----start---- pharmacology 1/19/98 Fluharty: sympathetic nervous system overview: today we'll review some properties of the SANS, anatomically and functionally, and review the mechanisms which govern catecholamine synthesis, release, and function. we'll do this b/c these are the NTs that are released from postganglionic neurons of the SANS, and that the drugs we use clinically really impact on catecholamine function. then we'll discuss the drugs that alter, eg enhance or reduce, sympathetic activity. we'll discuss how they work, when to use them, some of their limitations, and so forth. end overview. slide: figure that isn't in the handout. just a reminder about the ANS and the sympathetic part of the ANS. remember ANS split into SANS and PANS. in most tissues, the two systems have reciprocal/antagonistic activity. anatomically, the important thing to remember is that the effector side, eg the means by which the SANS regulates target tissues, involves two neurons in series. this is very very important. the first neuron is in the CNS (thoracolumbar cord) and is called the preganglionic neuron, because once they exit the cord, they synapse w/in the autonomic ganglia. all the neurons making up sympathetic outflow have cell bodies in thoracolumbar segments of the cord. ultimately, the preganglionic neurons synapse on the second neuron, whose cell body is in the ganglia, and whose processes innervate target organs. these are the postganglionic neurons, that release the catecholamines and regulate target tissue activity via NT release. preganglionics use ACH, not catecholamines. we're not going to talk about the preganglionic neurons to any extent; we're going to focus on postganglionic neurons. note regarding terminology: because the synapse we care about is the postganglionic neuron's synapse on the effector organ - this is where the drugs we use work - if a drug affects the terminals - say the synthesis or release of catecholamine - we call it a presynaptic adrenergic drug. presynaptic is not preganglionic, ok? we're talking about presynaptic with reference to the postganglionic synapse. when we talk about drugs that affect the postsynaptic side, the receptors, of the neuron/effector synapse we call them postsynaptic drugs. don't confuse presynaptic with preganglionic. also, the SANS has an endocrine component. when we look at the whole thing of how catecholamines regulate biological activity, we have to think of adrenal medulla, which releases catecholamines into circulation. it is innervated by preganglionic neurons of SANS, and when activated it releases catecholamines into the general circulation, not to a specific target organ. some of the SANS activity is a result of this release of NT into the blood. when the adrenal medulla releases catecholamines as part of an overall sympathetic event, we call it a sympathoadrenal response. release of epi and small amount of norepi via this endocrine loop is very important in stress response. b/c it is epi being released from adrenal medulla, we have to know what epi does relative to norepi. and any drug influencing target tissues, if it resembles epi more than neurepi will produce endocrine type response, if it resembles norepi more, will look more like neural response. why do we care about SANS? see table one p 2 of handout. the point is - all the major effector organs involved in cardiovascular and metabolic homeostasis - critically involve the sympathetic division of the ANS. heart, vasculature, bronchial smm muscle, GI, renal events. sympathetic innervation and function are required for normal function or for reestablishing regulation of these organs. this is why it is so important. also, the key to understanding drugs that affect the SANS is to memorize this table ** there is no substitute for this. learn and retain this information ** on the right are the receptor types listed. this means that if you know this table, and someone says "we're gonna use a beta 1 selective adrenergic agonist to tx a heart problem" you will know that this will increase HR, CO, force of contraction, and conduction rate. knowing this will help you know when to use selective agonists and antagonists. actions of SANS regulation of HR and contractility regulation of bv constriction regulation of sm muscle contraction regulation of secretory processes CHO and fat metabolism need to know how catecholamines work. why? they are what influence tissue activity in the body. therefore if a drug augments this activity, it augments sympathetic function, and if it inhibits it it will reduce sympathetic activity. review of catecholamines: think back to neuroscience. the synthesis of catecholamines. we're most interested in postganglionic neurons of SANS. these neurons have high affinity uptake mechanisms for tyrosine. then, tyrosine hydroxylase (rate limiting, regulatory step) changes tyrosine into DOPA. so the rate at which catecholamines are produced is directly determined by activity of tyrosine hydroxylase. as the rate limiting enzyme, it can undergo a lot of changes. if postganglionic neurons rae repeatedly stimulated, tyrosine hydroxylase activity can rapidly increase, to sustain catecholamine release. if you block the enzyme, eg with a drug, you interrupt the synthesis of catecholamines, which will deplete the tissues of a pool of catecholamines, shutting down the sympathetic nervous system. this would be a presynaptic adrenergic blocking drug, acting within the synapse and nerve terminal of the postganglionic neuron. so anyway - tyrosine is hydroxylated into DOPA. then, DOPA decarboxylase (a nonspecific enzyme seen throughout the body) will decarboxylate the DOPA, leaving you with dopamine (DA). in neurosci, we were very concerned about dopamine, because we know it is a critical CNS NT. however, in the ANS, and the SANS that regulates visceral function, dopamine plays a much lesser role. may be important in renal vasculature, and there may be other roles we aren't fully aware of, but overall it is a lesser player. it's more of a precursor (not entirely, but mostly). so dopamine is transported into vesicles and converted by DAbetahydroxylase into norepinephrine. now, we know that norepi will be the NT of the postganglionic neurons. it interacts with the alpha and beta receptors to produce the effects seen in table one. BUT in the chromafin cells of the adrenal medulla, norepi is converted to epi by phenylethanolamine N methyltransferase - epi is really methylated norepi. adding that methyl group to the terminal amine changes the biological activity of the NT very significantly in some tissues. epi is only derived from the chromaffin cells of the adrenal medulla. norepi, however, is mainly released from postganglionic neurons - although chromaffin cells may also release small amts of norepi. mostly the circulating catecholamine is epi, though. and norepi mostly found in synapses. drugs that interfere w/catecholamine synthesis are useful. if there is too much sympathetic activity, you can give a presynaptic adrenergic blocking drug, shut down synthesis, diminish release, reduce overall sympathetic activity. that's the plus. the downside of these drugs, and the reason they aren't commonly used, is that they're pretty nonspecific. they affect synthesis and release of catecholamines through the whole SANS. ideally, you want to control a particular target tissue. that tissue will probably express a catecholamine receptor that isn't shared by all other target tissues, so by blocking receptors you can get a lot more specificity. that's the main reason that drugs affecting postsynaptic receptors are more specific for tissues and have greater usability. drugs affecting synthesis and release are less useful, although still sometimes used. presynaptic adrenergic blockers - inhibitors of CA synthesis alpha methyl p tyrosine - interferes with tyrosine hydroxylase activity. is taken up into nerve terminals like tyrosine, b/c it looks like tyrosine. competitively inhibits tyrosine hydroxylase by acting as false substrate. because it is inhibiting the rate limiting enzyme, it's very useful for shutting down catecholamine synthesis. presynaptic adrenergic blocking drug. reduces pool of catecholamine available in the body. used in treatment of pheochromocytoma. this is a tumor of the chromaffin cells in adrenal medulla which causes excess release of catecholamines - huge increase. all the SANS activity is increased - HR way too fast, BP way too high, GI motility all weird. ideally, to treat this, you get rid of the tumor - but, one problem with the surgery is that physical manipulation of the tumor causes greater release of catecholamines (CA). so preoperatively, you use this drug. that will inhibit production of catecholamines everywhere, including the tumor, then you remove the tumor, stop the drug, and hopefully the patient returns to normal. alpha methyldopa- inhibits aromatic AA decarboxylase. interesting drug, still used in some cases to tx high blood pressure; not in animals but in people. it illustrates the complexity of these drugs. we think it's easy to understand, because it inhibits dopa decarboxylase - one step further down from tyrosine hydroxylase - so we thought it depleted tissues of catecholamines by a very similar mechanism to the drug described above. but it is really much more complex. what happens is (see cartoons at end of handout) it acts as a false neurotransmitter - alpha methyldopa gets converted to alpha methylnorepinephrine. see fig 7 pg 19. it not only interferes with norepi synthesis, but it also can be itself converted extracellularly to alpha methylnorepi. then, it gets taken up by nerve terminals and stored in the vesicles that normally would release norepi. so over time, vesicles lose norepi and get full of the false NT. the alpha methylnorepi functions as a false NT - that means that when the neuron is stimulated by an AP, after drug was given for a long time, it doesn't release norepi, but it releases the false NT. this alpha methylated norepi, which isn't epinephrine either, the methyl group on epi is on the terminal amine and here it is on the alpha carbon - anyway it produces less biological activity at the postsynaptic receptors than does regular norepi. so when false NT is released there is less biological activity produced in effector tissue. so that's another reason that sympathetic activity is reduced by alpha methyldopa - not just because it is blocking synthesis of norepi. it is still not the whole story, though. in the brain, alpha methyl norepi is rather potent at alpha 2 receptors, and so then you get decreased descending control of the sympathetic nervous system. so there is descending inhibition of sympathetic outflow as well. the reason this can happen is that there are multiple receptors for catecholamines, and alpha methyl norepi is less effective at the alpha 1, classic receptor, but actually more potent at the alpha 2 receptor in the CNS, which produces decreased sympathetic outflow. so those are two examplse of drugs with clinical utility, both of which interfere w/CA synthesis and are presynaptic adrenergic blocking agents. once CA are synthesized, they are stored in vesicles. vesicular storage is really important. by storing them this way you prevent degradation by monoamine oxidase which is intracytoplasmic in the neuron. if you don't store the CA in vesicles, it can be degraded by MAO. also, the generation of NE is dependent on vesicular storage, b/c dopamine beta hydroxylase is in the vesicles also, so you have to get the stuff in the vesicles so it can be synthesized. anything interfering with vesicular storage interferes with release of CA. anything not in vesicles is degraded by MAO, and isn't further converted to NE. inhibitors of catecholamine vesicular storage: reserpine - alkaloid from an Indian climbing shrub, serpentine type plant. blocks uptake of CA into storage vesicles. actually it also damages the vesicles irreversibly. this means that if you give reserpine, recovery from the drug is slow. after drug is removed, recovery isn't established until new vesicles are made. drugs that act this way, whether they inhibit enzymes or damage receptors or whatever, have slow recovery times and cumulative effects with repeated doses. as you might predict, this drug causes tissues to be depleted of norepi, b/c the norepi that's already been made is exposed to the MAO and readily deaminated. moreover, we interfere with synthesis of catecholamines, because the vesicles are damaged, dopamine doesn't get into them, so it doesn't become norepi. consequently, the SANS tries to compensate. this is very common. often, what you try to do clinically (reduce CA activity here) with a drug, is opposed by compensations within the NS. so now, postsynaptic receptors get very very sensitive. when postsynaptic receptors don't see normal amounts of NT, they tend to get very very sensitive. so you reduce amt of norepi released, but receptors get more sensitive. now, with this drug, this isn't a bigdeal, b/c the drug is so effective that very very little norepi will reach the receptors. however, once you remove the drug, the supersensitive receptors can be very important in recovery process, b/c normal norepi release will have too great an effect. this is an undesirable effect of the blocking drug, and is a common effect of drugs that block norepi. when you withdraw drugs like this, you have to be really careful to monitor recovery, because you might get a restoration of increases in BP, tachycardia, etc. These are called "withdrawal syndromes" where the NS overcompensates for things. reserpine also inhibits tyrosine hydroxylase action, so neuron makes more of it, and then when you remove drug, you end up with more of it working... Presynaptic adrenergic drugs - drugs affecting exocytosis once CAs are synthesized, they are stored in vesicles and released via exocytosis. the exocytosis is regulated by what we call presynaptic autoreceptors. for CAs, beta 2 receptors facilitate release of CAs, and alpha2 receptors inhibit release of CAs. so there is feedback of CA in synaptic cleft onto the presynaptic facilitory or inhibitory receptor. one way then that you could influence ongoing release of CAs would be by using alpha2 selective agonist (to inhibit CA release) or beta2 selective agonist (to increase CA release). so, strictly speaking, these would be presynaptic adrenergic drugs. but, since they directly interact with known receptors also seen on postsynaptic membranes, we call them receptor directed. Clonidine - interacts with presynaptic alpha2 receptors as an agonist, inhibiting norepi release. many other drugs do this by other methods, like guanethidine and bretylium, which interfere with arrival of APs within nerve terminal, acting a bit like local anesthetics, preventing release of CAs through a distinct mechanism not using the autoreceptors. for guanethidine, it frequently enters nerve terminals via high affinity uptake mechanism, and tends to displace norepi from vesicles once it gets in there. if a drug does this, it may have a transient period of increased sympathetic activity - as the drug displaces norepi and norepi gets released, it produces a transient peak in sympathetic activity - an "initial sympathomimetic action". then, over time, CA release is reduced, and you have longterm inhibition of CA release. ---break--- other drugs inhibiting norepi release Ok, now we've covered drugs regulating CA synthesis, storage, and release. obviously altering any of these things affect how CA influences target tissue. termination of CA action: there are two main ways to inactivate CA. one way uses uptake mechanisms to remove CA from the synapse after release; terminates ability for it to interact w/receptor by removing it from site of action. second way involves enzymatic transformation/degradation. enzyme way: there are two enzymes - these enzymes transform CA to inactive forms 1. Monoamine oxidase (MAO) which acts on epi and norepi, beginning to degrade it by removing the terminal amine. this amine is critical for interaction b/w NT and receptor. without terminal amine you have a very unstable compound which is quickly changed to an inactive metabolite. this is a mitochondrial enzyme, particularly important for neuronal degradation. it deaminates CA that isn't in vesicle. especially in the brain, it is highly associated with the nerve terminals themselves. but in the ANS, we know that MAO can be associated with nonneuronal tissues like the liver. if you use an MAO inhibitor (used to be used for depression in humans very commonly, and also for heart dz), you prevent this initial step in the enzymatic degradation of catecholamines, thereby augmenting the action of catecholamines. you increase release and the amount of receptor/NTinteraction. these drugs enhance sympathatic activity. 2. catechol-O-methyl transferase (COMT) - adds a methyl group, creating an unstable intermediate which is rapidly oxidized or reduced and no longer interacts with the receptor. this is mainly an extraneuronal enzyme, associated with effector organs and the circulatory system. most likely it influences circulating catecholamines released from adrenal medulla. right now no drugs are specifically related to blocking this enzyme. all drugs which augment catecholamine action by blocking enzymes are MAO inhibitors. uptake processes way - principal mechanism for termination of CA actions 1. once norepi is released, it can be recaptured by a high affinity uptake mechanism called uptake 1. blocking this leads to potentiation of catecholamines, either endogenous or exogenous, because you are preventing their removal from the synapse, prolonging interaction with receptor. this is a low capacity system. 2. uptake 2 - (note that these uptake mechanisms are both transporter proteins that bind catecholamine, and internalize it once they have it bound.) uptake 2 is associated with nonneuronal terminals, usually effector organs - a low affinity high capacity system, takes it up, so it can be degraded by COMT. because uptake 2 is so low affinity, most uptake related drugs inhibit uptake 1. uptake 1 inhibitors include cocaine - a powerful inhibitor of uptake 1 which greatly potentiates actions of norepi and epi, resulting in life threatening effects as well as enjoyable (for some) effects; and tricyclic antidepressants such as imipramine. these drugs also interfere with uptake 1 and potentiate catecholamine activity. nowadays, SSRI drugs are probably used more for tx of depression. these are also presynaptic adrenergic drugs. they augment catecholamine activity. they increase sympathetic activity. ultimately, everything catecholamines do is a result of their ability to interact with high affinity at receptor sites, and trigger biological activity at those sites at the effector organs. now, most autonomic pharmacology focuses on the receptors. go back to table 1. learn where the receptors are expressed and what biological effects they mediate. ultimately, if you're going to know what a beta 2 selective antagonist does, you have to know where those receptors are and what normally happens when catecholamines interact with them, then you can know what the antagonist will do. one way to do this is to take the table and reclassify it a bit by classifying target organs based on the receptors they express. note that beta 3 receptors were recently found in fat, and are important for metabolism of fat. alpha receptors are largely found in vascular smooth muscle. alpha and beta 2 receptors are found together in some tissues. this is a good way of figuring it out. know where the receptors are expressed, and what is the biological outcome of occupancy of the receptor. how do you classify these receptors? some guy Alquist in 1948 observed that catecholamines act in many target tissues, but the agonists do not always act identically in all the target tissues. he noticed that even though norepi and epi affect a large array of target tissues, the drugs we use to mimic them can be more tissue specific. isoproteronol mimics CA in the heart, incrases HR, Fc, and conduction rate. so isoproteronol in the heart is a beta selective agonist. but in vascular smooth muscle, it produces vasodilation. that's because alpha receptors are found in vascular smooth muscle. so this guy called receptors stimulated by isoproteronol beta receptors, and those that weren't, alpha receptors. phenylephrine is an alpha selective agonist. epi and norepi have alpha and beta effects, but we design drugs to be more selective. First we just had alpha and beta - and this was created totally on the basis of pharmacology, but they do in fact represent distinct receptor families. in fact, they can be subdivided into alpha1, alpha2, 1a, 1b, etc. and beta receptors can be similarly divided into beta1, beta2, and beta3. the key is that as we learn more about the unique tissue distribution of the receptors, we can start to design drugs selective for a receptor, and therefore for a given biological action. this is a big step forward compared to presynaptic acting drugs which affect the entire spectrum of CA activity. these drugs are very selective. a beta3 selective agonist will increase sympathetic activity only in adipose tissue. so you won't get unwanted cardiac and respiratory effects. sybtype classification alpha1 - in vasculature - high affinity for epi, a bit less for norepi, and none for isoproteronol alpha2 - presynaptic autoreceptors - epi and norepi about equal, isoproteronol ineffective beta1 - in heart - isoproteronol very potent, like epi, about the same as norepi beta2- isoproteronol very potent, then epi, and norepi hardly effective at all. you have to understand not only relative affinities of natural compounds, but also drugs. see p 4 of handout. once you know a drug's pharmacological spectrum of activity, and tissue distribution of receptors, and biological activity of natural compounds at the receptor site, you'll be able to figure this out. there is a bit of complexity here that isn't in handout - we know that alpha2 receptors are not only presynaptic autoreceptors - they are also in many blood vessels, but are away from the synapse, so probably respond to circulating catecholamines - more important with sympathoadrenal responses, we think. this is important b/c if you give an alpha2 selective compound, you will have some vasoconstriction resulting. but this will be an extrajunctional effect. so it isn't good enough to just know what tissue expresses a receptor, but also know if it is uniquely presynaptic, postsynaptic, or both. think about alphamethyldopa - it isn't so simple, like we originally thought. things rarely affect only one receptor subtype, and tissues rarely express only one receptor subtype. also remember, catecholamine receptors are G protein coupled receptors. this is important - it means virtually all catecholamines and related drugs interact with these receptors, that in turn influence second messenger production. so you have to generate things like adenylate cyclase activity, increase cAMP, or influence phospholipase 3, IP3, cause calcium release, activate kinases, etc. so maybe you can influence these things too. beta 2 receptors are found in airway smooth muscle, and cause relaxation of it when stimulated, via generation of cAMP. when norepi or epi or a drug like that interacts w/beta 2 receptor, it causes increased cAMP level in cell via G protein action, and then a series of events occurs leading to muscle relaxation. so to augment ability of beta 2 agonist to increase the relaxation, you could give another drug that potentiates the 2nd messenger pathway. eg, cAMP as an intracellular 2nd messenger, when production is driven by receptor occupancy, is a transient event - rapidly broken down by phosphodiesterases. so you increase it with your beta 2 agonist, and then also give a phosphodiesterase inhibitor, so your cAMP level stays elevated for much longer, producing a (theoretically) greater level of bronchial smooth muscle relaxation. so when you see combinations of drugs being used, this is probably why. another thing about these receptors - they aren't passive participants - they undergo receptor adaptations that influence activity. for example, if you give an agonist to CA receptors for a long time, you will eventually desensitize the receptors. this involves an uncoupling from the pathways leading to cAMP production. this is a problem b/c it minimizes the continued utility of an agonist. in extreme conditions, it necessitates a drug holiday (not a holiday with drugs; a holiday without them!!) this allows receptors to become resensitized to the agonist. the other side of the coin is supersensitivity as we already discussed - when you use antagonists, you get supersensitive receptors. as long as the antagonist is in the animal, this isn't a problem and in fact you don't even know about it but if you try to remove the antagonist, now your tissue is supersensitive, and you generate an overwhelming, increased response, a restoration in exaggerated form of whatever you were treating with the antagonist. this is what we discussed about reserpine. this is what leads to a withdrawal syndrome. lots of drugs that block CA receptors must be removed carefully because of this receptor adaptation - supersentization. that gets us finally to the first class of drugs to discuss: sympathomimetics: these activate the SANS by causing receptor activation. they come in two main forms, although the distinction isn't so clear biologically. - directly acting: isoproteronol - interacts directly with receptors of the beta type - a nonselective beta receptor agonist, with equal selectivity for beta 1,2,3, but no alpha activity. phenylephrine - nonselective alpha agonist, with no effect on beta receptors. norepi affects both alpha and beta receptors. these are all directly acting. these drugs can have unique tissue distribution of action, can be very selective, unlike indirect acting. -indirectly acting - drugs that cause release of CA - causing biological effect of the actual CA that is released. so the drug promotes release of endogenous substances that then react with receptor - amphetamine, ephedrine, tyramine. these can't have biological specificity like direct agonists, because the effect isn't the result of the drug but of the endogenous compounds, which are nonselective. many drugs are mixed - may initially cause displacement with minimal selectivity, then become direct acting with high selectivity. major factors determining actions of sympathomimetic amines: 1. relative potency of amine in activation of alpha or beta receptors. how selective is it? does it interact with both types? with only one? with only a subtype? note that this information is useless if you have not memorized table one. 2. proportion and density of the various receptors or receptor subtypes in effector organs. this is on table one. you must learn table one!!! 3. reflex or homeostatic adjustments which the organism will make in response to the actions produced by the amine. the body may act to counter the effects of the drug. if you use a drug to decrease BP, say an alpha1 antagonist, lowering vasoconstriction, then the baroreceptors in the vasculature may say "hey. what's going on" and cause a compensatory increase in HR (tachycardia) to try to bring BP back up. that's a homeostatic adjustment that impacts on ability of drug to do its job 4. refractoriness of receptor - will it get desensitized? when? how long? how much? partial agonists tend to desensitize less than full agonists. ---end---