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• W2066475453 abstract "The largest family of cell surface receptors involved in signal transduction, G protein coupled receptors (GPCRs), are one of the major targets for current drugs as well as new drug development. Ligands interacting with for e.g. adrenergic, histamine, adenosine, opioid, dopamine or serotonin receptors, constitute a large portion of currently used therapeutics. A common property of GPCRs is that upon activation (agonist binding) they transmit signals across the plasma membrane via an interaction with heterotrimeric G proteins (Stadel et al., 1997). The corresponding activated G protein subsequently interacts with an intracellular effector system, such as adenylate cyclase or phospholipase C, leading to a wide variety of distinct physiological responses. Recent evidence suggests that GPCRs have the potential to be ‘active’ even in the absence of an agonist. This exhibition of spontaneous receptor activity has led to the observation that various ligands, previously considered as antagonists with no intrinsic activity, actually can inhibit this spontaneous activity, appearing to possess ‘negative intrinsic activity’. This phenomenon has been termed inverse agonism and the corresponding ligands are referred to as inverse agonists. Although intrinsic constitutive receptor activity and inverse agonism have unequivocally been demonstrated in vitro, (patho)physiological consequences are far from self-evident. Thus, in this review we should like to focus on the expression of inverse agonism under more ‘physiological conditions’, since it appears timely to address the physiological relevance and consequences of this new concept, both in GPCR research and drug discovery. Traditional receptor theory has postulated on a single, ‘quiescent’ receptor state to which agonists bind inducing a conformational change of the receptor to an activated and ‘functional’ state. This view, initially formed in the early 1950s, was more clearly expressed by Del Castillo & Katz (1957), and stood as the foundation of receptor pharmacology for decades. It was believed that antagonists interact with the receptor, thereby preventing agonist binding, without having an effect on conformational changes of the receptor that remained in its ‘quiescent’ state. The first evidence of a ligand (‘antagonist’) producing opposite effects to those of an agonist, and thus not merely inhibiting agonist binding, stems from the GABA-benzodiazepine field. In Braestrup et al. 1982, reported on the discovery of an agent, DMCM (methyl 6,7-dimethoxy-4-ethyl-β-carboline-e-carboxylate), which in contrast to benzodiazepines, not only was a potent convulsant in vivo, but seemed to favour binding to benzodiazepine receptors that were in the non-GABA(agonist)-stimulated conformation. For the first time, it was elaborated that GABA-benzodiazepine receptors may perhaps exist in two conformations which are in equilibrium, an open chloride channel form (activated conformation) and a closed one (inactivated conformation) for which DMCM may have a high affinity and a tendency to stabilize, thereby decreasing binding of GABA to the activated conformation. Interestingly, the above concept of an ‘agonist-independent’ two-state receptor conformation, introduced initially for ion-channel-coupled receptors, soon found support from those studying the large family of GPCRs. Costa & Herz (1989) were pioneers in setting the grounds for what later would be referred to as ‘inverse agonism’. They demonstrated for the first time that some antagonists of the δ opioid receptor had ‘negative intrinsic activity’ in vitro, in contrast to others that lacked any intrinsic activity. Moreover, such ligands diminished even further the ‘basal’ or constitutive activity of the receptor defined as the activity of the receptor in the absence of any ligand. It was apparent that the concept of ‘negative intrinsic activity’ implied a pre-existing equilibrium between (at least) two states of a receptor. These two states could easily be defined as either a G protein-bound or a free form of the receptor, the first one active and the latter inactive. In their studies, basal GTPase activity in NG108-15 cell membranes was suggested to be due to stimulated activity resulting from a spontaneous interaction between empty or free receptors and G proteins. Their data lent support to the receptor model of Wregget & De Lean (1984) which predicted that ‘antagonists may be active by hindering the ability of receptors to associate spontaneously with G proteins in membranes’. Since then, and especially over the past few years, disclosure of negative intrinsic activity has corroborated even further this two-state model of GPCR activation. Hence, a large number of publications within this decade are concerned with, and demonstrate with a variety of systems or means, the phenomenon of constitutive receptor activity and its implication for inverse agonism. In this review we will first address some of the more seminal papers, showing that in most cases genetically engineered cell systems were pivotal for the development of this new concept. These and many more studies have been adequately and thoroughly reviewed recently by Milligan et al. (1997) and Leurs et al. (1998). We will then gradually move to more ‘physiological’ systems, in order to address the central issue of this review whether inverse agonism and spontaneous receptor activity are relevant phenomena in health and disease, and hence for drug discovery. It should be pointed out, however, that a classification of ligands based on their (negative) intrinsic activity is not an easy task. Due to the large influence of receptor systems and experimental conditions (whole cells versus membranes, stoichiometry of receptors/G protein, signalling proteins etc.) the same ligand may behave as an inverse agonist, a neutral antagonist or even a (partial) agonist. Inverse agonism on GPCRs is not always easily established, since basal receptor activity is generally not pronounced. Thus, various manipulations to increase basal (constitutive) receptor activity have been explored, such as construction and expression of constitutively active mutant receptors (CAM) or overexpression of either the receptor or the G protein to favourably change the receptor-G protein ratio (R : G). CAM receptors show higher agonist-independent activity and have been reported for various receptor subtypes. Since CAM receptors have a higher basal receptor activity, the effect of inverse agonists is more readily observed. Samama et al. (1993) were the first to describe a CAM receptor of the β2-adrenoceptor (AR); replacement of four amino acids of the third intracellular loop by the corresponding residues of the α1B-AR, led to agonist-independent activation of adenylate cyclase. Previous work by Cotecchia et al. (1990) had demonstrated that substitution of residues in the third intracellular loop of the β2-AR by the corresponding residues of the α1-AR, led to chimeric receptors that were coupled to PI hydrolysis, like the native α1-AR, instead of adenylate cyclase. This provided direct evidence that the third intracellular loop is important for G protein binding and activation. Since normal expression levels of wild type receptors do not always result in constitutive activity, numerous studies have been performed in various systems where overexpression of the wild type receptor has been induced. Examples of these are the expression of wild type β2-AR in Sf9 insect cells leading to receptor densities up to 40 pmol mg−1 protein (Chidiac et al., 1994) or the overexpression of the calcitonin receptor in HEK293 cells (Pozvek et al., 1997). In the latter case, two different clonal cell lines were selected, expressing 5×106 and 25×103 receptors/cell, respectively. Whereas the first cell line displayed an 80 fold increase in basal cyclic AMP production, the second was not constitutively active. Apparently, receptor density is positively correlated to spontaneous activity and various classes of GPCRs can display constitutive activity upon overexpression. Overexpression of the G protein involved may also lead to increased basal levels of second messengers. High levels of Gαq cotransfected with various muscarinic receptor subtypes in NIH3T3 cells resulted in increased basal activity of the receptors (Burstein et al., 1997). This induced constitutive activity of the receptors was reversed completely by the muscarinic antagonists tested, indicating that they behaved as inverse agonists. Thus, elevation of G protein levels favours formation of the active conformation of the receptor the fraction of receptors that are coupled to the G protein and provides a more sensitive means for the detection of inverse agonism. These genetic approaches even work in vivo, since transgenic animals overexpressing GPCRs proved another source of constitutively active receptors. Bond et al. (1995) described transgenic mice overexpressing the wild type β2-AR at various receptor levels. Baseline left atrial tension in these transgenic mice was increased 3 fold over control mice while the β2-selective ligand ICI-118,551, acting as an inverse agonist, decreased baseline tension. The inhibitory effect of ICI-118,551 was correlated with β2-AR densities, suggesting that it was a receptor-mediated event. Apart from these organ bath data, the effect of ICI-118,551 was also studied in vivo, where cardiac contractility was measured in both control and transgenic mice. ICI-118,551 decreased cardiac contractility in transgenic mice by approximately 70%, an effect that was associated with a fall both in heart rate and left ventricular systolic pressure, while the compound exhibited no effects on control hearts. Recently, the CAM β2-AR mentioned above, has also been overexpressed in mice (Samama et al., 1997). In this case, the mouse phenotype was not very different from normal, probably due to the rather modest overexpression (∼3 fold). However, long-term treatment with ICI-118,551 increased CAM β2-AR density, resulting in marked basal atrial tension and cardiac contractility. Finally, Nagaraja et al. (1999) described the effects of long term treatment of various β-adrenoceptor ligands on baseline left atrial tension in transgenic mice with modest β2-AR overexpression (50 fold compared to 200 fold β2-AR overexpression in the paper by Bond et al. (1995)). In these transgenic mice, inverse agonists such as ICI-115,881, carvedilol and propranolol increased baseline left atrial tension, whereas untreated or alprenolol-treated mice were unaffected. Baseline left atrial tension was not affected by any ligand in the hearts of wild type mice. This paper therefore showed the differential effects of a neutral (alprenolol) and inverse agonists (e.g. ICI-118,551) besides the importance of receptor density in the study of inverse agonism. Thus, constitutive receptor activity has been demonstrated for several GPCRs after some form of genetic manipulation. We will now review other types of studies that may have a more direct link to in vivo pharmacology and physiology. These studies include cell systems with ‘normal’ levels of receptor expression and tissue or organ bath preparations. The issue of ‘endogenous’ inverse agonists will also be discussed. Various authors have described inverse agonism and constitutive activity on wild type receptors expressed in artificial cell lines but at more or less ‘physiological’ levels of expression. Examples of such studies are listed in Table 1 and discussed below. The rat histamine H2 receptor stably transfected in CHO cells was studied by Smit et al. (1996). This receptor had pronounced basal activity, as indicated by an increase in basal cyclic AMP production. Although this basal cyclic AMP was further increased by the endogenous agonist histamine, the H2 blockers cimetidine and ranitidine were shown to decrease basal cyclic AMP production, therefore expressing inverse agonism in this system. Burimamide, on the other hand, did not alter basal cyclic AMP levels but was able to block both the histamine-induced increase and the cimetidine-induced decrease of basal cyclic AMP production. Hence, burimamide behaved as a neutral antagonist. Similar results were obtained for the human histamine H2 receptor (Alewijnse et al., 1998), although at this receptor burimamide behaved as a weak partial agonist, increasing basal cyclic AMP production by 16%, compared to the maximal response induced by histamine. Newman-Tancredi et al. (1997) studied the 5-HT1A subtype of the serotonin receptor expressed in CHO cells at a receptor density of 1.6 pmol mg−1 protein. Modulation of [35S]-GTPγS binding in a membrane preparation was used to discriminate between the various ligands tested. 5-Carboxamidotryptamine (5-CT) was classified as a full agonist increasing [35S]-GTPγS binding to the same extent as serotonin (5-HT), whereas spiperone was identified as an inverse agonist since it decreased basal [35S]-GTPγS binding by 30%. Meanwhile, WAY100,635 behaved as a neutral antagonist. It showed no effect on basal [35S]-GTPγS binding by itself, but was able to block both 5-CT-induced stimulation and spiperone-induced inhibition of basal [35S]-GTPγS binding. The effect of spiperone could not be explained by a simple displacement of endogenous 5-HT; not only had the membranes been extensively washed, but if basal activity had been due to 5-HT1A receptor activation by endogenous 5-HT, the antagonist WAY100,635 should have also blocked this activation. The behaviour of both subtypes of the human cannabinoid (CB1 and CB2) receptor was analysed by Bouaboula et al. (1997; 1999). CHO cells, stably expressing either the CB1 or the CB2 receptor, showed higher basal MAPK activity compared to untransfected CHO cells. In both transfected cell lines CP-55940, a non-selective cannabinoid agonist, further increased basal MAPK activity. The CB1-selective compound SR141,716A decreased basal MAPK activity in CHO cells expressing the CB1 receptor, thus behaving as an inverse agonist for this receptor (Bouaboula et al., 1997). SR141,716A also displayed effects opposite to agonists in a cyclic AMP-related luciferase assay. Instead of a decrease in luciferase activity induced by cannabinoid agonists, SR141,716A elicited an increase. Apparently, SR141,716A acted as an inverse agonist in two different signal transduction pathways, i.e. Gβγ-mediated MAPK-activity and Gα,i-mediated adenylate cyclase inhibition. Similar results were obtained with SR144,528, a CB2-selective ligand that behaved as an inverse agonist, decreasing both basal MAPK activity and [35S]-GTPγS binding on CHO cells expressing the CB2 receptor (Bouaboula et al., 1999). Furthermore, Landsman et al. (1998) reported on another inverse agonist for the human CB1 receptor, AM630, which decreased basal [35S]-GTPγS binding, in contrast to the cannabinoid agonist WIN55,212-2. Inverse agonistic effects were also shown at various dopamine receptor subtypes. Tiberi & Caron (1994) reported basal receptor activity of the dopamine D1A and D1B receptor, the human D1B receptor being linked to higher intracellular basal cyclic AMP levels, compared to the D1A receptor. Similar results were obtained for the corresponding rat receptors. Two dopamine antagonists, (+)-butaclamol and flupentixol, were able to decrease basal cyclic AMP levels; the inhibitory effect of these compounds, acting as inverse agonists, was more pronounced at the human D1B receptor, since the basal cyclic AMP level was higher. More recently, Griffon et al. (1996) showed that various antipsychotics inhibited [3H]-thymidine incorporation in NG108-15 cells expressing the recombinant human dopamine D3 receptor. Since dopamine agonists enhanced [3H]-thymidine incorporation, the antipsychotics tested (haloperidol, fluphenazine and chlorpromazine), behaved as inverse agonists. Nafadotride, a D3 receptor-preferring antagonist, had no effect of its own on [3H]-thymidine incorporation, therefore behaving as a neutral antagonist. The two isoforms of the human calcitonin receptor (referred to as hCTR-1 and hCTR-2) activate adenylate cyclase, while in addition one of them (hCTR-2) is also able to activate phospholipase C to generate inositol phosphates (IP). Cohen et al. (1997) described constitutive receptor activity of human calcitonin receptors. Both hCTR-1 and hCTR-2 receptors expressed in COS-1 cells were constitutively active as shown by an increase in basal cyclic AMP production, although to a different extent. However, the hCTR-2 receptor did not show an increase in basal IP production. Apparently, spontaneous activity of this receptor was more readily observed for activation of adenylate cyclase. Addition of salmon calcitonin (sCT), a calcitonin receptor agonist, increased cyclic AMP production further, whereas the analogue Nα-acetyl-sCT-(8-32)amide did not elicit an effect. The paucity of available ligands prevented a further demonstration of inverse agonism. A last example is the human formyl peptide (FP) receptor, a chemoattractant GPCR, studied by Wenzel-Seifert et al. (1998). This receptor was expressed in Sf9 and HEK293 cells at receptor densities of approximately 1 pmol mg−1 protein. These receptor levels are in the same range as the receptor density in HL-60 cells that endogenously express the FP receptor. It was shown that basal [35S]-GTPγS binding increased in the presence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), a FP receptor agonist. On the contrary cyclosporin H decreased basal [35S]-GTPγS binding, thereby behaving as an inverse agonist. It seems, therefore, that at physiological receptor levels and expressed in different cell lines, the human FP receptor is constitutively active. A prerequisite for considering the physiological relevance of inverse agonism, is its study in experimental conditions that are as close to physiological as possible. Data related to inverse agonism obtained in intact animals (in vivo) are in most cases difficult to acquire, unless animals are genetically altered (e.g. transgenic mice) as described previously. Thus, apart from potential ‘in vivo’ data, other more or less ‘physiological studies’ may include data from cell lines endogenously expressing the receptor of interest, as for example the previously mentioned NG108-15 cells expressing δ opioid receptors (Costa & Herz 1989), or from tissue preparations such as cardiac or brain cortex membranes. Both latter methods may provide ex vivo data of wild type receptors at physiological or pathophysiological levels. Various examples of such studies in which potential inverse agonism was detected are summarized in Table 2. Hilf & Jakobs (1992) described a decrease in G protein activation by antagonists of the muscarinic receptor in porcine atrial membranes. With this membrane preparation, enriched by sucrose density gradient centrifugation to contain ∼1.4 pmol receptors per mg protein, inhibition of both basal and carbachol-induced [35S]-GTPγS binding by atropine was shown. The presence of endogenous acetylcholine (ACh) was ruled out in this study by pretreatment of the membranes with 10 μM atropine or 100 μM GDP and subsequent washes, both compounds displacing all ACh possibly present. Jakubik et al. (1995) showed inverse agonism in rat cardiomyocytes expressing the M2 receptor. Atropine and QNB, both muscarinic antagonists, increased basal as well as forskolin-induced cyclic AMP production, an effect that was opposite to that of agonists. Similar results were obtained for three other subtypes of the muscarinic receptors, M1, M3 and M4, albeit in a ‘non-physiological’ setting, i.e. in CHO cells stably transfected with the human receptor gene. To detect inverse agonism in H.E.L. 92.1.7 cells endogenously expressing the human α2A-AR, Jansson et al. (1998) used two different assays. Effects of various α2-adrenoceptor ligands on both intracellular levels of Ca2+ ([Ca2+]i) and forskolin-stimulated cyclic AMP production were investigated. The ligands used were thus classified from full agonists to inverse agonists. Both assays gave similar indications of intrinsic activities, showing that different assays and/or signalling pathways can sometimes be used to detect and classify ligands as inverse agonists. Quite remarkable in these studies were the opposite effects of the enantiomers of medetomidine; while dexmedetomidine acted as a partial agonist, increasing [Ca2+]i and decreasing forskolin-stimulated cyclic AMP production, levomedetomidine behaved as an inverse agonist by decreasing [Ca2+]i and increasing cyclic AMP production. Other adrenoceptor subtypes have also been studied to observe inverse agonism in such ‘physiological studies’. The α2D-AR, endogenously expressed in RIN5AH cells and heterologously expressed in PC-12 cells, revealed inverse agonistic effects of rauwolscine in [35S]-GTPγS binding assays (Tian et al., 1994). Accordingly, isoprenaline increased GTPγS-induced adenylate cyclase activity via the β-AR expressed in turkey erythrocytes, while both propranolol and pindolol showed a decrease, thus behaving as inverse agonists (Götze & Jakobs 1994). A single cell preparation from cardiac tissue was used in the following two examples. Mewes et al. (1993) recorded L-type calcium currents (ICa) on both guinea-pig and human ventricular myocytes as a means to study the effects of the β-AR antagonists atenolol and propranolol. The myocytes were first superfused with 0.5 μM forskolin, to make the cells more sensitive to receptor-mediated changes of ICa. Application of both antagonists led to a decrease in forskolin-stimulated ICa in all myocyte preparations. Successive exposures to atenolol, a hydrophilic compound that could be readily washed away, resulted in similar changes in ICa, suggesting that these inhibitory effects were not due to a potential competition with endogenous agonist but to inverse agonistic activity of these antagonists. Hanf et al. (1993) performed a similar study also using cardiomyocytes, but from two different species, frog and rat. They studied ICa regulated by muscarinic receptors. To increase basal ICa, isoprenaline was added during the experiments with frog ventricular cells (Figure 1). Addition of ACh resulted in a decrease of this basal ICa, whereas addition of atropine had the opposite effect, namely an increase of ICa. A similar effect of atropine was also noticed in the absence of Ach. This effect was dose-dependent and reversible. Results obtained with rat myocytes did not require the addition of isoprenaline since basal ICa was already high enough to observe the effects produced by the agonist ACh and the inverse agonist atropine. Atropine behaves as an inverse agonist on calcium currents (ICa) in frog ventricular cells (reproduced with permission from Hanf et al., 1993). Organ bath preparations have also been used to detect inverse agonism (Noguera et al., 1996). Helically cut strips of rat thoracic aorta were prepared and the effects of α1-adrenoceptor antagonists on the regulation of the resting tone, induced by noradrenaline or Ca2+ exposure, were studied. Benoxathian and WB4101 not only inhibited the increase in resting tone induced by noradrenaline, but also blocked the response to noradrenaline in calcium-free medium. An explanation for these results was given within the concept of inverse agonism; it was suggested that both benoxathian and WB4101 acted as inverse agonists, decreasing the proportion of the receptor population in the active state (R*). Moreover, both compounds inhibited the increase in resting tone in the absence of agonist, providing a simple model for analysing inverse agonism in functional studies. Other receptor types showing inverse agonism under ‘physiological conditions’ are the bradykinin BK2 receptor and the δ opioid receptor. Briefly, different bradykinin antagonists decreased basal IP production in rat myometrial cells endogenously expressing the BK2 receptor, while increasing concentrations of bradykinin increased basal IP production (Leeb-Lundberg et al., 1994). Furthermore, ICI-174,864 was shown to behave as an inverse agonist at the δ opioid receptor, endogenously expressed in NG108-15 cells, in both GTPase activity measurements and [35S]-GTPγS binding (Costa & Herz, 1989; Szekeres & Traynor, 1997). It may be concluded from the above examples, that even in ‘classical’ organ bath or cell preparations inverse agonism may be readily demonstrated. This led us to examine older studies that show indirect evidence of some form of inverse agonism ‘avant la lettre’. Within the concept of a (simplified) two-state receptor model (Figure 2), ligand efficacy may be redefined as the differential affinity of the ligand for the two conformational states (R, R*) with full agonists exhibiting higher affinity for the ‘active’ conformation (R*) and full inverse agonists for the ‘inactive’ conformation (R) (Monod et al., 1965; Leff, 1995). This differential affinity may be detected in radioligand binding studies with the use of appropriate ‘modulators’ of the receptor state. For example, in several receptor systems it is known that guanylyl nucleotides (e.g. GTP in the μM–mM range) uncouple the G protein from the receptor leading to a ‘low’ affinity state of the receptor for agonists (Chang & Snyder, 1980; De Lean et al., 1980; Stiles, 1988; Lohse et al.,1984). Thus, ‘GTP-shifts’ have been extensively used, in some GPCR fields, as a parameter to discriminate full from partial agonists due to the differential affinity of these ligands for the two receptor states (Ijzerman et al., 1996; Ehlert et al., 1985). Simplified model representing the two conformational states of a receptor (inactive R, and active R*) and their differential affinities for ligands. However, extrapolation of this concept to antagonists that are able to discriminate between the free and G protein-bound form of the receptor may reflect, and even correlate with, the extent of inverse agonistic properties (negative intrinsic activity) of these antagonists. Although a regulatory role of GTP on antagonist binding, which is inverse to the role of GTP on agonist binding, has been previously suggested (Burgisser et al., 1982; Ströher et al., 1989) we should like to point out its significance and consequence that seems to have escaped attention somewhat. Examples of guanylyl nucleotide enhancement of binding of several antagonists (as have been reported in literature) are shown in Table 3. Both affinity and Bmax values of A1 adenosine receptor antagonists [3H]-XAC and [3H]-DPCPX, on rat adipocyte and guinea-pig brain membranes, respectively (Ramkumar & Stiles, 1988; Ströher et al., 1989), were increased significantly in the presence of GTP, indicating a preference of these antagonists for the uncoupled receptor state. A similar increase in binding was observed with two muscarinic cholinergic antagonists, [3H]-QNB on frog heart membranes (Burgisser et al., 1982) and [N-methyl-3H]-scopolamine methyl chloride on rat heart membranes (Berrie et al., 1979). Accordingly, differential increase in binding of antagonists has also been observed in pertussis-toxin-mediated uncoupling of receptor and G protein; Costa & Herz (1989) were once more the first to show a leftward shift (increase in binding) upon toxin treatment in the competition curve of the δ opioid receptor antagonist ICI-174,864. The absence of any shift of antagonist MR2266 indicated a potential correlation of the negative intrinsic activity of ICI-174,864 with its greater affinity for the uncoupled form of the receptor. Along these lines, interaction of sodium ions with several GPCRs is presumed to result in a stabilization of the low affinity (R) conformation of receptors (Green, 1984; Nunnari et al., 1987). It is believed that this effect of Na+ ions is linked to an aspartate residue in transmembrane helix II, conserved in virtually all GPCRs (Horstman, 1990). This effect of Na+ ions has been shown to be correlated with intrinsic activity of ligands (Tsai & Lefkowitz, 1978), the largest Na+-induced reduction in binding being observed for full agonists. However, although Na+ ions in almost all cases inhibit strongly agonist binding, their effect on ‘antagonist’ binding has been variable, ranging from no effect to an increase in binding (Chang & Synder, 1980; Pert & Snyder, 1974; Nunnari et al., 1987; Green, 1984). Although no explanation was given for this variability of effect among the different ‘antagonists’, it is now tempting to speculate that the inverse agonist behaviour of some may be responsible for the degree of ‘Na+-shift’ they exhibit. Hence, upon retrospective consideration, extrapolation of this concept may shed a new light on the observed effects of Na+ ions. The increase in binding of some antagonists in the presence of Na+ ions, such as [3H]-naloxone to the μ opioid receptor (Pert & Snyder, 1974), ICI-174,864 to the δ opioid receptor (Appelmans et al., 1986), [3H]-yohimbine to the α2-AR (Nunnari et al., 1987) or [125I]-epidepride to the D2 receptor (Neve et al., 1990), may be indicative of the inverse agonistic properties of these ligands. The fact that naloxone has been proposed to act as an inverse opiate agonist in a guinea-pig ileum preparation (Cruz et al., 1996) is further support for this correlation. Finally, one may note that apart from differential binding of ligands, the effect of Na+ on the R⇆R* equilibrium is also apparent in the reduction of basal levels of [35S]-GTPγS binding (Szekeres & Traynor, 1997) or GTPase activity (Gierschik et al., 1989; Costa et al., 1990) in in vitro assays. This reduction is not only similar to the effect of inverse agonists themselves on these assays (decrease of basal receptor activity), but confirms the presence of constitutively active receptors in these assay systems. Thus, although the presence of Na+ ions in radioligand binding assays may help to i" @default.
• W2066475453 created "2016-06-24" @default.
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• W2066475453 date "2000-05-01" @default.
• W2066475453 modified "2023-10-13" @default.
• W2066475453 title "Inverse agonism at G protein-coupled receptors: (patho)physiological relevance and implications for drug discovery" @default.
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• W2066475453 doi "https://doi.org/10.1038/sj.bjp.0703311" @default.
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