----start---- pharmacology 1/16/98 Kotlikoff Review: Postsynaptic receptors: Muscarinic cholinergic - location, coupling, subtypes Nicotinic cholinergic - location, structure Drugs: 1. muscarinic agonists - already discussed 2. muscarinic antagonists - already discussed 3. cholinesterase inhibitors (anticholinesterases) - start this today. where do agents that inhibit ACHesterase work? what are the sequelae of these drugs? these are active at all cholinergic synapses, on both muscarinic and nicotinic receptors 4. neuromuscular blockers - start these later today. these are really nicotinic antagonists/agonists. these are very potent compounds that act on Nm at the neuromuscular junction. -- ACHesterase inhibitors: if you return to the cholinergic junction, and we're discussing ALL cholinergic synapses right now, although the slide is of a neuromuscular junction, we could be talking about a ganglionic synapse or other ACH synapse like a parasympathetic effector synapse, or something, anyway, looking at the synapse, in all cases, ACHesterase exists in the synaptic cleft. Part of the normal termination of neurotransmission is that the released ACH is immediately deesterified and broken down by the junctional ACHesterase. remember, we talked about a forest of stalks with ACHesterase molecules on them,filling the cleft, and the ACH has to get through to the other side. at neuromuscular junction, only aout 10% of the ACH reaches postsynaptic membrane. there are two kinds of ACHesterase - nonspecific, which is found in plasma, and which hydrolyzes ACH without being specific for it, and there is acetylcholinesterase which is specific for ACH, found at the synapse. the point is that if you give a muscarinic agonist medication, it will be exposed to the nonspecific pseudocholinesterase in the plasma, and may be broken down before reaching site of action. the drugs we discuss really interact with ACH at the synaptic site, and they have a specific mode of action that relates to the structure of the ACHesterase molecule. So that the ACHesterase molecule has an anionic site on it that binds the charged N+ of ACH and similar molecules. recall we discussed this charge on ACH - it binds the anionic part of the esterase, and orients the ACH on the enzyme such that it exposes the oxygen to the esteratic site where there is oxygen on the enzyme. the molecule is cleaved very rapidly, 150 microseconds (.15 milliseconds), and then choline floats off, and acetate floats off, and you have a reavailable enzyme to break down another ACH molecule. so this is important b/c as you will see, the ACHesterase inhibitors use either the anionic site or esteratic site to block enzyme function. 1. ACH + charge binds to anionic part of enzyme 2. ester bond is broken down, choline leaves, leaving acetylated enzyme 3. acetate floats off also 4. enzyme can start over that's what normally happens. now, before going into specific drugs, think about the effect of inhibition of ACHesterase. this causes you to swamp the postsynaptic membrane with ACH.the effect is to provide more ACH at this nicotinic receptor, and at muscarinic receptors. again, refer to comments from early on when we discussed postsynaptic receptors - muscarinic is G protein coupled - increased ACH will bind it, activate G proteins, keep it activated. more ACH at nicotinic receptor will effectively inactivate the receptor, because you have an ion channel on some membrane (postsynaptic), and you put in ACH, which opens the channel - if you flood with ACH, channel is always open and membrane stays depolarized and can't generate new end plate potentials - so the membrane is inactivated - or additionally, you have channel inactivation. so effectively, you get initial stimulation, disorganized muscle contractions, tetany, fasciculations (not coordinated at all), and then you get passive atony - no tone in your muscles - flaccid paralysis. ACHesterase inhibition - signs of this are flaccid paralysis and SLUD. signs of muscarinic toxicity and neuromuscular paralysis, and CNS effects to the extent that you get inhibition of ACHesterase in the CNS as well. history of cholinesterase inhibitors: these are very potent drugs. physostigmine is found in some plant, was given by african natives to people as an ordeal drug, in that if they took it and lived they were innocent, if they died they were guilty. these drugs were used as potent toxins and the real clue to the site of action came when it was found that part of the effects were antagonized by atropine. kinds of cholinesterase inhibitors: there are three to know 1. reversible: edrophonium 2. slowly reacting or carbamylating agents - physostigmine 3. irreversible - organophosphates note on cholinesterase inhibitors and clinical use - two main therapeutic uses - one is for glaucoma as discussed (slow acting/carbamylating type), and one is for myasthenia gravis, where the NMJ is structurally impaired by Ab to proteins on NMJ that disrupt postsynaptic membrane structure. the endplate potential is subthreshold so you give the inhibitor and increase the available amt of ACH. Reversible agents: edrophonium. it has a positive charge and it binds negative site on enzyme in a noncovalent way, and competitively inhibits ACH from binding to the enzyme. if you give excess ACH, you then compete off the edrophonium, so this is reversible. but, this drug is effective short term in getting more ACH to the postsynaptic membrane, and is used for diagnosis of myasthenia gravis. it's used basically for diagnostic testing. given by injection. if the animal improves after the drug is given, it has MG, most likely. this is a short acting drug - not used therapeutically, but can be used to adjust the dose of a longer acting drug like physostigmine, eg to test to see if animal will improve further. you have to fine tune it because you don't want to overstimulate receptors. if you start getting paralysis, you can reverse it. Slowly reacting substances - physostigmine, neostigmine, something-else-stigmine. these agents are used in agriculture, flea control, - they are carbamylating agents. there is a lot of toxicity associated with them. They all have a net positive charge and all act like ACH in binding to anionic site of esterase, covalently bonding with esteratic site, then their base agent floats off but leaves the carbamyl group on the enzyme. it's estimated that it takes about 30 minutes for tthe carbamyl group to leave, so the enzyme is inhibited that whole time. but then it does eventually come off. physo and neo stigmine differ a bit - neostigmine is a quaternary amine, physo is tertiary, gets into CNS more, etc. toxicity - poisoning that occurs lasts hours/several hours, not too long. we aren't mentioning, either, the muscarinic effect. the tx here would be to tx skeletal muscle weakness and paralysis with artificial respiration if needed, but also with atropine from excess muscarinic reception. toxicity signs are weakness, paralysis, and SLUD. irreversible agents - organophosphates. generally a phosphate with long organic groups hanging off it. the phosphate binds the esteratic site of enzyme, forming strong covalent bond, and never really comes off. takes longer to come off than for enzyme to break down. irreversibly destroys the ACHesterase molecule. a number of these drugs weremade by Schraeder in germany for use as nerve gases earlier in the century. we talked about antidotes to these before. a group of very toxic agents, but there are a lot of different ones, like malathion, parathion used as insecticides, or against the medfly as wide area sprays - they use organophosphates with little permeability into people. the nerve gases, though, are very permeant, do access CNS, etc. one thing about these compounds, b/c they break down the enzyme permanently you may see progressive toxicity. with low level exposure you don't see toxicity until excess esterase is removed. this may take some time to occur if it happens at say 1% a day. mechanism - depolarization, contraction of muscle and then flaccid paralysis. paralysis of diaphragm and other resp mm. if these drugs ge into the cns, it is very bad. so the therapy for toxicity is continued dilatory support (?), continued support for paralysis, and continued tx with muscular antagonist - like systemic atropine. one sort of out here kind of thing. there is a drug that reverses organophosphate interaction with ACHesterase, and that is called pralidoxine. if you have an organophosphate on the esteratic site, and you do not have the drug, you end up with an "aged" enzyme, which doesn't work anymore. before that happens, you can give pralidoxine, which has a charge and will interactict with the enzyme and kick off the toxin. but this antidote must be given quickly, before the aged enzyme occurs. if you know what the exposure is and you get it fast, you can reduce effective exposure. desert storm people had this stuff. how much time do you have before you inject it? less than an hour. of course, we never see our patients that fast. *sigh* we use these drugs for glaucoma (long acting local) and myasthenia gravis (long acting for tx, short acting for dx), ileus, bladder paralysis (rarely for those two) ....MUST.....GET....COFFEEEEEEEEEEEEEE..... SUMMARY---summary---SUMMARY we discussed cholinesterase inhibitors 1. site of action 2. mechanism - remember the difference b/w effects on muscarinic and nicotinic receptors, that being that providing more ACH to the receptor causes continued stimulation of muscarinic and initial stimulation and then blockage of nicotinic receptors 3. types of ACHesterase inhibitors - 1. reversible (edrophonium) 2. slowly reacting/carbamylating (physostigmine) 3. organophosphates/irreversibles - mainly toxins 4. antidotes - 2PAM aka pralidoxine Rx to treat toxicity with ACHesterases is to block the muscarinic effects with atropine, and to provide ventilatory support for the animals to override paralysis. ----break---- into the home stretch... a question was asked during break about myasthenia gravis and why ACHesterase inhibition is a good treatment. what happens is at the NMJ the ACH receptors are destroyed, the end plate is destroyed, and extra ACH receptors proliferate away from the MEP . normal amounts of ACH don't reach the peripheral receptors. the idea is, if you stop breaking down the ACH, more of it will reach those proliferating receptors on the sides, so you get more effective neuromuscular transmission in the face of a normal amount of ACH release. BUT if you totally wipe out the ACHesterase, you get too much ACH, and you block the receptors and get paralysis. so you ahve to carefully titrate your dose. final group of compounds - neuromuscular blockers very potent group of compounds shrouded in wonderful history. curare - found when the first europeans went to amazon basin - indians there used arrows tipped with a poison that killed the person you shot. it wasn't clear how this worked, and the indians wouldn't give them samples, so there was a long series of interactions where one guy befriended the Indians, and the medicine man shared the info, and then they took it to europe, and claude bernard worked out the mechanism - curare is purified from the strychnose plant (?) which here contains curare, and in africa contains the GABA inhibitor strychnine. these drugs bind and block the nicotinic receptor. recall the image of the MEP and the NMJ. these drugs bind the Nm receptors on the postsynaptic membrane. recall that two alpha subunits on Nm bind ACH, and the nicotinic antagonists bind those binding sites, preventing ACH from binding. there are two classes of the neuromuscular blockers - those that bind to the receptor and don't open it but that competitively inhibit ACH action - these are: competitive/non-depolarizing blockers, represented by pancuronium, a curare derivative - doesn't open channel, but binds to nicotinic sites. depolarizing blockers: represented by succinylcholine (anectine) which is really two ACH molecules linked together. binds the channel and opens it. all these drugs are charged and have structures similar to ACH. generally have poor lipid and CNS permeability, and are used as peripheral Nm blockers. vary a bit wrt affinity for Nn - they all have some variable affinity for Nn as well, though, which is present at ganglia and CNS. They have no affinity for CNS. They all have much higher affinity for Nm than Nn though. Nm is the main target. competitive blockers - normally, you start with closed nicotinic channel, ACH binds it and opens it, then pops off, gets broken down, channel closes. with a competitive/nondepolarizing blocker, it binds to the ACH binding site, but doesn't open the channel. depolarizing blocker - binds the binding site, opens the channel - acts more like having too much ACH - causes a depolarizing blockade. also the clinical features of these groups of drugs are a bit different. the competitive blockers (pancuronium, gallamine), which are nondepolarizing, are most commonly used in the clinics. they are competitive antagonists at Nm and they cause flaccid paralysis - they prevent skeletal muscle excitation. See handout for other drug names. They have effects on both postjunctional nicotinic receptors and prejunctional nicotinic receptors, which isn't completely understood. electrophysiological effects - don't worry if you don't fully understand, just try to grasp clinical features. looking at force production in response to neural stimulation of the muscle - a tetanic stimulus, a stimulus which evokes the maximal response, is given. then a regular stimulus is given and you have a post-tetanic increase in the amount of force - post-tetanic potentiation. Ok, so now you give pancuronium and the normal twitch is about 1/2 of the normal force - there is muscle weakness. a prominent feature of these drugs is "tetanic fade" - tetanus is not sustained as it normally is - and it is felt that normally during tetany, you bind prejunctional receptors to cause more ACH release, but now those are blocked and you therefore can't sustain the tetany. Now, you also still do get the post-tetanic potentiation, and we're not sure why, that may not be due to prejunctional stimulation but due instead to changes in the muscle. so if you have an animal anesthetized, and you test the degree of NM blockade, you'll look for this tetanic fade. no one really understands this but it is there. it is felt that the prejunctional receptors are blocked, preventing the normal positive feedback which would cause the relase of more ACH during continued stimulation. a more clearly understood feature of nondepolarizing, competitive blockers, is if you look at the combination of one of these and an ACHesterase inhibitor - you do your twitch survey, provide curare, and you see the strength of contraction fall as you block more and more nicotinic receptors. then, if you add neostigmine, an ACHesterase inhibitor, ACH will compete off the curare, and that's why it's called competitive - it competes it off and the action is reversed. it happens because when you give the esterase inhibitor, more ACH is available in the synapse, and the effect can be reversed. contrast this with the depolarizing drugs like succinylcholine which is really the only one used clinically and even so is used rarely which depolarize and open the Nm ion channel. There is a marked distinction b/w the effect of an ACHesterase inhibitor's action on receptors blocked by these drugs. after giving succinylcholine, force of contraction decreases as more Nm are blocked/opened - then, if you put on neostigmine, increasing amt of ACH available, you exacerbate the block. already, you have the equivalentof too much ACH there, because you've opened all the channels and depolarized the end plate - so this isn't a competitive situation. the ACH can't compete off the succ. the ACH will just try to bind receptors and open them - but that just makes it worse. that's really the fundamental difference. when you first apply the depolarizing type blockers, you initially have an excitatory phase marked by fasciculations, then you get flaccid paralysis. ok, this gets a bit more complicated - the depolarizing drugs have two phases of block - phase one, as described, where it binds, depolarizes end plate, causes generalized fasciculations, followed by flaccid paralysis. then, while channel is open, muscle becomes depolarized, stays that way, and is unavailable for activation - HOWEVEr, what happens now is that the channel inactivates. the Nm do not like being held open. so after phase 1, which doesn't last that long - a few minutes - the channel inactivates, the postsynaptic membrane repolarizes, and we have the succinylcholine still sitting there, but if you compete it off, you can open the channel, so phase two mimics a competitive blockade. this complicates it only in the sense that phase 1 is a depolarizing one, but after that the muscle repolarizes and you have a competitive block, phase 2. so in phase 2, it's reversible by increased ACH. clinically, this phase 1 is undesirable because you do not know how long it will last, it is unpredictable, and you don't want to try to reverse it and end up getting worse because you are still in phase 1, so you tend to use the competitive agents more often. summary competitive depolarizing phase 1 phase 2 initial effect none fasciculations none ACH reverses augments reverses ACHase inhibitors reverses augments reverses recovery rate 30-60 min 4-8 min >20 min channel config closed open closed NOTE: in handout there is an ERROR. ** in phase 1, the channel is open ** in phase two the channel is closed ** this is a big mistake in the handout!! ignore the handout on this. i have it correct here. a few words about systemic effects. all neuromuscular blockers cause histamine release, which really sucks, and this is why gallamine is preferable to some other drugs (i guess it causes less?). there are cardiovascular effects of rapid injection of D-tubocurare because it affects Nn. the predominant cardiovascular tone is adrenergic. blocking both limbs causes hypotension and tachycardia. Pancuronium has some ganglionic stimulation and causes hypertention via vascular smooth muscle contraction, and other effects. cautions: agents commonly used clinically have some neuromuscular blockade - inhaled anesthetics have effects at NMJ, so be careful about using these other drugs with them, have to reduce doses, etc. aminoglycosides also cause some neuromuscular blockade. anesthesiologists will always find out what other drugs are goign to be used in patients. clinical uses are mainly during artificial ventilation, during surgery, to paralyze respiratory muscles to allow efficient mechanical ventilation; also during orthopedic procedures, where you need to get a lot of relaxation of muscle tension, also ophthalmic surgery, and you don't want to use that much anesthetic, because you don't want the negative cardiovascular effects. you can paralyze the muscles with the NM blocker and use less anesthetic - that's safer. However - you have to remember that these drugs do not block pain. they just paralyze. you must maintain appropriate anesthesia. the animal can't struggle, cry out, etc - you have to be careful. historically, people have used these drugs to paralyze/rapidly immobilize wild animals, and then doing a procedure like a castration - and this is very painful! you can't do that. it's inhumane. you must monitor cardiovascular response for signs of excitement or pain. the animal can't move. now, you can use these drugs to knock down an animal, then immediately sedate it, and anesthetize, or something. that's ok. clinical reversal by ACHesterase inhibitors is sometimes used, but more often they just allow blocker to wear off, generally that's felt to be safer. cholinesterase inhibitors are never used with depolarizing blockers b/c you can't really predict the effect. summary--SUMMARY--summary 1. Nm blockers a) competitive (nondepolarizing) eg pancuronium - don't open channel, marked by "fade of tetany" which can be used to check for presence of competitive blockade. can be reversed by ACHesterase inhibitors b) depolarizing eg succinylcholine - open channel phase I, closed phase II (phase 1 augmented by ACHesterase inhibitors)(phase II mimics competitive blockade) 2. effects of these agents - cardiovascular effects, histamine release, no sedation or analgesia. only immobilizing agents. do NOT cause pain/stress without using other pharmacological agents as well. ----end----