarizing blockers.
- d-Tubocurarine or curare for short, is a natural compound.
- Gallamine and pancuronium are synthetic compounds.
- They are classical competitive antagonists at nicotinic acetylcholine receptors.
- They bind to the receptors and block receptor action.
- Muscle contractility is partially impaired when 75 to 80% of the nicotinic receptors are occupied and completely blocked when 90 to 95% are occupied.
- Curare, derived from plant extracts, is used by South American Indian tribes. Plant extracts are applied to arrow tips for hunting and warfare.
19
Q
Curare Effects on Muscle Contraction
A
- This shows the specificity of the competitive non-depolarizing blockers. In this slide we are recording muscle contractions which are produced by alternate stimulations of the nerve or muscle.
- At point A we add curare and at B we discontinue application. When we add curare, one set of lines (i.e. muscle contractions) disappears because we block contraction due to nerve stimulation.
- Curare doesn’t block depolarizations due to direct stimulation of the muscle. So one set of contractions is constant.
- Curare and related drugs are generally administered IV, they are not active with oral administration. This why the South American Indians can use game captured by curare arrows or darts.
- The effect of the neuromuscular blockers is to block nicotinic receptors, and they are relatively specific for nicotinic receptors in skeletal muscle.
- There is a related nicotinic receptor in the autonomic ganglia, the neuromuscular blockers can also block these receptors and produce some autonomic effects including cardiac arrhythmias and decrease blood pressure in some patients.
- Curare can also cause histamine release resulting in bronchospasms and hypertension, increase BP.
20
Q
The toxic effects of neuromuscular blockers are:
A
- Respiratory paralysis, by blocking the muscles involved in respiration
- Cardiovascular collapse, increase in heart rate and blood pressure.
- Because these drugs are highly charged, are expected not to act on the CNS, and there are no known CNS effects of curare.
- Because these drugs are competitive antagonists, you should be able to treat the toxic effects of these drugs with anti-choline esterases, thereby increasing the amount of ACh at the synaptic cleft.
21
Q
Depolarizing Blockers
A
- A second class of neuromuscular blockers are known as depolarizing blockers,
- decamethazoinum-C10
- succinyl choline
- they are partial agonists.
- They bind to the receptors, activate them but antagonize ACh action.
- This causes twitching, blocks nerve-evoked contraction by this general activation of the receptor.
- Succinyl choline is not degraded by acetyl choline esterase and produces receptor occupation for a prolonged period of time.
- Therefore, in the continued presence of the depolarizing blockers they cause desensitization.
22
Q
Duration of Succinyl Choline Action
A
- Succinylcholine normally has a t1/2 of a few minutes; it is hydrolyzed by an enzyme in the serum called pseudo- or plasma-choline esterase, which is similar to the ACh choline esterase at synapses.
- This is often used as an example of pharmacogenetics because there are mutants among us 1/3000 have very low levels of pseudo or plasma choline esterase.
- With a normal patient, breathing will rapidly continue when succinylcholine application is stopped.
- With some patients, they have prolonged apnea (stop breathing) Because they don’t degrade succinyl choline as fast as normal people.
- There is clearly a difference in how the toxic effects of competitive (non-depolarizing) blockers such as curare and succinylcholine are treated.
- Toxic effects of curare can be overcome by treatment with anti- acetylcholine esterases, this increases ACh and overcomes the competitive inhibitors.
- In contrast, if you have a patient who has very low levels of pseudo or plasma choline esterase and you treat with succinylcholine and they experience apnea-suspension of breathing, you do not give them anti- acetylcholine esterases, increasing ACh will enhance the desensitization due to depolarizing blockers such as succinyl choline.
- Artificial respiration is therefore used to overcome apnea cause by succinylcholine or other depolarizing blockers.
23
Q
Acetylcholinesterases
A
- Acetylcholinesterases, which are located on the basal lamina, terminate the action of ACh at the neuromuscular junction.
- Most of the acetylcholine released into the synapse never reaches the receptors.
- If you inhibit the activity of acetylcholinesterase, you cause large increases in the concentration of ACh in the synapse.
- There are a number of different classes of inhibitors of the acetylcholinesterases which differ in their mechanisms of action.
24
Q
Active Site Model for Acetylcholinesterase
A
- The active site of this acetylcholinesterase has two sites:
- an anionic site that interacts with the quaternary ammonium group of ACh
- a site for ester hydrolysis, often referred to as the esteratic site.
- The esteratic site contains a histidine and a serine.
- Anti-cholinesterases interact with one or both of these sites.
25
Mechanism for Acetylcholine Esterase
* The esteratic site contains a serine which functions as a nucelophile to attack the ester bond of ACh.
* The histidine functions as a general base and facilitates the removal of the hydrogen from the active site serine, thereby enhances its nucleophilicity.
* The enzyme goes through an Acyl-enzyme intermediate in which the acetyl group is temporarily esterified to the active site serine.
* Subsequently, this is hydrolyzed to yield unmodified enzyme and acetate.
* This is one of the fastest enzymes know with a turnover number of 10,000/sec
* We recognize three general types of cholinesterase inhibitors:
1. noncovalent and reversible
2. Covalent but reversible
3. Covalent and irreversible
26
Structure of Edrophonium
* The first class of drugs is readily reversible because they form a noncovalent complex with the enzyme at the anionic site.
* The classical example of this class of drugs is edrophonium, a compound that contains a quaternary ammonium function.
27
Complex of Edrophonium
* This slide shows the complex between edrophonium and acetylcholine esterase. It is hypothesized that the drug not only interacts with the anionic site of acetylcholine esterase but that there is also a hydrogen bond with a histidine in the esterase.
* Edrophonium has a relatively short half-life and is readily eliminated via the kidneys. The half-life is a couple of minutes....effective for about 10 minutes.
28
Structures of physostigmine and neostigmine
* The second class of acetylcholine esterase inhibitors, the carbamate esters, form a covalent complex with the enzyme, but the covalent complex is slowly reversible.
29
Mechanism of Action for the Carbamate Esters
* These compounds which include physostigmine and neostigmine, carbamylate the active site serine and covalently inactivate the enzyme.
* However, the carbamylated enzymes turn over with a half-life of about 30 minutes.....effective for 3 to four hours.
30
Mechanism of Action of Organophophates with DFP as an Example.
* The third class of drugs, the organophosphates, form a covalent complex with acetylcholine esterase, includes the prototypic drug DFP (diisoproplyflurophosphate), parathion and echothiophate.
* DFP is nerve gas. It forms a phosphorylated enzyme intermediate which is very stable.
31
Aging of phosphorylated AChE
* A further conversion, called aging occurs.
* During aging the covalently modified enzyme loses one of the alkyl groups that are on this phosphate.
* When it undergoes this aging process the enzyme-covalent intermediate is extremely stable and cannot be reactivated.
* The time for aging varies with the drug; with DFP it is 2 to 3 days with Soman, a nerve gas, it is 3 min.
32
Reversal by Pralidoxime
* Before aging takes place, the phosphorylated enzyme can be reactivated with added nucleophiles
* e.g. 2-PAM (pralidoxime) which frees the enzyme and forms an oxime-phosphonate with the drug.
* So if you have been poisoned with an organophosphate you can treat with 2-PAM right away and reverse the action of the organophosphate. The aged enzyme is resistant to 2-PAM.
33
Physiological Effects of Anticholinesterases
* Generally amplify the effects of acetylcholine at all sites throughout the nervous system, not just at the NM junction.
* They activate nicotinic receptors at the NM junction and in sympathetic and parasympathetic ganglia. In skeletal muscle low does increase the force of muscle contraction. High doses cause receptor desensitization and blockade.
* Anticholinesterases indirectly activate muscarinic receptors in the parasympathetic nervous systems and in sweat gland.
* They increase GI smooth muscle motility.
* They decrease heart rate.
34
Myasthenia Gravis Muscle Contraction ± Neostigmine.
* Myasthenia gravis is an autoimmune disease in which the patients make Abs against their own nACR in muscle.
* This interferes with neuromuscular transmission and causes neuromuscular weakness and the patient readily experiences muscular fatigue.
* From this slide you can see that these patients normally show a diminished or decremental response upon continued muscle stimulation.
* When you apply an acetylcholine esterase inhibitor, in this case neostigmine, you prolong the time the patient can produce muscular contractions.
* This occurs because the drug increases the lifetime and effective concentration of ACh at the neuromuscular junction.
* Muscular endurance can be easily measured in the lab using tests such as sustained handgrip test.
* Other anticholinesterase drugs are used for the treatment of Myasthenia gravis, but these drugs will also stimulate ACh levels in the autonomic nervous system (ANS).
* Therefore, one also treats with atropine which is a muscarinic antagonist to suppress this effect.
* For diagnosis of MG, one uses a short-acting inhibitor of acetylcholine esterases such as edrophonium.
* When administered IV to a MG patient there will be a transient increase in strength lasting several minutes.
35
Toxicity of Acetylcholine esterase inhibitors, e.g. in pesticides, dog and cat flea collars.
* Death from these drugs is most commonly because of respiratory paralysis. ACh goes up, you desensitize nicotinic R, respiratory paralysis.
* Also in the ANS, can have cardiac collapse due to decrease in heart rate and lowering of BP.
* Treatment of the toxicity of organophosphate poisoning is by treatment with 2-PAM to reactivate ACh esterase and atropine to block muscarinic effects arising from elevated ACh in the ANS.
Phcol 402
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