---start--- pharmacology 1/23/98 fluharty handout on nitric oxide was distributed. so far, we've been discussing only biogenic amines as far as NTs go. we discussed ACH, epi, norepi...now, we're going to talk about the AA NTs, that are probably the predominant type, both inhibitory and excitatory, and we'll also discuss peptide NTs. as we begin to prepare ourselves for the CNS pharm lectures, we need to know this stuff because these NTs are affected by tranquilizers, analgesics, and anesthetics. AA neurotransmitters: amino acids can be inhibitory or excitatory. It has to do with the postsynaptic receptor they interact with. they are the predominant type of NT in the CNS. inhibitory: GABA - gamma amino butyric acid - very important in clinical action of anxiolytics (benzodiazapines). This is an inhibitory AA NT. it is widely distributed through the CNS - unlike many other NTs that are generally found only in certain regions of the brain, this is all over - throughout cortex, brainstem, and subcortical regions. this is because it functions as an important inhibitory NT for interneurons. some areas have more of it - the substantia nigra has a lot of GABA (in all species that have been studied). this is important b/c GABA inhibits dopaminergic neurons involved in regulation of motor control and basal ganglia function. it is the analysis of GABAs inhibitory action in substantia nigra on dopaminergic neurons that has helped us to understand how gaba functions, and was first exp't setting in which mechanism of benzodiazepine function at a cellular level was clearly worked out. helps us understand CNS control of movement. GABA neurons projecting from striatum have inhibitory synapses on the dopaminergic neurons in substantia nigra, and this is where benzodiazepine action was explored. it is also important in controlling movement. so GABA like many AA NTs has fairly wide distribution in the brain. it is synthesized from glutamate or glutamic acid by glutamic acid decarboxylase -GAD- the only and therefore the rate limiting enzyme in its synthesis. highly regulated enzyme. there are many forms of GAD - there is a smaller mw form and a higher mw form. the significance isn't fully understood. But it's GAD65 or 67 the presence of GABA takes a cell that might have used glutamate for excitatory transmission and turns it into a cell using GABA for inhibitory transmission. GABA is stored in vesicles and released by exocytosis as other NTs we've discussed. the opening of calcium channels in response to depolarization is again what causes docking and fusion of vesicles. once released, GABA interacts w/a bunch of receptors. how is GABA inactivated? it can be taken back up by high affinity uptake mechanisms like the monoamines - this terminates the interaction of GABA with its receptors, shutting down its biological activity. so uptake is important for this function. there is also GABA transaminase, GABA T, a mitochondrial enzyme in nerve terminals and postsynaptic cells, so there is also enzymatic degradation, which converts GABA back into glutamic acid. GABA receptors: now we see increasing levels of complexity. for a while, we thought all the receptors were ligand gated ion channels. all the early literature talked about it binding a ligand gated ion channel with high affinity, initiating a Cl- conductance, hyperpolarizing the postsynaptic cell, lowering resting membrane potential. the story seemed complete with that. there were other complexities that emerged. one big one was, how many of these channels were there? cocktail conversation tidbit: these ligand gated ion channels are almost always pentameric proteins - made of 5 distinct protein subunits that when assembled form a channel that can be in Open or Closed configuration, depending on receptor occupancy. For the GABA A receptor, this is true. it has 5 subunits (a,b,g,d) that assemble, form a channel, and when GABA binds, it opens, allowing Cl- conductance. but then, molecular biologists started looking into things. it turns out there are 16 distinct subunits that have been cloned for GABA A receptor. if you take those and put them together in a combination of 5 - any 5 of those 16, 5 as, 5 bs, 3 as and 2 gs, or whatever - you put these into any cell, in every instance, you end up with the GABA receptor that will bind and promote Cl- conductance. so if you look at 16! it's 524,000 possible GABA A receptors. there is no evidence in any tissue or any spp that there are actually that many receptors expressed. this may simply be an artifact of the combinatorial experiments. but it raises the possibility that the degree of receptor heterogeneity for GABA A receptor may be huge, bigger than any other described to date. how many GABA A receptors have been actually described? ssomewhere around 5 to 10, not anywhere close to that. so it is a pentameric protein, made of 5 subunits, any of which assembled together will give GABA binding and Cl- conductance - but might not act just like the actual ones that are expressed do. also, over last ten years, we've found other GABA receptors. there are also GABA B receptors. these are not ion channels, they are G protein coupled receptors. GABA binds them, promoting an interaction b/w occupied receptor and GTP binding protein, which activates an intracellular enzyme. these receptors have been reported to be coupled to regulation of cAMP and IP3. important point: it turns out that GABA is inhibitory regardless of which receptor type it interacts with. it's easy to understand how the GABA A part works. what about GABA B? well, when level of 2nd messenger is changed in cells that express GABA B, it's a slower development of inhibition where 2nd messenger feeds back to regulate K+ channels, increasing flow of K+ out of the cell, hyperpolarizing the membrane. so the basic fact of inhibition is maintained. the difference is the time frame - A is rapid, B is slower. also, A is Cl- conductance, B is K+ conductance. Ok, back to GABA A and benzodiazepines - in human med, long history of use and abuse as anxiolytics. were touted as magic bullet, because early on no one considered the abuse potential of these drugs. they peaked in usage around the 1970s when they were in the top 3 prescribed drugs, used for all kinds of things. it turned out to represent a far too indiscriminate use of these drugs. dr klide will discuss their use in vet med. what are benzodiazepines? well, they act on the GABA A receptor. the GABA inhibition of dopaminergic neurons in substantia nigra was studied. the first suggestion for action of benzos was that they acted on the GABA A receptors by increasing the Cl- conductance that GABA itself caused. the idea was maybe they were just GABA agonists, and like GABA acted at high affinity to promote Cl- conductance, and that enhanced inhibition in CNS would reduce anxiety. well, clearly, benzos facilitate GABA mediated Cl- conductance - but benzodiazepines themselves are not GABA agonists. they do not, in the absence of GABA, promote Cl- conductance. they depend on GABA. this was easy to show, because in this synapse, the only way a benzodiazepine would promote inhibition of dopamine was if the presynaptic cell was releasing GABA. but it did enhance inhibition when the first neuron was releasing GABA. so, maybe the benzodiazepine was an indirect agonist, promoting release of GABA? no. benzos don't enhance GABA release. the interaction is occuring at the receptor. it involves GABA and benzodiazepines together. a model was put forth: this model is purely hypothetical. there is not evidence to support the whole thing. it is an attempt to create a construct in which benzodiazepines might work to enhance GABA transmission. idea is that the GABA A receptor, that is made of protein subunits, has certain functional domains, one of which is high affinity GABA binding site, another of which promotes a conformational change after binding GABA, and now there is a proposed regulatory subunit, normally occupied by gabamodulin, an endogenous molecule that when bound, retarded or limited in some way the ability of GABA to drive Cl- conductance. the benzodiazepines were proposed to compete for this site with gabamodulin, and when they took over the site, increased ability for GABA to promote Cl- conductance. see diagram p 2. now, why propose this model? well, why would you think that there would be a binding site for an artificial exogenous substance like benzodiazepines that aren't made by the brain? the brain must make something that interacts with the site that the benzos interact with, otherwise why is the binding site there, right? That's why we propose the gabamodulin molecule. now, why is gabamodulin proposed to be inhibitory? well, benzos increase Cl- conductance, right? so probably something else was inhibiting it and when inhibition is removed by benzos, Cl- conductance is facilitated. sounds feasible. has an understanding of the subtypes provided insights into the validity of this model? no, not really. we do know that of the subunits making up a GABA A receptor, the gamma subunit is critical for benzodiazepine action. if you construct a GABA A receptor as discussed before, without including the gamma subunit, your artificial GABA A receptor will not show the benzodiazepine facilitation of Cl- conductance. so, maybe the gamma subunit is the benzo binding site? nope. you can show clearly that if you have only gamma subunits, benzos don't bind to those either. it's something about how gamma subunit interacts with other subunits that confers benzodiazepine action. why is this useful information? well, it suggests that as we probe the brain, and try to understand benzo action, we can figure anywhere GABA A receptors with gamma subunits exist, benzodiazepines will act, and anywhere where GABA A receptors don't have gamma subunits, benzo won't act. moving on. Glycine: unlike GABA, this isn't the product of an enzyme. also, although GABA is the prototype AA NT, it's not really an AA, it's a decarboxylated AA. but glycine is an endogenously expressed AA like other AA NTs, product of ongoing metabolic events in the cell. we call this the metabolic pool of AA - when a cell captures some of this NT and packages it into the functional reservoir - eg vesicles - then the neuron has the capacity to actually release it during exocytosis. there is no enzymatic conversion. there is compartmentalization of metabolic pool of AA into a functional, vesicle packaged reservoir. glycine is an important inhibitory NT, but has more limited sphere of action. found mainly in medulla and cord inhibitory interneurons. released during exocytosis from a functional, sequestered pool. inactivated by high affinity uptake systems, but not by enzymes. glycine interacts with ligand gated ion channels, and that may be exclusively true - there is debate about this. all physiological actions of glycine clearly involve ligand gated ion channels that promote Cl- conductance, though. one thing we know involving glycine receptor is the action of strychnine, the poison. it turns out that it's a competitive inhibitor for glycine ligand gated ion channels. glycine usually produces inhibition. strychnine competitively inhibits the inhibition. this shifts the dose response curve to the right, without changing the maximal response. as a consequence for this (see diagram p 3), you remove some of the inhibition produced by these interneurons, and you create convulsions and death due to excessive neural excitation in critical brainstem areas. so strychnine is targeted highly for the receptor itself, not the Cl- conductance. excitatory AA NTs: important for primary afferent transmission into cord via DRG comment: there is a handout that was given to us today with 3 figures on it. this is stuff that got left out of the main handout. glutamate: the only excitatory AA NT we'll really discuss. however, generally, all the excitatory AA NTs are like all the AA NTs, in that they're shifted from metabolic pool, stored in functional pool of vesicles, released by exocytosis, interact w/receptors that are usually pentameric ligand gated ion channels, and removed by high affinity uptake systems. for excitatory AA NTs, they bind cation channels that promote cation influx. remember ligand gated ion channels are somewhat less selective than the more specific voltage gated ion channels which allow only one particular ion to get through. one place we know excitatory AA NT is important is in spinal cord excitatory afferent transmission via DRG. glutamate important esp in monosynaptic synapses. diagram p 4 - long monosynaptic synapse onto ventral horn neuron - uses glutamate. if it is disynaptic, NT is often aspartate. another excitatory NT. inhibitory NT also involved - when inhibition is established in cord, usually uses GAB for feedback inhibition, or glycine for direct postsynaptic inhibition. actions of these NTs on CNS are really important too - role of glutamate in CNS is very important. probably glutamate has gotten most attention by researchers. new material: there is a phenomenon known as "long term potentiation" which refers to the fact that in some areas of the brain, you can stimulate a neuronw with a high frequency of stimulation, and for all intents and purposes permanently change the firing properties of that cell and its postsynaptic cell. after this, the neuron and postsynaptic targets respond with enhanced activity to stimulation. this has been demonstrated in many areas, but especially in the hippocampus which you should have come across in the neuroscience course. the hippocampus is interesting b/c damage to hippo produces memory deficits, long lasting memory deficits. now, when you see long term potentiation in hippocampla neurons, and you know that hippocampal damage produces memory deficits, it isn't a huge leap to suggest that maybe long term potentiation is a cellular model for memory.maybe it's a way to encode memory, by altering firing responses. this phenomenon has revolutionized neuroscience. it is really energizing efforts to understand complex brain functions like learning and memory. these are important things. the quality of our lives depends on these. if you lose memory, you lose your identity. now, how does long term potentiation relate to glutamate? it is dependent on a glutamate receptor - actually on multiple glutamate receptors, one of which is called the NMDA receptor. this is the second figure in the handout we were given - p 153. this is an attempt to diagram long term potentiation (LTP) in hippocampal layer Ca1.how is the NMDA receptor involved? well, if you use antagonists for NMDA receptors you block LTP. the NMDA receptor is a ligand gated ion channel that promotes Ca++ entry into cells - consistent with role as excitatory AA NT receptor. but it has a peculiar property - at the resting membrane potential, the NMDA receptor even when occupied by NMDA won't promote Ca++ entry. this is because there is ionic blockade of the NMDA receptor by Mg - called Mg blockade. as long as Mg is bound to sites on inner surface of receptor, glutamate binding won't promote Ca++ influx. the Mg block is only removed by another type of glutamate receptor, coexpressed by these neurons, known as the AMPA receptor. so as glutamate is released from hippocampal neuron during LTP, it interacts with AMPA and NMPA receptors. NMDA stays quiescent, utnil AMPA allows influx of cations and depolarization of postsynaptic neuron, removing the Mg block. now, the NMDA recpetor can promote Ca++ influx. this produces activation of postsynaptic cell. So, then, what does the Ca++ do? it turns out that LTP is dependent upon both changes in the firing properties of the presynaptic cell,and the sensititivity of the postsynaptic cell. so this amplifies a circuit. the presynaptic cell changes firing response to subsequent stimulus, as well as postsynaptic cell altering response to stimulus. so how does the presynaptic cell get amplified? this led in the CNS to discovery of molecules now known as retrograde messengers. we've discussed 2nd messengers that are generated inside cells but we've confined our discussion to what happens in the repsonsive cells -not considering the possibility that it might diffuse out and affect nearby cells. but there are freely diffusible molecules generated in postsynaptic cells that can feedback to nearby cells - and these are called retrograde messengers. so then we had to find the retrograde messenger involved in LTP. we now believe that it is NO (nitric oxide) - see first page of supplemental handout. NO is one of those freely diffusible retrograde messengers that was so important in understanding LTP. where does it come from? well, it turns out that NO can be produced in a variety of cells when Ca++ levels are elevated. this is b/c the enzyme that makes NO from argenine, known as NOS (nitric oxide synthase) is a Ca++ regulated enzyme that is turned on by Ca++. so when Ca++ binds calmodulin, it increases NOS activity, producing NO from argenine. Glutamate, when it binds the NMDA receptor, elevates Ca++ levels in all neurons. so, if NOS is present in a postsynaptic cell, it will make NO, which freely diffuses out of cells. now we have the retrograde messenger. the people studying LTP thought they now had the whole story. glutamate NMDA receptors and NO became the big molecules of neurosci. NO was named molecule of the year by Science magazine. it turns out though that NO had already been discovered by people studying vascular biology - it was endothelium derived relaxing factor, EDRF. this is the stuff made in endothelial cells that promotes relaxation of vascular smooth muscle. vascular biologists knew that EDRF entered the muscle cell and activated guanylate cyclase, similar to adenylate cyclase, which comes in membrane bound and soluble forms. like adenylate cyclase, it acts on nucleotide (GTP) and makes cGMP. NO activates the soluble guanylate cyclase and generates cGMP, relaxing vascular smooth muscle. we know now that EDRF is NO. so NO has changed a lot of our thinking about vascular biology. It turns out that the importance of NO can't be overstressed. there are many physiological actions of NO not limited to neuro or vascular biology, but in many tissues, NO has this kind of action. so we can expect, as we try to understand the functioning of many NTs coupled to Ca++ mobilization that a role for NO will be present. it turns out that in understanding actions of anesthetics in CNS this pathway has been implicated. summary - understand that in all areas of pharmacology, new pathways of cellular activity are emerging, and one of the newest, with respect to compounds that increase Ca++ is the realization that when NO is engaged, its action affects many neighbor cells due to freely diffusable characteristic. this is very true for effects of glutamate. ---break--- one more word about NO - the point was to introduce another pathway by which we understand NTs to act. we'll hear more in Klide's lectures; hopefully that will give us a clinical context. also from Fluharty later when we discuss autacoids. now we're shifting gears to discuss peptides, a whole other class of NTs with a long history in neurosci. we've known for a while that peptides play a big role in neuroendocrinology. peptides regulate/underly hypothalamic regulation of pituatary gland - hypothal releases peptides to regulate anterior pituitary, and synthesizes peptides that are released from posterior pituitary. but, peptides are also found widely distributed throughout brain and periphery - when we figured this out it ushered in a whole new era of research. peptides in terms of synthesis are totally different from other NTs. you start with big precursors and cleave them into smaller molecules. the synthesis of any neuropeptide starts with gene transcription - gene codes for a preproprotein, or preprohormone, a large precursor molecule. you have signal sequence cleaved off proprotein and then it undergoes postranslational modifications like phosphorylation, acylation, glycosylation. in the case of peptide NTs, another event occurs - peptidase enzymes start to cleave out of the structurally modified proprotein the actual peptide NTs. see fig 6 in handout p 5. you have initial gene transcription, followed by mRNA translation, giving you precursor proteins ranging in size from 150 to 300 AAs. contained within them are several NTs or neural hormones that have to be cleaved out by the peptidases. the peptidases are sometimes carboxypeptidases - they cleave AA bonds present in carboxy terminus of molecule - somatostatin, for example, was originally considered an inhibitory peptide for GH secretion, and is now considered to be an important NT throughout the brain. it is made by a carboxypeptidase cleaving it out of a large precursor. in other instances, the NT is cleaved out of the aminoterminal by an aminopeptidase. vasopressin is cleaved out in this way. vasopressin is not only a regulator of renal function - it is found elsewhere in the brain and functions as an NT. it's made from a large precursor and like somatostatin has multiple forms. then, there are endopeptidases that act in the middle of the precursor to cleave out NTs like enkephalins, endogenous opiates. the small 5 AA enkephalin molecules are cleaved out of proenkephalin by endopeptidases. so this is really different from the way the other NTs are made, in that you start with gene transcription and make these precursors. peptides also represent the largest families of NTs that we know of. while AAs are most prevalent NTs in CNS, peptides are most diverse family, meaning structurally diverse and also the large numbers of compounds that hae been suggested to function as peptide NTs. there is a big table in the handout, don't memorize it :). just get a sense of how many molecules that are peptides have been suggested to function importantly in the CNS. most of them had/have relatively well accepted physiological roles and only recently have been recognized as NTs - so names can be misleading. VIP was originally found in GI system, where it is vasoactive. now we know it is also in the brain. VIP isn't that useful when you describe its CNS activity, but there you go. there are other examples - glucagon, somatostatin, many misnomers. we're going to discuss several with important roles in pain and analgesia - substasnce P associated with pain conduction, some endogenous opiates like enkephalins, endorphins, dynorphins. very large family with structural diversity. another interesting thing about peptide NTs - they are often colocalized with other transmitter molecules. this is very important b/c it allows them to modulate the activity of other more classical NTs. think of the rules governing neurotransmission - then when you have peptide colocalized with other NT in same neuron, you could get differential effects depending which transmitter is released when cell is excited. it's now very clear that peptide NTs when colocalized with other NTs are segregated into different vesicular populations. each NT is in separate vesicle. also, release of the two types of molecules in their different vesicles is almost always frequency dependent. so vesicular segregation results in frequency dependent differential release. so, in a neuron where ACH is colocalized with VIP, for example - low frequency stimulation releases only the classic NT, high frequncy stimulation releases both. this occurs in many neurons with different NTs. if you increase stimulation of neuron past a point, postsynaptic effects are modulated by the peptide NT - may be enhancement or inhibition of classic NT effects. this is another way the neural system translates different firing patterns into altered chemical signalling. fig 7 p 6 - we're going to talk about substance P and pain: substance P was isolated from the GI tract, where like VIP, it has potent effects on smooth muscle. but its also an NT. one place you see it is in unmyelinated C fibers - small diameter, unmyelinated fibers which have slow conduction velocity.these fibers are involved in nociception and in particular, slow burning pain. when they are activated, you see substance P release via exocytosis from vesicles. sub P interacts with postsynaptic receptors which activate spinothalamic projections to somatosensory areas involved with pain reception. there are sub P antagonists being developed. so sub P is important in primary afferent nociceptive transmission. a sub P antagonist might be one way to try inhibiting pain. or you could try to reduce release of sub P during nerve stimulation, to reduce nociception and produce analgesia. it turns out the endogenous opiates do that in the spinal cords of many animals. endogenous opiates and analgesia: as klide will explain, ther ei s a lot to know about these things. they are really important in treating pain. are subject to spp variability. we'll only discuss mechanisms today. endogenous opiates are called that b/c their activity is similar to that of opium. opium is very old drug known to have abuse potential and powerful analgesic effects. esp morphine. morphine has given rise to more and more opiates over time, that produce varying degrees of analgesia with different loci of action. also produce euphoria, leading to abuse potential. discovery - story is similar to gabamodulin/benzo story. almost at same time two teams figured out that brain and enteric ns in gut expressed morphine receptors. if you tagged morphine you could see it binding in areas of the gut and the brain. why would this happen? why do you have a receptor for a foreign compound? well, you usually don't. it implies a natural compound in the body that binds that site. so they looked in the 70s for these endogenous opiates, and it turned out there are many. not only did the brain produce morphine like compounds, but there were multiple classes of endogenous opiates, multiple receptor families, and some of these compounds were more potent than morphine. so the CNS makes its own analgesics. if one could learn to harness them, to promote their release, it might obviate the need for the exogenous ones. three families of endogenous opiates exist b/c there are three preproproteins - three initial gene products - proopiomelanocortin (POMC), proenkephalin, and prodynorphin. POMC gives rise to the endorphins, alpha beta and gamma endorphin, each about 30 AA, released by carboxypeptidases. ACTH also comes from POMC, indicating gene conservation. proenkephalin gives rise to a whole host of small 5 AA sized enkephalins, called met or leu enkephalins. there are many of these, varying b/w spp. more recently we've found prodynorphin, which includes dynorphin A and dynorphin B, which are very very potent. so there are a lot of these and they produce varying degrees of analgesia in varying tissues. in addition to vast # of ligands, there are three main receptor families - mu, kappa, and delta. clearly, as Dr. Klide will explain, the action of endogenous and exogenous opiates to produce analgesia, are at the mu and kappa receptor. there are sometimes distinctions made b/w relative role of mu and kappa in different types of analgesia, eg it's argued that mu is more important in supraspinal analgesia, and kappa more important in local spinal analgesia - but it isn't that clearcut for most of the drugs used. there is reason to believe that a full understanding involves interactions b/w mu and kappa receptors, and you can't really make the distinction. what is role of delta receptor? it doesn't appear to be as important wrt analgesic effects, but may be more important for euphoric effects. virtually all morphine like compounds we use have abuse potential. if we could remove the mood elevating effects we might remove abuse potential. morphine as a compound is pretty nonselective for all the receptors, and so are most of the endogenous opiates. but drugs, can be very selective. so you could make a drug that is more potent at mu or at kappa or whatever. depending on type and location of analgesia you need, this can dictate (along with major issues of spp variability) what drug to use. mechanisms of analgesia: one that's fairly worked out is a return to this substance P. it's thought that enkephalin containing neurons can inhibit release of substance P. so if you use a drug that mimics action of enkephalin, you could establish spinal analgesia. there's also evidence that receptor on presynaptic terminals is probably kappa receptor coupled to cAMP that reduces ability of cell to make substance P - presynaptic inhibition. another way to acheive this could have involved enkephalin synapsing on postsynaptic neuron, and trying to reduce it's responsiveness to substance p - postsynaptic inhibition, that occurs as well. then there is a third type - inhibitory modulation. in many brain pathways as discussed, peptides are colocalized with other NTs. as the neuron is stimulated at low levels, it activates postsynaptic cell by releasing classic NT. when neuron is stimulated at higher degree, it starts also releasing peptide - which may start to reduce release of classical NT from presynaptic neuron, and also may act on postsynaptic cell. in some instances, endogenous opiate is in the same neuron as the transmitter. frequency dependent release and frequency dependent analgesia occurs. see p 9. summary: point was to introduce other NTs, AAs and peptides, and their important roles in CNS pharmacology. wrt veterinary medicine, it turns out that some of these figure prominently in analgesic, anesthetic, and tranquilizing drugs. ---end----