Agonist signalling properties of radiotracers used for imaging of dopamine D2/3 receptors
© van Wieringen et al.; licensee Springer. 2014
Received: 14 August 2014
Accepted: 23 September 2014
Published: 7 October 2014
Dopamine D2/3 receptor (D2/3R) agonist radiopharmaceuticals are considered superior to antagonists to detect dopamine release, e.g. induced by amphetamines. Agonists bind preferentially to the high-affinity state of the dopamine D2R, which has been proposed as the reason why agonists are more sensitive to detect dopamine release than antagonist radiopharmaceuticals, but this theory has been challenged. Interestingly, not all agonists similarly activate the classic cyclic adenosine mono phosphate (cAMP) and the ?-arrestin-2 pathway, some stimulate preferentially one of these pathways; a phenomenon called biased agonism. Because these pathways can be affected separately by pathologies or drugs (including dopamine releasers), it is important to know how agonist radiotracers act on these pathways. Therefore, we characterized the intracellular signalling of the well-known D2/3R agonist radiopharmaceuticals NPA and PHNO and of several novel D2/3R agonists.
cAMP accumulation and ?-arrestin-2 recruitment were measured on cells expressing human D2R.
All tested agonists showed (almost) full agonism in both pathways.
The tested D2/3R agonist radiopharmaceuticals did not exhibit biased agonism in vitro. Consequently, it is likely that drugs (including psychostimulants like amphetamines) and/or pathologies that influence the cAMP and/or the ?-arrestin-2 pathway may influence the binding of these radiopharmaceuticals.
The dopamine (DA) system plays a central role in several neuropsychiatric disorders including Parkinson's disease and schizophrenia. DA D2 receptor (D2R) antagonists are used to reduce psychotic symptoms, whereas D2R agonists are commonly used in the treatment of Parkinson's disease. Consequently, radiopharmaceuticals targeting D2Rs are of value to obtain insight in the pathophysiology of these brain disorders.
The D2R is a subfamily within the superfamily of G-protein-coupled receptors (GPCRs) and contains several subtypes including some in the D2-like subfamily, the D2 (splice variants D2short (D2S) and D2long (D2L) ), D3 and D4 receptors . They primarily couple to the Gi/o type of G-proteins to inhibit the enzyme adenylyl cyclase in producing cyclic adenosine mono phosphate (cAMP) . Like other GPCRs, they exhibit interconvertible high- and low-affinity states for agonists in vitro . In the high-affinity state, the receptor is coupled to the G-protein and this is considered to be the active state of the receptor. In the low-affinity state, the receptor is uncoupled and consequently inactive.
Dopamine D2/3 receptor (D2/3R) agonist radiopharmaceuticals for positron emission tomography (PET) have been developed successfully (e.g. 11C-NPA (N-propylnorapomorphine) and 11C-PHNO ((+)-4-propyl-9-hydroxynaphthoxazine)), and such agonists are more sensitive in detecting DA release in humans compared to antagonists like 11C-raclopride -. It has been proposed that the reason for this increased sensitivity may be that D2/3R agonist radiopharmaceuticals bind preferentially to the high-affinity state of the dopamine D2R, whereas antagonists do not differentiate between the high- and low-affinity states -. However, this proposal depends upon the existence of two affinity states in vivo and the results of recent studies challenged this theory -.
Activation of the ?-arrestin-2 pathway may play a role in the increased sensitivity to detect dopamine release in vivo. Activation of this pathway eventually leads to the regulation of glycogen synthase kinase 3 (GSK3), a protein that is involved in many DA-dependent behaviours . Many drugs (antipsychotics, antidepressants, lithium) affect this cascade, and recently compounds, based on the aripiprazole scaffold, were discovered that are functionally selective for the ?-arrestin-2 pathway ,. Consequently, it is important to characterize pharmacologically the intracellular pathways that DA D2/3R agonist radiopharmaceuticals act on. This information is not only relevant when the radiopharmaceuticals are used to evaluate actions of novel drugs which may show biased agonism but also because ?-arrestin-2 itself may be involved in the desensitization of DA receptors and as such in the detection of DA. Literature about biased signalling of currently available agonist PET radiotracers 11C-NPA and 11C-PHNO at cloned DA receptors does not exist, and therefore, we tested unlabelled (`cold¿) NPA and PHNO for agonism in both the cAMP and ?-arrestin-2 assays.
Additionally, and in a broader perspective, we are developing new DA D2/3R agonist radiotracers for PET and single-photon emission computed tomography (SPECT) imaging, where the investigated compounds are based on the 2-aminomethylchromane (AMC) scaffold. We have tested agonism for our AMC compounds by means of a cAMP assay and have found that most of these compounds showed full agonism for this canonical pathway . As for NPA and PHNO, also for these new AMC tracers, we were interested to evaluate if they would show biased agonism. Finally, the D2/3R antagonist SPECT tracer 123I-iodobenzamide (123I-IBZM) is used in many studies including studies on Parkinsonism  and on amphetamine-induced DA release . Based on early studies in rats showing that IBZM was a potent inhibitor of an apomorphine-induced syndrome (apomorphine is a D2R agonist) of hyperactivity and stereotypy, it was concluded that IBZM was a DA antagonist . As no other research examining the functional agonism or antagonism of IBZM has been reported, we also tested IBZM for biased agonism. Of note, studies with other receptors show that antagonists for one signalling pathway of a receptor may be agonists for another pathway.
Accordingly, in this study, we have tested if clinically used as well as recently developed D2/3R radiopharmaceuticals show biased agonism.
cAMP accumulation assay and receptor binding assay
As described in detail earlier , cAMP accumulation was measured in HEK 293 cells stably expressing the human DA D2L receptor with an assay from PerkinElmer (Waltham USA). Receptor binding experiments were done on the membranes obtained from the above mentioned cells using 3H-spiperone as radioligand.
?-arrestin-2 recruitment assay
The PathHunter¿ eXpress human DA receptor D2L CHO-K1 ?-arrestin-2 GPCR assay from DiscoveRx (Fremont, CA, USA) was used according to the manufacturer's protocol. Cells were seeded at a density of 8,000 cells/well in a 96-well plate in 100 ?l PathHunter¿ CP reagent and incubated 48 h in a humidified atmosphere at 5% CO2 at 37°C. After 48 h, the compounds dissolved in Hank's balanced salt solution supplemented with 5 mM HEPES and 1.1% DMSO were added to the wells in duplo. Then a detection mixture was made and added to the wells. The cells were incubated for 60 min at room temperature, and subsequently chemiluminescence was measured on a Wallac Victor (PerkinElmer, Zaventum, Belgium).
Radioligand binding and functional activities of different compounds*
The potencies of all tested compounds, as measured in the two functional tests, correlated (r =0.80; Spearman's rank correlation).
We found that all agonist compounds tested that were active in the cAMP assays were also active in ?-arrestin-2 recruitment with an efficacy similar to that of DA. Moreover, the potencies of all agonists in the two functional assays were well correlated with a slope close to unity (1.046?±?0.3965). However, their potency was consistently higher in the cAMP than in the ?-arrestin-2 assay, especially for DA and PHNO. This may be explained by the presence of a receptor reserve in the cells used in the cAMP experiments causing signal amplification while the arrestin recruitment assay measures a more 1:1 interaction of the receptor to arrestin. All in all, the present data do not support biased agonism for these compounds.
Skinbjerg and colleagues recently demonstrated that the striatal binding potential (BPND) of the agonist 11C-MNPA and the antagonist (or inverse agonist; for discussion see below) 18F-fallypride was not different in wild-type mice and ?-arrestin-2 knockout mice . The results of this study suggest that a modified arrestin pathway does not affect radiotracer binding during `baseline¿ conditions, independent of agonist signalling properties of the radiotracer. A modification in the arrestin pathway is more likely to be demonstrated using a pharmacological challenge, as shown in the amphetamine studies by Skinbjerg and co-workers . The current work, therefore, appears most relevant to drug interaction studies, and consequently may shed new light on the reason why agonist D2/3R radiopharmaceuticals are more sensitive to detect DA release than antagonists -. More specifically, it has been demonstrated that arrestin desensitizes the D2R and prepares it for internalization by hindering the coupling of the G-protein to the receptor. Consequently, the desensitized receptor is in the low-affinity state . So, after agonist binding, the receptor is switched to the low-affinity state and kept in that state by arrestin; thus, theoretically the density of D2 receptors in the high-affinity state decreases after binding with an agonist that recruits arrestin.
DA release is measured with PET or SPECT by assessing the difference in binding potential for radiotracers that label DA D2/3Rs after a dopaminergic challenge as compared to a baseline condition ,,. DA can displace the radiotracer from the receptor (competition model), but the lower binding potential after a DA challenge may also be explained partly by internalization of DA D2/3Rs. In a typical PET/SPECT study, DA release is measured 1 h after a DA challenge ,, and internalization of DA D2/3Rs has been reported in a 11C-raclopride PET study in cats already 1 h after the administration of the DA releaser amphetamine . Moreover, Guo and co-workers  demonstrated a lower binding affinity of agonist and antagonists radiotracers for internalized DA D2/3Rs, possibly reflecting a poorer accessibility than to surface receptors. Since, the ?-arrestin pathway plays an important role in the internalization of DA D2/3Rs, it is important to know if a radiotracer is biased towards or away from ?-arrestin translocation. Radiotracers that are biased towards cAMP may not promote ?-arrestin-mediated receptor internalization and in theory may be more accurate tools to detect DA release. Recently cAMP-specific compounds have been developed for the dopamine D2R and may offer the opportunity to be developed further to radioligands in future investigations . These propositions may have implications for the interpretation of results from clinical studies with agonist tracers, particularly pharmacological challenge studies, e.g. with DA-releasing psychostimulants.
Since all tested agonists activate the cAMP as well as the ?-arrestin-2 pathway, it is likely that they will also activate both GSK3 (via ?-arrestin-2) and protein kinase A (via cAMP). This information is of importance when these radiopharmaceuticals are used to study or evaluate (novel) drugs. Dopaminergic drugs, like antipsychotics, but also drugs affecting other monoamine systems like the serotonin releaser fenfluramine, exert actions on the ?-arrestin-2 pathway. Lithium, a drug used in the treatment of bipolar disorder, was found to have a direct inhibiting action on GSK3 . Finally, compounds based on the aripiprazole scaffold were recently discovered that are functionally selective for the ?-arrestin-2 pathway and that show robust antipsychotic effects in rodents ,. So, it is likely that drugs that influence the canonical and/or the non-canonical pathway, may influence indirectly the in vivo binding of our presently tested D2/3R agonists, including the radiotracers 11C-NPA and 11C-PHNO, which are frequently used in human studies ,.
Our novel finding that IBZM showed inverse agonism in the cAMP assay is in line with recent findings that the antipsychotics nemonapride and sulpiride (similar to IBZM, benzamides), and also antipsychotics from other chemical classes (e.g. butyrophenones like haloperidol), that were initially believed to be DA receptor neutral antagonists are now re-classified as inverse agonists by the results of studies using functional assays . It is alleged that inverse agonists bind preferentially to the inactive state of the receptor , so this might indicate that in vivo123I-IBZM predominantly binds to striatal D2Rs in their inactive low-affinity state. Since 11C-raclopride PET and 18F-fallypride are frequently used to assess DA release in vivo,,, it may be of relevance to test whether raclopride and fallypride are also inverse agonists for cAMP instead of antagonists. If our proposition is true that benzamide radiopharmaceuticals in vivo bind predominantly to DA D2/3Rs in their low-affinity state, this will extend the discussion on why benzamides tracers are less sensitive than agonists D2/3R radiopharmaceuticals (like 11C-PHNO) to detect DA release in vivo,.
Nevertheless, more functional pharmacological characterization studies on agonist and antagonist (or inverse agonists) D2/3R radiopharmaceuticals are needed to fully understand the mechanism how dopamine release can be detected in vivo using such radiotracers.
In this study, we focused on the D2R. Since all developed radiopharmaceuticals to image this receptor also bind to the D3R, it may be of interest to evaluate in future studies also how these radiotracers act on intracellular signalling of the D3R.
The agonist compounds tested exhibited agonism for both the cAMP and the ?-arrestin-2 pathways, and no evidence was found for biased agonism. This information is crucial for the interpretation of findings with these tracers, especially in drug interaction studies. In addition, our data demonstrate, for the first time, that the benzamide IBZM is an antagonist for both ?-arrestin-2 recruitment and cAMP formation, displaying inverse agonist properties for the latter.
Work in the authors' lab has been supported by a grant from the Dutch Technology Foundation STW (grant 10127).
- Usiello A, Baik JH, Rouge-Pont F, Picetti R, Dierich A, LeMeur M, Piazza PV, Borrelli E: Distinct functions of the two isoforms of dopamine D2 receptors. Nature 2000, 408: 199–203. 10.1038/35041572View ArticlePubMedGoogle Scholar
- Jackson DM, Westlinddanielsson A: Dopamine receptors: molecular-biology, biochemistry and behavioral-aspects. Pharmacol Ther 1994, 64: 291–370. 10.1016/0163-7258(94)90041-8View ArticlePubMedGoogle Scholar
- Sibley DR, De Lean A, Creese I: Anterior pituitary dopamine receptors. Demonstration of interconvertible high and low affinity states of the D-2 dopamine receptor. J Biol Chem 1982, 257: 6351–6361.PubMedGoogle Scholar
- Ginovart N, Galineau L, Willeit M, Mizrahi R, Bloomfield PM, Seeman P, Houle S, Kapur S, Wilson AA: Binding characteristics and sensitivity to endogenous dopamine of [11C]-(+)-PHNO, a new agonist radiotracer for imaging the high-affinity state of D2 receptors in vivo using positron emission tomography. J Neurochem 2006, 97: 1089–1103. 10.1111/j.1471-4159.2006.03840.xView ArticlePubMedGoogle Scholar
- Narendran R, Mason NS, Laymon CM, Lopresti BJ, Velasquez ND, May MA, Kendro S, Martinez D, Mathis CA, Frankle WG: A comparative evaluation of the dopamine D2/3 agonist radiotracer [11C](-)-N-propyl-norapomorphine and antagonist [11C] raclopride to measure amphetamine-induced dopamine release in the human striatum. J Pharmacol Exp Ther 2010, 333: 533–539. 10.1124/jpet.109.163501PubMed CentralView ArticlePubMedGoogle Scholar
- Narendran R, Hwang DR, Slifstein M, Talbot PS, Erritzoe D, Huang Y, Cooper TB, Martinez D, Kegeles LS, Abi-Dargham A, Laruelle M: In vivo vulnerability to competition by endogenous dopamine: comparison of the D2 receptor agonist radiotracer (-)-N-[11C]propyl-norapomorphine ([11C]NPA) with the D2 receptor antagonist radiotracer [11C]-raclopride. Synapse 2004, 52: 188–208. 10.1002/syn.20013View ArticlePubMedGoogle Scholar
- Shotbolt P, Tziortzi AC, Searle GE, Colasanti A, van der Aart J, Abanades S, Plisson C, Miller SR, Huiban M, Beaver JD, Gunn RN, Laruelle M, Rabiner EA: Within-subject comparison of [11C]-(+)-PHNO and [11C]raclopride sensitivity to acute amphetamine challenge in healthy humans. J Cerebr Blood Flow Metabol 2012, 32: 127–136. 10.1038/jcbfm.2011.115View ArticleGoogle Scholar
- Laruelle M: Imaging synaptic neurotransmission with in vivo binding competition techniques: a critical review. J Cerebr Blood Flow Metabol 2000, 20: 423–451. 10.1097/00004647-200003000-00001View ArticleGoogle Scholar
- Seneca N, Finnema SJ, Farde L, Gulyas B, Wikström HV, Halldin C, Innes RB: Effect of amphetamine on dopamine D2 receptor binding in nonhuman primate brain: a comparison of the agonist radioligand [11C]MNPA and antagonist [11C]raclopride. Synapse 2006, 59: 260–269. 10.1002/syn.20238View ArticlePubMedGoogle Scholar
- Skinbjerg M, Namkung Y, Halldin C, Innis RB, Sibley DR: Pharmacological characterization of 2-methoxy-N-propylnorapomorphine's interactions with D2 and D3 dopamine receptors. Synapse 2009, 63: 462–475. 10.1002/syn.20626PubMed CentralView ArticlePubMedGoogle Scholar
- Finnema S, Halldin C, Halldin C, Bang-Andersen B, Gulyas B, Bundgaard C, Wikström HV, Farde L: Dopamine D2/3 receptor occupancy of apomorphine in the nonhuman primate brain- a comparative PET study with [11C] raclopride and [11C] MNPA. Synapse 2009, 63: 378–389. 10.1002/syn.20615View ArticlePubMedGoogle Scholar
- McCormick PN, Kapur S, Seeman P, Wilson AA: Dopamine D2 receptor radiotracers [11C](+)-PHNO and [3H] raclopride are indistinguishably inhibited by D2 agonists and antagonists ex vivo. Nucl Med Biol 2008, 35: 11–17. 10.1016/j.nucmedbio.2007.08.005View ArticlePubMedGoogle Scholar
- McCormick PN, Kapur S, Reckless G, Wilson AA: Ex vivo [11C]-(+)-PHNO binding is unchanged in animal models displaying increased high-affinity states of the D2 receptor in vitro. Synapse 2009, 63: 998–1009. 10.1002/syn.20671View ArticlePubMedGoogle Scholar
- Michel MC, Alewijnse AE: Ligand-directed signaling: 50 ways to find a lover. Mol Pharmacol 2007, 72: 1097–1099. 10.1124/mol.107.040923View ArticlePubMedGoogle Scholar
- Beaulieu JM, Gainetdinov RR: The physiology, signaling, and pharmacology of dopamine receptors. Pharmacol Rev 2011, 63: 182–217. 10.1124/pr.110.002642View ArticlePubMedGoogle Scholar
- Beaulieu JM, Gainetdinov RR, Caron MG: The Akt-GSK-3 signaling cascade in the actions of dopamine. Trends Pharmacol Sci 2007, 28: 166–172. 10.1016/j.tips.2007.02.006View ArticlePubMedGoogle Scholar
- Chen X, Sassano MF, Zheng L, Setola V, Chen M, Bai X, Frye SV, Wetsel WC, Roth BL, Jin J: Structure-functional selectivity relationship studies of ?-arrestin-biased dopamine D2 receptor agonists. J Med Chem 2012, 55: 7141–7153. 10.1021/jm300603yPubMed CentralView ArticlePubMedGoogle Scholar
- Allen JA, Yost JM, Setola V, Chen X, Sassano MF, Chen M, Peterson S, Ydav PN, Huang XP, Feng B, Jensen NH, Che X, Bai X, Frye SV, Wetsel WC, Caron MG, Javitch JA, Roth BL, Jin J: Discovery of beta-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. Proc Natl Acad Sci U S A 2011, 108: 18488–18493. 10.1073/pnas.1104807108PubMed CentralView ArticlePubMedGoogle Scholar
- van Wieringen JP, Shalgunov V, Janssen HM, Fransen PM, Janssen AGM, Michel MC, Booij J, Elsinga PH: Synthesis and characterization of a novel series of agonist compounds as potential radiopharmaceuticals for imaging of dopamine D2/3 receptors in their high-affinity state. J Med Chem 2014, 57: 291–410.View ArticleGoogle Scholar
- Wenning GK, Donnemiller E, Granata R, Riccabona G, Poewe W: 123I-?-CIT and 123I-IBZM-SPECT scanning in levodopa-naive Parkinson's disease. Mov Disord 1998, 13: 438–445. 10.1002/mds.870130311View ArticlePubMedGoogle Scholar
- Booij J, Korn P, Linszen DH, van Royen EA: Assessment of endogenous dopamine release by methylphenidate challenge using iodine-123 iodobenzamide single-photon emission tomography. Eur J Nucl Med 1997, 24: 674.PubMedGoogle Scholar
- Depaulis T, Kumar Y, Johansson L, Ramsby S, Florvall L, Hall H, Angebymoller K, Ogren SO: Potential neuroleptic agents. 3. Chemistry and antidopaminergic properties of substituted 6-methoxysalicylamides. J Med Chem 1985, 28: 1263–1269. 10.1021/jm00147a025View ArticleGoogle Scholar
- Skinbjerg M, Liow JS, Seneca N, Hong J, Lu S, Thorsell A, Heilig M, Pike VW, Halldin C, Sibley DR, Innis RB: D2 dopamine receptor internalization prolongs the decrease of radioligand binding after amphetamine: a PET study in a receptor internalization-deficient mouse model. Neuroimage 2010, 50: 1402–1407. 10.1016/j.neuroimage.2010.01.055PubMed CentralView ArticlePubMedGoogle Scholar
- Seeman P: Are dopamine D2 receptors out of control in psychosis? Progr Neuro-Psychopharmacol Biol Psych 2013, 46: 146–152. 10.1016/j.pnpbp.2013.07.006View ArticleGoogle Scholar
- van de Giessen E, Celik F, Schweitzer DH, van den Brink W, Booij J: Dopamine D2/3 receptor availability and amphetamine-induced dopamine release in obesity. J Psychopharmacol 2014, 28: 866–873. 10.1177/0269881114531664View ArticlePubMedGoogle Scholar
- Ginovart N, Wilson AA, Houle S, Kapur S: Amphetamine pretreatment induces a change in both D2-receptor density and apparent affinity: a [11C]raclopride positron emission tomography study in cats. Biol Psychiatry 2004, 55: 1188–1194. 10.1016/j.biopsych.2004.02.019View ArticlePubMedGoogle Scholar
- Guo N, Guo W, Kralikova M, Jiang M, Schieren I, Narendran R, Slifstein M, Abi-Dargham A, Laruelle M, Javitch JA, Rayport S: Impact of D2 receptor internalization on binding affinity of neuroimaging radiotracers. Neuropsychopharmacol 2009, 35: 806–817. 10.1038/npp.2009.189View ArticleGoogle Scholar
- Free RB, Chun LS, Moritz AE, Miller BN, Doyle TB, Conroy JL, Padron A, Meade JA, Xiao J, Hu X, Dulcey AE, Han Y, Duan L, Titus S, Bryant -Genever M, Barnaeva E, Ferrer M, Javitch JA, Beuming T, Shi L, Southall NT, Marugan JJ, Sibley DR: Discovery and characterization of a G protein-biased agonist that inhibits b-arrestin recruitment to the D2 dopamine receptor. Mol Phamacol 2014, 86: 96–105.Google Scholar
- Hall DA, Strange PG: Evidence that antipsychotic drugs are inverse agonists at D2 dopamine receptors. Br J Pharmacol 1997, 121: 731–736. 10.1038/sj.bjp.0701196PubMed CentralView ArticlePubMedGoogle Scholar
- Rang HP, Dale MM, Ritter JM: How drugs act: general principles. In Pharmacology. 5th edition. Edited by: Hunter L. Churchill Livingstone, London; 2003:17.Google Scholar
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