Cholinergic control at the NMJ

Structure and function of skeletal muscles Skeletal muscles are under voluntary control Therefore, they will contract when we make a conscious decision to move They need to conduct information from the brain to the muscle quickly for proper control The fibres leading to muscles are myelinated for fast conduction of action potentials The motor neurones in the spinal cord use glutamatergic synpases Remember: NMDA and AMPA in the spinal cord will open their ion channels in response to glutamate for FAST depolarisation They need to be fast, because they tend to be the longest cells in the body Needs to reach from the brain down to all the muscles Again, myelin is used to increase conduction speeds within neurones Schwann cells in the periphery are responsible for wrapping their membranes around the axon of the nerve to cover it in lipids Keeps the axon insulated against the leakage of ions, allows for faster connections There are regular breaks in the myelin wraps though, these are call the nodes of Ranvier Dense in ion channels, causes the influx of ions at these points to continue the conduction of the action potential If these neurones are to become demyelinated, the control of skeletal muscles can be lost Multiple Sclerosis (MS) is one of them Incurable Motor nerves leave the spinal cord from the ventral roots Anterior Each nerve will activate a few muscle fibres at the same time This is called a motor unit Having a greater 'nerves to muscle fiber' ratio leads to better control of the muscle i.e. if one nerve controls every 3 muscle fibres, then you can have fine control, because you can turn on muscle fibres in multiples of 3, allowing for a wide range of forces generated by the overall muscle But if one nerve controls 200 fibres, it's more difficult to have fine control because you can only activate muscle fibres in multiples of 200, so it's hard to specifically control how much force is generated Skeletal muscles will use ACh (acetylcholine) as the transmitter at the neuromuscular junction (NMJ) Neuromuscular Junction (NMJ) This is where the nerve meets the muscle fibre Tends to be located at the middle of fibres Allows the muscle to contract evenly, because the depolarisation spreads from the middle out If it were to be on the end, then one end will contract faster than the other end, leading to poor co-ordination between muscles The process at the NMJ is: Action potential reaches the nerve terminal, and that opens the voltage gated calcium channel Calcium will allow the vesicle to undergo exocytosis SNARE proteins are used to anchor the vesicle onto the membrane for
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 SNARE proteins are used to anchor the vesicle onto the membrane for exocytosis They are cut by the botulinum A toxin, which prevents exocytosis and causes paralysis Once ACh is released into the cleft, it can travel to the motor endplate and bind to Nicotinic Cholinergic receptors i.e. these receptors will bind strongly to nicotine and ACh This leads to opening of the channel, which causes sodium influx to depolarise the muscle Called the excitatory post synaptic potential (EPSP) Must reach the potential threshold as well to trigger a muscle-wide depolarisation and contraction Depolarisation leads to opening of the voltage gated calcium channels of the sarcoplasmic reticulum This causes an increase in calcium, which leads to contraction Meanwhile, the ACh in the cleft is broken down into choline Choline is rapidly taken back up into the neurone It is metabolised into ACh by CAT using Acetyl CoA The ACh is repackaged into vesicles via a fast and specific transporter located on the vesicles

Cholinergic receptors ACh is quite commonly used around the body Nicotinic ACh receptors (nAChR) NMJ (as seen above) Adrenal gland Ganglia of both the sympathetic and parasympathetic systems High dose nicotine leads to initial sympathetic activation followed by parasympathetic activation Causes initial increase in blood pressure and heart rate, followed by a decrease in blood pressure and heart rate Therefore, it's not a good idea to have a non-selective nACh agent as it will cause a whole lot of side effects Muscarinic ACh receptors Mostly at the organ of the parasympathetic system
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Mostly at the organ of the parasympathetic system Some glands under sympathetic control We will be focusing on nAChR as they are used in the NMJ There are 11 different types of subunits 5 subunits per receptor Each subunit contributes their helix to the ion pore in the middle Different types of receptors (muscle, CNS and ganglionic) uses different mixes of subunits Allows for specific targeting of the receptors (but not 100% selectivity, so there is some cross-reactivity at high concentrations) Muscle uses 2 alpha-1 subunits, 1 beta-1 subunit, 1 gamma subunit and 1 eta subunit In muscle the two alpha subunits are important as they have the binding sites for ACh Both binding sites must have ACh attached to open the channel The binding of ACh is very fast Short distance to diffuse across It comes off very fast ACh esterase (AChE) will quickly break down ACh into choline and acetate Even if it stays on for 1ms it has an action A lot of sodium can pass into the muscle in that time to cause depolarisation

Blockers at the NMJ There are some therapeutic uses of blocking actions at the NMJ One of them is to cause paralysis during surgery to prevent the patient from moving around Reflexes aren't shut down, they will spasm if cut This can cause major damage during surgery But there is a huge problem with blocking actions at the NMJ The diaphragm is an important muscle in breathing It is under voluntary control Therefore, blocking actions at the NMJ causes paralysis of the muscle, which stops the patient from breathing Blockers must be used with artificial ventilation and careful supervision ACh esterase inhibitors The AChE can be inhibited for three reasons Treatment of Myasthenia Gravis Autoimmune reaction, antibodies made against the nACh receptors in the muscle endplate Prevents their activation by ACh, which causes muscle weakness If we block AChE, then there will be more ACh in the cleft to trigger more nAChR to increase muscle strength War agent/pesticides Organophosphates will covalently bond to the AChE to inactivate them Leads to too much ACh being in the cleft The ACh receptors in the motor endplate will stop reacting to ACh, because the ion channels are kept open (see below for a phase I block) Remember: the diaphragm is under voluntary control, organophosphates will lead to paralysis so you stop breathing Why doesn't paralysis occur in the treatment of myasthenia gravis? We don't cause too much activation. If you happen to cause too much activation, then that causes the paralysis
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too much activation, then that causes the paralysis Antidote to non-depolarising block agents (see below) Non-depolarising block These agents are antagonists at the receptor Causes paralysis by preventing ACh from having an effect at the ion channel BUT pain and other senses are not affected, if people aren't knocked out completely, then they can hear and feel themselves being cut apart Curare-based compounds were found to be non-depolarising blockers All of them have a quarternary ammonium, therefore, they are pretty much positively charged Very low (if any) bioavailability These relaxants all have a slow onset and recovery Slow onset because 70-80% of the receptors need to be blocked before paralysis occurs Called the safety-factor of transmission which is natural protection against neuromuscular blocking agents Slow recovery because they bind tightly to the receptor and stay there We can try to reverse this by using cyclodextrins (rings of sugars) to try and 'suck out' the drug of the receptor to stop their action The blocker will cause paralysis to certain parts of the body first Eyes, then the face, then the limbs etc. But importantly, the respiratory muscles are the last to be paralysed, which also gives us some protection from death if a blocker was administered Once the agent is metabolised, the order is the same but in reverse (respiratory muscles first, eyes last) Older agents can actually block ganglionic nACh receptors Not selective enough Causes hypotension Because this is competitive antagonism of the nAChR, we can give the person a AChE to increase ACh to outcompete the blocker to restore normal function

Depolarising block These agents are agonists at the receptor Causes initial contraction, then paralysis Initial contraction comes from the activity before the phase I block sets in There are two phases which are important to the action of these agents Phase I The ion channels are kept open by the agent Therefore, depolarisation isn't possible, because it's already depolarised (kept at 0mV) The increase in voltage (depolarisation) is important, not the absolute value So ACh release has no effect on the endplate (already depolarised) Phase II Tachyphylaxis/sensitisation The ion channels naturally close due to a safety mechanism, causing hyperpolarisation again (cell voltage drops back to -70mV) They won't open again in response to ACh for a while now

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 Suxamethonium (Sux) is a clinically used agent Two ACh molecules linked together Metabolised by a cholinesterase (but not AChE) Not as quickly compared to ACh, half-life of 4-6 minutes compared to a second at most Liked by surgeons due this short period of action Give a large initial bolus dose (quick paralysis) followed by IV infusion to maintain for the duration of the surgery Causes both Phase I then Phase II block These agents CANNOT be reversed by AchE AChE just allows more agonists to be present Actually makes the paralysis WORSE!

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Medchem
ACh and SAR ACh is quite a simple molecule (shown top) Simple ester on the left Susceptible to hydrolysis (shown bottom) Quaternary nitrogen on the right Always ionised Requires an active transporter to get it across the membrane Same goes for choline as well

AChE inhibitors May be reversible or irreversible But both are competitive. i.e. will bind at the active site instead of ACh Edrophonium chloride is a reversible AChE inhibitor

This class of inhibitors all have a carbamate ester instead of a simple ester Carbamate esters are resistant to hydrolysis Stable in water (Stays for awhile in the cleft) Stable in the enzyme (the enzyme-inhibitor complex takes a few minutes to clear (compared to a few milliseconds with ACh) This is because the positive charge on the carbon attached to the serine means it's easier to remove the residues of the agent In ACh, this positive charge is quite high, which is why it's kicked off so quickly In the blocker, the positive charge on the carbon is reduced due to resonance with the nitrogen atom

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 Physostigmine is able to work in the eye to contract the pupil Contains a carbamate No positive quarternary nitrogen though Allows access to the Canal of Schlemm for optic drainage to counter glaucoma, which is where the pressure inside the eye is too great Under muscarinic ACh control Is also studied for its effects to improve cognition in Alzheimers disease

Irreversible AChE inhibitors These are phosphate esters Organophosphates The reason why they are so effective is due to the strength of bonding of the residues to the serine If you go back above, it is mentioned that the positive charge on the carbon attached to serine determines how quickly it is kicked off the enzyme The irreversible inhibitors undergo a process of aging, where it loses its ester groups while it's attached This further reduces the positive charge on the phosphate, meaning it can't be kicked off, so the active form of the enzyme can't be regenerated

Non-depolarising agents Tubocurarine is a non-depolarising agent Notice it has two quaternary nitrogen groups, which are always charged Also notice how ACh also has a quaternary nitrogen group, which is important in binding to the receptor Remember: charge not needed to bind to AChE though The distance between the two nitrogens is about 1.4nm, or about a 10-12 carbon chain This distance is very important The spacer between them is not as important

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 Pancuronium and Vecuronium are aminosteroids They use a steroid as a spacer between the nitrogens Why a steroid? The plant just makes them like that. If it works, it works. Rapid onset and medium-long acting Renally eliminated, but metabolised in the liver If either the liver or kidney are damaged, then clearance is reduced Why? Because having at least one ester intact (coloured as red) means it's still active. Therefore, since it has an active metabolite, both metabolism and excretion are important factors in clearance

Atracurium is quite interesting due to its elimination Non-enzymatic Hoffman elimination Very good, doesn't rely on the patient's organ function for elimination Plus it has a shorter half-life Instead, it is base catalysed (we still got some OH- in water remember?) Note: I don't know if we have to know the mechanism, but it's quite simple There is an alpha carbon located next to the esters These esters may be cleaved but the more important thing is the alpha carbon next to it The alpha carbon has a surprisingly acidic hydrogen due to the positive charge on the carbonyl carbon And the positive charge from the nearby nitrogen helps too

Then, this reaction occurs (called the Hoffman elimination reaction)

Depolarising agents Same as above, but these are connected by a flexible chain
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 Same as above, but these are connected by a flexible chain The most commonly used one is suxamethonium (AKA Succinylcholine) Notice it has two simple esters Rapidly cleaved by a cholinesterase (but not AChE, which is why it lingers around for longer) If a person lacks this esterase, then they are paralysed for longer (apnea) Short half-life (desirable)

Estrogen receptor modulators Raloxifene (pictured below) is an Antagonist at the breast and uterus Prevents side effects (e.g. tender breasts, abnormal bleeding etc) Agonist at osteoblasts and osteoclasts Prevents the breakdown of bone

Vitamin D Important as a hormone in its final form (calcitriol or 1,25dihydroxycholecalciferol) May be obtained from: The conversion of 7-Dehydrocholesterol to cholecalciferol by UV light falling on the skin Dietary intake of cholecalciferol Dietary intake of ergrocalciferol Obtained from UV irradiated mushrooms, actually works pretty good Converted from cholecalciferol to calcidiol (or 25-hydroxycholecalciferol) in the liver Converted to its final calcitriol form in the kidneys Calcitriol will bind to the DNA after it binds to a intracellular receptor (it's a steroid) to cause transcription, especially for calcium binding protein (or calbindin, seriously who names these things? Give him/her a medal for making calcium-related things so obviously easy!) Calbindin is used in the lumen of the GI tract to bind calcium to make it easier to absorb into the body

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Bisphosphonates Have a very simple SAR: Two phosphate groups R1 is almost always OH (clodronate is the only exception, uses Cl) R2 can be modified to change its activity

Normally in the bone, we have pyrophosphate, which looks very similar to bisphosphonates Just the middle carbon is an oxygen

Overall, the absorption of the bisphosphonates is low due to its polarity and the half-life in the plasma is short But this is offset by the fact that it accumulates nicely in the bone This is due to the phosphate groups and the OH group will chelate to calcium WARNING: this chelation means it must NOT be taken with magnesium, iron or calcium containing products. Otherwise they'd chelate and prevent either being absorbed This is REALLY important because the people taking bisphosphonates may be taking a calcium supplement as well. Tell them to take bisphosphonates at least 30 minutes before the calcium supplement There are three generations to consider: First generation Least potent Only has a simple carbon sidechain for R2 Second generation
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 Second generation More potent (x100) Has a simple nitrogen-containing sidechain for R2 Third generation Even more potent (x10,000) Has a heterocyclic ring containing sidechain for R2 Notice how all three generations have OH for R1 Except for clodronate (uses Cl, is first generation)

Their mechanisms of actions slightly differ But both rely on the fact that osteoclasts break down bone, which releases the bisphosphonate for the osteoclast to absorb First generation: Metabolised into compounds which compete with ATP to cause osteoclasts to apoptose Second generation: Inhibits farnesyl diphosphate synthase (FPPS) in the HMG-CoA reductase pathway (AKA melvonate pathway) Causes changes in the cells' GTPases which affects normal function (e.g. prevents the ruffled border formation with the bone) and cell survival and generation (make less cells, they survive shorter periods of time) There's another class of drugs which inhibits the HMG-CoA reductase pathway, and that's the statins Debate about its effectiveness in osteoporosis Not thought to be effective because it just doesn't build up in bone like bisphosphonates

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Pain (again) and inflammation

Note Very similar to the lecture given in Oncology The notes for this lecture assumes you understand everything from that lecture This lecture may reinforce or add extra details However, we will focus mainly on the drugs used specifically in this module instead so it's not a complete waste of time

Revision of the terms (some may be new) Nociceptive pain Caused by the activation of receptors in response to a stimulus Somatic pain- Easy to describe and locate as it's mapped to a certain part of the brain Visceral pain- Hard to describe and locate as it's not as accurately mapped in the brain Neuropathic pain Caused by the spontaneous activation of nerves without a response to a stimulus Acute pain Occurs from a specific identifiable incident (i.e. you knew it happened), which goes away within days to weeks. Caused due to nociceptive pain Chronic pain Not easy to figure out where the pain is coming from. May keep going indefinitely. Can be nociceptive or neuropathic A-delta-fibres Small, myelinated fibres for fast conduction. Used for physical and thermal pain to cause sharp pain C-fibres Unmyelinated fibres for slow, chronic conduction. Causes burning pain Pain pathways

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