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Receptor Multiplicity

Receptor Multiplicity Receptor Multiplicity By Christian George Over the past three decades the diversity and selectivity of ligands has increased and it has become clear and evident that multiple subtypes of receptors exist. That is subtypes within many previously defined classes of receptors. Due to the advances in scientific research especially over the past decade with molecular cloning, there has been a revelation in the presence of several closely related subtypes of receptors where only a single species was thought to exist, and some receptor subtypes have been shown to be differentially expressed during development, differentially regulate physiological systems, regulate transmitter release, be involved in permanent changes in the brain via integration. Knowledge of receptor subtypes is of interest to the researcher, at present many studies are being carried out into the investigation of new receptors and the mechanisms of known receptors. Manipulation of the receptors has now been achieved facilitating the search for new receptors. There is a huge diversity and receptor multiplicity involving subtypes and a few examples are going to be discussed in this essay involving some history and identification of subtypes, various imaging of subtypes, and some of the more detailed roles that they play in our physiological systems with pharmacological experimental evidence to support this. The term “receptor multiplicity” was mentioned above from carrying out research there is no general definition of “receptor multiplicity”, however there are examples which display “receptor multiplicity”. The Opioid receptors have been subtyped, and there is evidence for the existence of  receptor multiplicity. The evidence is supported by the fact that high affinity 1 receptors not only bind morphine but enkephalins as well, in addition to 2 receptors that preferentially bind morphine. This demonstrates that receptor subtypes in the  type opioids exist defining receptor mutiplicity, it means a class of receptors say adrenergic has subtypes within that class. These adrenergic subtypes allow endogenous noradrenaline to bind and activate the receptor resulting in a biological change, but specific ligands can also activate a selective subtype in that class of receptors without affecting the other subtypes for example salbutamol is an agonist of the 2 adrenoceptor only and produces bronchodilation, it is used in the treatment of asthma and produces little side affects due to it’s selectivity. Different subtypes of the same receptor class can also mediate different biological responses at different locations in the body, the nicotinic cholinergic receptor for example, when antagonised in the ganglia to control blood pressure does not affect the nAChR in skeletal muscle paralysing it. Other agents such as tubocurarine has the ability to antagonize the action of acetylcholine at the neuromuscular junction and is confined to this location. These subtypes of receptor are analogous to tissue specific isozymes of an enzyme. There are two main mechanisms of receptor G –protein coupled receptors and ligand gated ion channels. These two types can occur in the same family for example the serotonin receptor class are all G protein coupled receptors apart from 5HT3 which is a ligand gated ion channel. The differences in these two type will be explained later on(Figs. 1 and 2). The mechanisms of action of some receptor subtypes may be very similar, differing only slightly in kinetics or regulatory activity, but other subtypes the G protein coupled receptors display fundamental differences in their biochemical and cellular regulatory activities. For example 1 and 2 adrenergic and M1 and M2 muscarinic cholinergic subtypes. All four subtypes regulate G proteins, the 1 Figure 3adrenergic and the M1 and M3 muscarinic receptors initiate Ca2+ signalling via Gq, in contrast the 2 adrenergic and M2 and M4 muscarinic receptors regulate other signalling pathways via GI and another GTP binding protein, G0 (Fig. 2). Expression of such different receptor subtypes allows a single agonist to evoke unique responses in specific cells or tissues. Figure 1. G-Protein coupled receptor Figure 2. Ligated ion channel receptor In the recent decade advances in scientific research have accomplished the art of cloning. Using these techniques in combination with radioligand binding and autoradiography, novel receptors have been identified and implemented using these molecular biological techniques. If a tissue or cell expresses more than a single subtype of receptor or when only insufficiently selective drugs are available identification of the specific signal that is generated by an individual receptor requires more direct approaches. These include expression of the cloned cDNA for the receptor in a well characterised cell where it’s signalling activities can be studied in detail, the expression and purification of the recombinant receptor for direct biochemical analysis of it’s functions, or the use of antisense strategies to evaluate which signal transducing pathway is necessary for agonist effects. Through the use of identification methods immuncytochemical staining western, northern and southern blots a immense amount of receptor subtypes have been discovered here are some of the findings for the glutamate receptor (Fig 4.) and the Classic neurotransmiters of the CNS (Fig 5.) Figure 4. A structured tree displaying the glutamate subunits with binding sites Figure 5 A structural tree of the Classical neurotransmitters and there receptor subtypes To identify receptor subtypes in tissues a unique method has been adopted in using radioligands to enable tracing it’s binding to receptors. A good example of a radioligand binding experiment using the recent availability of a whole range of potent and selective prostaglandin agonists and antagonists is determining the pharmacology of [3H]Prostaglandin E1/[3H]Prostaglandin E2 and [3H]Prostaglandin F2 Binding to EP3 and FP Prostaglandin Receptor Binding Sites in Bovine Corpus Luteum. The procedure carried out involved grinding brain BCLM homogenates, centrifuging and placing in an incubator. After this the [3H]PGE1 and [3H]PGE2 binding assay was carried out. Then the same method was carried out using [3H]PGF2 binding assay. These were then mounted onto slides then autoradiographed. The study demonstrated the presence of high affinity and specific binding sites for [3H]PGE1, [3H]PGE2 and [3H]PGF2 in washed total particulate BCLM homogenates. Scatchard analyses indicated that [3H]PGE1 and [3H]PGE2 bound to a single population of nanomolar affinity receptor sites (fig 7a,7b). Both [3H]PGE1 and [3H]PGE2 labelled the same population of EP- receptor sites in the tissue because Kd and Bmax values obtained for both radioligands were very similar. (fig 7a,7b).The pharmacological specificity of both radioligands in the BCLM was essentially identical (Fig 8c). [3H]PGF2 labelled a high affinity (nM Kd) and an apparent low affinity (M Kd) site in the washed BCLM homogenates as determined from several experiments (Fig 7c). The major aim of this study was to define the PG receptor subtypes binding sites present in the BCLM using the latest pharmacological tools. Current studies have demonstrated that both [3H]PGE2 and [3H]PGE1 selectively label EP3 receptors in the BCLM due to potent highly selective EP3-receptor prostaglandins, such as GR-63799,SC-46275, sulprostone and enprostil competing for specific [3H]PGE2 and [3H]PGE1 binding with nanomolar affinities, while prostaglandins selective for other prostaglandin receptor subtypes were weak competitors. (fig 8a, 8b, 8c).The pharmacology of the[3H]PGF2- labelled high affinity receptor sites in the BCLM clearly indicates the identification of an FP receptor binding site. Pharmacological eveidence for this conclusion is that traditional prototypic FP- selective prostaglandins such as fluprostenol, Latanoprost acid, cloprostenol and 17 – phenyl-PGF2, were potent competitors of specific [3H]PGF2 binding to the washed BCLM homogenates exhibiting nanomolar affinities, although prostaglandins selective for (DP) BWA868C, (DP) ZK-118182, (EP) enprostil, (EP) sulprostone, (IP) PGI2 and (TP) U46619 all had micromolar affinities. Emulsion autographic studies found a lower level of [3H]PGE2-labelled sites than those labelled by [3H]PGF2 in the BCLM sections.(Fig 6) Figure. 6 Autoradiographic localization of FP receptors in BCLM sections. The top figure depicts total binding of [3H]PGF2 and the lower figure depicts nonspecific binding of [3H]PGF2 defined in the presence of 100 �M unlabeled fluprostenol. Similar degree of non-specific binding was obtained when 1-100 �M unlabeled fluprostenol or PGF2 were used. Note the high specific binding and high density of [3H]PGF2 binding sites associated with the granulosa cells but the very low density on connective tissue and blood vessels. CT, Connective tissue; GC, granulosa cells; BV, blood vessel. Magnification, �4 of original. Figure. 7 Scatchard plots of specific [3H]PGE1 (a), [3H]PGE2 (b) and [3H]PGF2 (c) binding to BCLM membranes. The plots shown for [3H]PGE1 and [3H]PGE2 are from one representative experiment, while the data for [3H]PGF2 are from 11 experiments; the mean � S.E.M. of the Kd and Bmax data from 3 to 11 experiments are also shown for these radioligands. Figure. 8 Competition curves for various PGs displacing specific [3H]PGE2 (a and b) binding and correlation of pharmacology of [3H]PGE1 and [3H]PGE2 binding (c) to BCLM membranes (r = 0.973; P * 0.001). d depicts a correlation plot of [3H]PGE2 binding to BCLM and [3H]PGE2 binding to the cloned EP3 receptor from the mouse (Kiriyama et al, 1995) (r = 0.798, P * 0.0099). Data are mean � S.E.M. from three to six experiments for the BCLM.The molecular basis of receptor subtypes is very similar an example the 4 transmembrane receptors in the Nicotinic Cholinergic receptor subtype are made up of 5 subunits ranging from 420 to 550 amino acids it is a pentameric complex. These subunits exhibit sequence identities from 25% to 75% with a similar distribution of hydrophobic and hydrophilic domains. Following the recent advances in technology molecular cloning has resulted in identifying muscle nAChR subunits 1, 1, , ,  (Fig. 10) and the structurally related neuronal subunits 2 to 9 and 2 to 4 with the agonist site normally located on the  site. The 2 to 4 subunits can assemble with the 2 to 4 subunits to generate a functional heteromeric receptor subtype, whereas the 7 to 9 subunits can generate functional homomeric receptors. Four glycine receptor subunits have been identified: three  subunits and one . Figure 10. Displaying the five subunits of the nAChR In order to demonstrate the similarity in receptor subtypes (Fig. 11) depicts a 4 transmembrane segment when 5 of these are assemble it forms a pentameric complex (Fig. 12) these five subunits that make up the nicototinic cholinoceptor have names as mentioned previously depicted in (Fig. 13). Figure 11 4 TM subunit Figure 12 Cross section of nAChR Figure 13 Pentameric nAChR As illustrated in the table different combinations of 5 subunits make up different receptor subtypes (Fig 14). The structure of subunits that can make up a subtype differs some what to allow differentiation in binding properties of the specific receptors example (Fig. 15 & Fig. 16). The end result is a combination of subunits creating a specifically shaped receptor allowing only certain ligands to bind (Fig. 17). Figure 14 A Table comprising some of the combinatorial subunits needed to assemble a receptor subtype Figure 15 Four TM subunit Figure 16 Three TM subunit plus pore Figure 17 Assembled subunits The structures of receptors can be extremely complex to view as demonstrated by the glycine receptor (Fig. 18) Figure 18 The molecular structure of a glycine receptor Below, the evolutionary tree for the rhodopsin-like receptors indicates that they have evolved both as a consequence of selection for coupling to different G proteins and selection for reaction with different ligands. However these two developments would appear to have occurred independantly and through different mechanisms. Subtypes of receptors which bind the same ligand generally have evolved within a given branch of the tree through ordinary divergent evolution. (see figure D2, D3, D4 dopamine receptors and also the muscarinic receptors). However subtypes of receptors are also frequently found in separate branches of the tree. Aminergic (ie binding monoamines) receptors are examples of how receptors that couple to a particular G protein, but bind different ligands, can be more homologous to each other than receptor subtypes which bind to the same transmitter. Thus it appears that the ability to bind, for example dopamine has evolved in different evolutionary branches of G protein coupled receptors, which through divergent evolution had already segregated from each other (D1 and D5 versus D2, D3 and D4 in the figure). Similarly, histamine H1 and H2 receptors are only approximately 20 % identical in their transmembrane segments and are in fact, more similar to receptors that bind acetylcholine and adrenergic ligands, respectively. Apparently, receptors have picked up the same ligand in different evolutionary branches, convergent evolution. Figure Part of the evolutionary tree for rhodopsin-like 7TM receptors. All rhodopsin like receptors are at least 15 to 20 % homologous, the shaded area equals 70 % sequence identity which coveres most receptor subtypes (From Donally, D., Findlay, J.B.C., and Blundell, T.L., Receptors and Channels, 2,61,1994, and supplementary material from D. Donally) Figure 19 A dopaminergic neuron in which dopamine receptor subtypes can be seen at different locations on pre and post synaptic terminals. By using extremely advanced techniques it is possible to achieve computerised images pinpointing the distribution and location of certain receptor subtypes here subtypes have been imaged in the brain by using selective radioligands and advanced autoradiograpy, providing this remarkable imagery of the different distributions and locations of dopamine subtypes in the brain. These two sets of images (Fig. 19) can allow a clinician to diagnose a patient with an illness and treat more accurately, it also provides priceless research in psychoneuropharmacology in treating disease states such as schizophrenia and parkinsons disease in which dopamine receptor subtypes are heavily involved. In this image the majority of D2 receptor subtype can be located in the caudate putamen and the hippocampus and all subtypes apart from D1 can be found in the hippocampus. Not only can the actual location in the brain or body be different for subtypes but different receptor subtypes can be found on the presynaptic neurons as well as the postsynaptic neurons this may help regulate the transmission levels (Fig. 20) Figure 20. Images produced by autoradiography displaying the distribution and location of dopamine receptor subtypes Adenosine is a purine nucleoside produced by all cells that plays an important role in cellular metabolism and functions as an extracellular physiological regulator. Intracellular adenosine is then transported into the local extracellular space where it activates P1 purinergic receptors, which include A1 and A3 which couple with Gi and Go respectively A2A and A2B couple to Gs. During fetal life adenosine is very important, fetal plasma levels are fourfold greater than maternal levels. A1 receptors have been found in rat hearts as early as post conception 7.5 days when the developing heart is a primitive tube that has not initiated rhythmic beating. A1 are amongst the earliest expressed G - protein coupled receptors in mammalian heart. In mature hearts a1 activation has been shown to slow atrioventricular conduction and alter the pacemaker current. Adenosine receptor subtypes have been characterised which the adenosine acts upon to regulate cardiac activity. The procedure for investigating this was produce fetal culture conditions, a heart rate assessment, statistical analysis, use of various ligands. The results attained included; influences of A1A agonists on heart rates, P2 purinergic receptor ligand studies, adenosine influences on heart rates, influences of Gi/Go and cAMP on A1 action and influences of ion channel blockade on A1 action. Dose response curves generated using a variety of adenosine agonists revealed that A1 activation potently regulated fetal heart rates. Agonist n6-cyclopentyladenosine, inhibited heart rate and stopped fetal cardiac contraction in 63% of preparations. A2A & A2B receptor activation did not alter heart rates, activation of A3 produced modest declines in heart rate. Endogenous adenosine acts tonically to supress fetal heart rates demonstrated by the use of A1 antagonists 1,3-dipropyl-8-cyclopentylxanthine, increasing heart rates while adenosine reuptake inhibitors dipyridamole lowered fetal heart rates. The pertussis toxin treatment blocked A1 action displaying A1 was G-protein mediated. Drugs that alter cAMP levels and ion channel action displayed a1 action involves events mediated by cAMP, ATP-dependant K, L-type calcium, sodium and chloride channels, and the pacemaker current. Adenosine A1 potently regulate mammalian heart rates via multiple effector systems at very early stages of prenatal development. The findings also raise the possibility that activation of a1a during early embryonic development may result in fetal death, as factors that adversely affect cardiac output will result in fetal demise. Intrauterine hypoxia or stress triggers a cascade of a1ar mediated events resulting in bradycardia and assytole at embryonic development.(HOFMAN et al 1997) Two systems influence vascular tone and growth of smooth muscle cells. The effects of the pressor substances of these two systems, angiotensin II (ANG II) and noradrenaline (NA), are triggered by their interaction with specific receptors on the vascular wall. It has been demonstrated that the 1-adrenergic receptor mediates sympathetic vasoconstriction of the blood vessel, whereas most vascular ANG II receptors in all species studied to date are mainly of the type 1 ANG II receptor (AT1) that mediates contractile and growth effects of ANG II in vascular smooth muscle. "There are multiple interactions between the renin-angiotensin system and the sympathetic nervous system. For example, ANG II facilitates sympathetic neurotransmission at several sites, including the central nervous system, adrenal medulla, sympathetic ganglia, and presynaptic noradrenergic nerve terminals. Studies demonstrate that continuous infusion of NA without alteration of the blood pressure decreases AT1 mRNA levels in the aorta of the rats. Conversely, infusion of a nondepressor dose of prazosin increases aortic AT1 mRNA content. Studies also show that NA has a negative effect on both AT1 mRNA expression and AT1 receptor density in cultured VSMC. This inhibitory effect of NE can be prevented by the 1-adrenoreceptor antagonist prazosin, but not by the 2-adrenoreceptor antagonist yohimbine. This is evidence showing that NE negatively regulates AT1 receptor expression in the vascular tissue through an 1-adrenergic receptor-mediated mechanism. In opposition to these positive interactions,results from studies indicate that the sympathetic nervous system negatively regulates the vascular AT1 receptor in vivo and in vitro. For example, it has been shown in brain neuronal cultures of Wistar-Kyoto rats that NE decreases AT1 receptor density and its gene expression. Also, renal denervation or sympathetic blockade with guanethidine increases glomerular ANG II receptor density in normotensive and hypertensive rats, suggesting that the sympathetic nervous system exerts an inhibitory effect on AT1 receptor expression in glomeruli. These studies provide clear evidence for heterologous downregulation of the AT1 receptor by NE. The mechanisms by which NE induces downregulation of the AT1 receptor in the vascular tissue are that NE activates 1-adrenergic receptors and, through modulation of one or more signaling pathways, downregulates AT1 receptor gene expression.support for this come from studies where blockade of the 1-adrenoreceptor with prazosin increases AT1 mRNA levels in the aorta of the rat; this increase does not appear to be an artifact of the effect of DMSO due to preliminary experiments displaying no significant difference in aortic AT1 mRNA levels between DMSO and saline-infused rats, and 2) inhibitory effects of NE on both AT1 mRNA and receptor protein in cultured VSMC were prevented by cotreatment with prazosin but not with the 2-adrenoreceptor antagonist yohimbine. It is possible that yohimbine at 0.1 �M may not be sufficient to completely block 2-adrenoreceptors. However, the fact that yohimbine had no effect whatsoever does serve to rule out involvement of this receptor type. We have shown that a nonpressor dose of ANG II infusion (25 ng � kg-1 � min-1) for 2 wk downregulates AT1 mRNA levels in both the aorta and mesenteric resistance arteries.This homologous downregulation of the AT1 receptor in the vascular tissue seems to be mediated by both transcriptional and posttranscriptional mechanisms. The results from the present study showed that the changes in AT1 receptor density parallel the changes in AT1 mRNA levels in cultured VSMC, indicating that NE-mediated downregulation of AT1 receptors occurs, at least partially, via a diminished AT1 receptor mRNA level. Although it is unknown whether NE-induced heterologous downregulation of AT1 receptors occurs at transcriptional or posttranscriptional levels, the data represent a clear example of cross talk between the two plasma membrane receptors. The physiological and pathophysiological significance of NE on AT1 receptor expression in the circulatory system of the rats is that the vascular AT1 receptor is a central component of the renin-angiotensin system, and regulation of its expression is likely to be important in cardiovascular responsiveness. a positive interaction between ANG II and NE through an action on the vascular AT1 and 1-adrenergic receptor to produce a greater vasoconstriction than that produced by either alone. At the same time, persistent increased ANG II and NE will downregulate the AT1 receptor, providing a "negative feedback" mechanism to attenuate the action of ANG II. Thus cyclic upregulation of ANG II and NE, a "feed-forward" mechanism, followed by downregulation of AT1 receptor by a negative feedback mechanism may play an important homeostatic role between the renin-angiotensin and sympathetic nervous systems in blood pressure regulation in the normotensive rat. Conversely, in hypertensive rats, disruption of the negative feedback mechanism will prevent downregulation of AT1 receptor. This ultimately leads to hyperactive renin-angiotensin and sympathetic nervous systems that may contribute to blood pressure increase in hypertensive rats. Studies supporting this show that inhibitory effects of NE on AT1 receptor expression in brain neurons of Wistar-Kyoto rats is absent in neurons of spontaneously hypertensive rats."(Ad Verbatum) Figure. 21 a serotoninergic neuron displaying 5-HT1A Autoreceptors Due to the dual location of 5-HT1A receptors(Fig. 21), regulation of serotoninergic transmission occurs. 5-HT1A located on serotoninergic neurones acting as somato – dendritic autoreceptors, and on targets of serotoninergic projections which correspond to post synaptic receptors. Two opposing effects occur from the stimulation of 5-HT1A in the raphe nuclei compared to regions such as the limbic system. Agonists of 5-HT1A acting at the somato – dendritic autoreceptors inhibit electrical activity of the serotinergic neurones therefore reducing the level of serotoninergic neurotransmission. However in contrast to this occurrence, where serotoninergic projections target the post synaptic level, agonists of 5-HT1A reproduce the effect by transmitting 5-HT from the serotoninergic terminals, overall enhancing the receptor dependant 5-HT1A neurotransmission. If an 5-HT1A antagonist is administered blockade of serotoninergic neurotransmission mediated through the post synaptic 5-HT1A receptors occurs but an increase in electrical activity of serotoninergic neurones also occurs due to a blockade at the somato-dendritic 5-HT1A autoreceptors. Not all 5-HT1A antagonists increase electrical activity of serotoninergic neurones this is due to their variable intrinsic efficacies in receptor blockade. An experiment carried out to demonstrate this (FORNAL et al. 1994a,b) spiperone and WAY100635 significantly increased the serotonergic neuronal firing rate in the dorsal raphe nucleus, this prevented endogenous 5-HT acting tonically at the somato-dendritic 5-HT1A autoreceptors. From the description here it can be deduced that the agonists of 5-HT1A have a dual effect on the 5-HT1A dependant mechanisms, these are inhibition of serotoninergic neurone firing and transmission via the somato-dendritic autoreceptors and stimulation serotoninergic neurotransmission due to the activation on post synaptic targets. Experiments conducted on rats show that the significant inhibition of serotoninergic neurotransmission via stimulation of the somato-dendritic autoreceptors in the anterior raphe nuclei stimulates food consumption, similar effects to this can be achieved by post synaptic blockade of 5 –HT2C receptors located on satiety mediating hypothalmic neurones) using cyproheptadine (KENNET 1999). Figure 22 (left) a resting potential (right) depolarization occuring on the post synaptic neuron LTP was first observed in mid 1970’s, it is a form of synaptic plasticity and is considered to be linked with learning and memory in the mammalian central nervous system. It displays the integration of glutamate receptor subtypes in the initiation of long term potentiation. LTP is achieved when glutamate released from a presynaptic terminal binds to two types of glutamate receptor NMDA and AMPA subtypes. The molecular basis of this achievement is explained using the biophysical properties of NMDA receptor and AMPA receptor subtypes. The key elements of this mechanism are the voltage gated blockade of NMDA receptor channel by magnesium ions, and the unusual permeability of calcium ions through the depolarised NMDA channel. In low frequency synaptic transmission, glutamate is released from axon terminals and binds to both NMDA and AMPA receptor subtypes. NMDA channels are blocked by a magnesium ion at negative membrane potentials therefore the current produced from the low frequency transmission flows through the AMPA channels which are permeable to Na+ only producing a resting membrane potential (Fig 22). Magnesium blockade of the NMDA channel is still present during the resting membrane potential, but disappears as soon as the cell is strongly depolarised due to excess Na+ entering the cell. Due to the voltage gated dependency of the NMDA gate depolarisation results in a release of the magnesium ion from it’s channel allowing current to flow through (Fig. 22). In comparison with AMPA, NMDA channels are far more permeable to calcium, this is an important second messenger and is essential for the establishment of LTP (Fig. 23). It can be deduced from this description that the NMDA receptor subtype acts like a molecular “AND” gate, the channel opens only when glutamate is bound to NMDA receptors and the cell is depolarised by glutamate bound AMPA allowing LTP to occur. Therefore if a weak stimulus is present glutamate will be released but depolarisation will not occur. (PURVES et al 1997) Figure 23 Influx of Calcium mediating LTP In the late 1970’s the platelet 2 adrenergic receptor was first radiolabelled, this peripheral receptor has been utilised as a marker for central 2 receptors. Radioligand binding techniques have been used to investigate 2 adrenergic receptors. Some studies have suggested an increase in total 2 adrenergic receptor numbers and others, and demonstrated increased affinity states or changes in ratio of affinity states, affinity of ligand, or direct demonstration of an altered receptor effector efficiency in various mental illness states. Taken as a whole these studies do suggest some altered peripheral 2 receptor/effector functioning in affective disorders. Appropriate binding studies have been designed to facilitate the complexity of 2 adrenergic receptor/effector systems and their regulation. The platelet 2 adrenergic receptor is comprised of many multiple membrane components a receptor/recognition site (R) , agonist and antagonist binding sites, N component a protein heterotrimer which links (R) to the third component, the second messenger (Ni linkage to catalytic moiety of Adenylate Cyclase). The intact system, these multiple components of receptor/effector system are in dynamic steady state influenced by factors such as amount of agonist present, pH, temperature and the prevalence of certain nucleotides and metal ions. In radioligand binding studies carried out in subjects presumed not to have affective disorders an antagonist tritiated rauwolscine was used, this radioligand labels affinity states for the 2 receptor more efficiently than an agonist. High affinity component of rawoulscine binding is the free R (2 (L) affinity state) in contrast low affinity component of rawoulsine binding is R-Ni complex(2 (H) affinity state) this measures the total number of receptor binding sites of both R and R-Ni in the membrane. The ratio’s of the number of free R relative to ‘coupled’ R-Ni sites 2(L)/ 2(H). Multi affinity state ratio reflects the efficiency of receptor coupling, higher ratio’s of R/R-Ni suggest 2 receptor recogition protein is less efficiently coupled to the second messenger. Patients with affective disorders in particular PTSD radioligand saturation studies and competition studies were carried out. Saturation studies demonstrated fewer total platelet 2 adrenergic receptor binding sites in PTSD than in control. A computer analysis of the data exhibited multiple components of rawoulscine saturation and two sites of interaction for both control and PTSD were displayed(2(H) and 2 (L)). The decrease in the number of rauwolscine binding sites was due to fewer 2(H) in the PTSD subjects. Consequently the affinity state ratio was also much higher in PTSD subjects. Adrenaline competition studies displayed that adrenaline was a less potent inhibitor of rauwolscine specific binding, the competition curve was steeper and shifted to the right. Computer analysis of this data also revealed two sites of interaction, more of the total number of 2 adrenergic receptors were in the low (2 (L)) and fewer in the higher (2 (H)) affinity state in the PTSD compared to controls. This accounted for a decrease in adrenaline potency. Fewer membrane binding sites and altered ratio of affinity states has been observed in other adrenergic/noradrenergic systems exposed to chronically high levels of endogenous agonist in the CNS. Similarly down regulation and uncoupling of platelet 2 receptors has been demonstrated in clinical states associated with increased circulating catecholamines: congestive heart failure, aging and hypertension. High levels of catecholamines and a secondary down regulation of platelet adrenergic receptors are consistent with an overactive sympathetic nervous system in PTSD. Chronic exposure to agonist decreased the total number of receptors and changed the steady – state between affinity sites (R and R-Ni). The number of receptors present in the membrane and accessible to agonist depends upon a variety of complex intracellular processes. Regulation of receptors in the membrane and receptor-mediated signal transduction is a complex process of receptor internalisation. Following agonist binding the receptor recognition protein is taken into the cell for recycling. A certain percentage of the receptor population is inside the cell. The rate of internalisation is dependant on a variety of factors including agonist concentration, duration of exposure, rate of degradation and rate of phosphorylation. Further dysregulation studies have been carried out to investigate these internalisation processes. It has been found that platelet 2 adrenergic receptors are desensitised to agonist in PTSD. It appears that chronic agonist administration uncouples the receptor and second messenger resulting in an accelerated mechanism for down regulation, that is a higher percentage of membrane receptors are taken out of the membrane and internalised following any agonist exposure. In PTSD there appears to be a more rapid and extensive apparent internalisation following agonist incubation which is also accompanied by a large increase in the ratio R is less likely to react with Ni. Chronic exposure to increased agonist level results in an receptor – effector system less capable of responding to agonist increases associated to subsequent stressors that is the platelet 2 adrenergic receptor-effector system was “overloaded” and easily ”fatigued”. It is clear that permanent intraneuronal changes which lead to alterations in expression of receptor proteins do take place with agonism in mature neuronal systems. This can be related to the molecular bases for memory and learning, in this regard the dissociative and flashback symptoms of PTSD can be conceptualised as physiological memories. (GILLER et al. 1990). This demonstrates the ability of an individual receptor to alter transmission rates long term resulting in actual dysregulation. The catecholamine neurotransmitters adrenaline and noradrenaline mediate their physiological responses through the family of adrenergic receptors. Three subfamilies of adrenergic receptors have been identified: 1 2 and . Within these subfamilies are subtypes including the subtypes of 2 adrenergic receptors: 2A 2B 2C. During an investigation on the mechanisms of down-regulation in the  adrenergic subtypes, an endogenous agonist noradrenaline was used in one of the procedures. Rat 2B adrenergic subtypes were transfected into a Chinese hamster ovary cell line. When 0.3M of noradrenaline is administered the 2B adrenergic receptors there is a down regulation by 50%. The 2C adrenergic receptor endogenously expressed in the opposum kidney cell line also down regulates by approximately 50% after 0.3M of noradrenaline is administered. In contrast down regulation of the 2A adrenergic subtype stably transfected into the Chinese hamster ovary cell line reaches a 50% control only when 30M of noradrenaline (BYLUND et al 1997). This is a hundred fold greater than that required to down regulate the 2B or 2C subtypes. The significance of this observation is not fully understood, but it may represent a fundamental difference in the way in which receptors are regulated. From the this experiment it can be deduced that subtypes within the same receptor subfamily have different affinities to an endogenous agonist. When concentrations of this endogenous agonist are changed for example increased it can have profound effects pharmacologically and physiologically as observed in the PTSD study. Dysregulation can occur, and this is the centre of many illnesses particularly in the brain, for example Schizophrenia.Nausea and vomiting associated with chemotherapy and radiation in the cancer patient seriously reduces the quality of life, and may be so severe as to cause the patient to discontinue therapy. (MINER et al.1986) and (COSTALL et al. 1986) were first people to demonstrate that the 5-HT3 receptor antagonists can inhibit emesis. 5-HT3 antagonists block emesis by 5-HT3 antagonism at the central sites, i.e. in the nucleus tractus solitarius, area postrema and dorsal motor vagal nucleus, also in the periphery at 5-HT3 receptors on the afferent vagus nerve terminals. According to research chemotherapy and radiation affect serotoninergic function in the gut, causing a release of 5-HT from the enterochromaffin cells which stimulate 5-HT3 receptors on the vagus nerve, 5-HT agonists such as m-chlorophenylbiguanide have been reported to induce emesis in animals, which is blocked by 5-HT3 antagonists. Granisetron is a selective 5-hydroxytryptamine3(5-HT3) receptor antagonist with little or no affinity for other serotonin receptors, including 5-HT1; 5-HT1A; 5-HT1B/C; 5- HT2; for alpha1-, alpha2-, or beta- adrenoreceptors; for dopamine-D2; or for histamine-H1; benzodiazepine; picrotoxin, or opioid receptors. Animal studies demonstrate that, in binding to 5-HT3 receptors, granisetron blocks serotonin stimulation and subsequent vomiting after emetogenic stimuli such as cisplatin. In the ferret animal model, a single granisetron injection prevented vomiting due to high-dose cisplatin or arrested vomiting within 5 to 30 seconds. In most human studies, granisetron has had little effect on blood pressure, heart rate or ECG. No evidence of an effect on plasma prolactin or aldosterone concentrations has been found in other studies. Another such 5-HT3 antagonist ondansetron has been of particular use in the treatment of post operative nausea and vomiting, it indicates an ability to prevent emesis in the presence of multifactorial aetiologies and interactions with other drugs. The findings display preliminary evidence that prevention of nausea induced by antibiotics and SSRI’s (COSTALL et al. 1997). Other drugs that are similar acting also used in the treatment of nausea from chemotherapy complications is tropisetron. This demonstrates the selectivity that drugs can have and increase the ratio of effective treatment : side effects. Bibliography: Ashby B. (1999). Prostaglandin and Related compounds. Pharmacology Quick Look (First Edition). 42 - 44. Bylund D B. (1999). Adrenergic Receptors. 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