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Pharmacological characterisation of the agonist radioligand binding site of 5-HT2A, 5-HT2B and 5-HT2C receptors

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Abstract

In the present study we compared the affinity of various drugs for the high affinity “agonist-preferring” binding site of human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors stably expressed in monoclonal mammalian cell lines. To ensure that the “agonist-preferring” conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([3H]-ketanserin for 5-HT2A receptor and [3H]-mesulergine for 5-HT2B and 5-HT2C receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([125I]-DOI for 5-HT2A receptor binding and [3H]-5-HT for 5-HT2B and 5-HT2C receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the “agonist-preferring” subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT2 receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT2A, 5-HT2B and 5-HT2C receptors respectively). There remains, however, a lack of highly selective agonists. (−)DOI is potent and moderately selective for 5-HT2A receptors, BW723C86 has poor selectivity for human 5-HT2B receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT2C receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT2 receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT2 receptor pharmacology.

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References

  • Akiyoshi J, Isogawa K, Yamada K, Nagayama H, Fuji F (1996) Effects of antidepressants on intracellular Ca2+ mobilization in CHO cells transfected with the human 5-HT2C receptors. Biol Psychiatry 39:1000–1008

    Article  CAS  PubMed  Google Scholar 

  • Barnes NM, Sharp T (1999) A review of central 5-HT receptors and their function. Neuropharmacology 38:1083–1152

    Article  CAS  PubMed  Google Scholar 

  • Baxter GS, Murphy OE, Blackburn TP (1994) Further characterisation of 5-hydroxytryptamine receptors (putative 5-HT2B) in rat stomach fundus longitudinal muscle. Br J Pharmacol 112:323–331

    CAS  PubMed  Google Scholar 

  • Baxter GS, Kennett GA, Blackburn TP, Blaney F (1995) 5-HT2 receptor subtypes, a family reunited? Trends Pharmacol Sci 16:105–110

    Article  CAS  PubMed  Google Scholar 

  • Bentley JM, Mansell HL, Roffey JRA, Adams DR, Davidson JEP, Gaur S, Monck NJT, Cliffe IA, Dawson CE, Pratt RM, Bebbington D, Duncton MAJ, Hamlyn RJ, Giles PR, George AR, Bickerdike MJ, Vickers SP, Benwell K, Misra A, Quirk K, Malcolm CS, Knight AR, Kennett GA, Dourish CT (2004) Indoline derivatives as 5-HT2C receptor agonists. Bioorg Med Chem Lett 14:2367–2370

    Article  CAS  PubMed  Google Scholar 

  • Bickerdike MJ (2003) 5-HT2C receptor agonists as potential drugs for the treatment of obesity. Curr Top Med Chem 3:885–897

    CAS  PubMed  Google Scholar 

  • Bonhaus DW, Bach C, DeSouza A, Salazar FHR, Matsuoka BD, Zuppan P, Chan HW, Eglen RM (1995) The pharmacology and distribution of human 5-hydroxytryptamine2B (5-HT2B) receptor gene products: comparison with 5-HT2A and 5-HT2C receptors. Br J Pharmacol 115:622–628

    CAS  PubMed  Google Scholar 

  • Borman RA, Tilford NS, Harmer DW, Day N, Ellis ES, Sheldrick RLG, Carey J, Coleman RA, Baxter GS (2002) 5-HT2B receptors play a key role in mediating the excitatory effects of 5-HT in human colon in vitro. Br J Pharmacol 135:1144–1151

    CAS  PubMed  Google Scholar 

  • Bos M, Jenck F, Martin JR, Moreau JL, Sleight AJ, Wichmann J, Widmer U (1997) Novel agonists of 5-HT2C receptors. Synthesis and biological evaluation of substituted 2-(indol-1-yl)-1-methylethylamines and 2-(indeno[1,2-b]pyrrol-1-yl)-1-methylethylamines. Improved therapeutics for obsessive compulsive disorder. J Med Chem 40:2762–2769

    Article  CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Bromidge SM, Dabbs S, Davies DT, Davies S, Duckworth DM, Forbes IT, Gadre A, Ham P, Jones GE, King FD, Saunders DV, Thewlis KM, Vyas D, Blackburn TP, Holland V, Kennett GA, Riley GJ, Wood MD (1999) Model studies on a synthetically facile series of N-substituted phenyl-N′-pyridin-3-yl-ureas leading to 1-(3-pyridylcarbamoyl)indolines that are potent and selective 5-HT2C/2B receptor antagonists. Bioorg Med Chem Lett 7:2767–2773

    Article  CAS  Google Scholar 

  • Cheng, YC, Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108

    Article  CAS  PubMed  Google Scholar 

  • Clemmett DA, Punhani T, Duxon MS, Blackburn TP, Fone KCF (1999) Immunohistochemical localisation of the 5-HT2C receptor protein in the rat CNS. Neuropharmacology 39:123–132

    Article  Google Scholar 

  • Czoty PW, Howell LL (2000) Behavioral effects of AMI-193, a 5-HT(2A)- and dopamine D(2)-receptor antagonist, in the squirrel monkey. Pharmacol Biochem Behav 67:257–264

    Article  CAS  PubMed  Google Scholar 

  • Damjanoska KJ, Muma NA, Zhang Y, D’Souza DN, Garcia F, Carrasco GA, Kindel GH, Haskins KA, Shankaran M, Petersen BR, Van De Kar LD (2003) Neuroendocrine evidence that (S)-2-(chloro-5-fluoro-indol-l-yl)-1-methylethylamine fumarate (Ro 60-0175) is not a selective 5-hydroxytryptamine2C receptor agonist. J Pharmacol Exp Ther 304:1209–1216

    Article  CAS  PubMed  Google Scholar 

  • De Vry J, Schreiber R (2000) Effects of selected serotonin 5-HT1 and 5-HT2 receptor agonists on feeding behavior: possible mechanisms of action. Neurosci Biobehav Rev 24:341–353

    Article  PubMed  Google Scholar 

  • Dourish CT (1995) Multiple serotonin receptors: opportunities for new treatment for obesity? Obes Res 3:449S–462S

    CAS  PubMed  Google Scholar 

  • Duxon MS, Flanigan TP, Reavley AC, Baxter GS, Blackburn TP, Fone KC (1997) Evidence for expression of the 5-hydroxytryptamine-2B receptor protein in the rat central nervous system. Neuroscience 76:323–329

    Article  CAS  PubMed  Google Scholar 

  • Egan CT, Herrick-Davis K, Miller K, Glennon RA, Teitler M (1998) Agonist activity of LSD and lisuride at cloned 5-HT2A and 5-HT2C receptors. Psychopharmacology 136:409–414

    Article  CAS  PubMed  Google Scholar 

  • Fitzgerald LW, Conklin DS, Krause CM, Marshall AP, Patterson JP, Tran DP, Iyer G, Kostich WA, Largent BL, Hartig PR (1999) High-affinity agonist binding correlates with efficacy (intrinsic activity) at the human serotonin 5-HT2A and 5-HT2C receptors: evidence favouring the ternary complex and two-state models of agonist action. J Neurochem 72:2127–2134

    Article  CAS  PubMed  Google Scholar 

  • Flanigan TP, Reavely AC, Carey JE, Leslie RA (1995) Evidence for expression of the 5-HT2B receptor mRNA in rat brain. Br J Pharmacol 114:369P

    Google Scholar 

  • Glennon RA, Metwally K, Dukat M, Ismaiel, Abd M, De Los Angeles J, Herndon J, Teitler M, Khorana N (2002) Ketanserin and spiperone as templates for novel serotonin 5-HT2A antagonists. Curr Top Med Chem 2:539–558

    CAS  PubMed  Google Scholar 

  • Havlik S, Peroutka SJ (1992) Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]-mesulergine in a clonal 5-hydroxytryptamine1C cell line. Brain Res 584:191–196

    Article  CAS  PubMed  Google Scholar 

  • Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PPA (1994) International Union of Pharmacology classification of receptors for 5-hydroxy tryptamine (serotonin). Pharmacol Rev 46:157–203

    CAS  PubMed  Google Scholar 

  • Humphrey PPA, Hartig P, Hoyer D (1993) A proposed new nomenclature for 5-HT receptors. Trends Pharmacol Sci 14:233–236

    Article  CAS  PubMed  Google Scholar 

  • Ismaiel AM, De Los Angeles J, Teitler M, Ingher S, Glennon RA (1993) Antagonism of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane stimulus with a newly identified 5-HT2- versus 5-HT1C-selective antagonist. J Med Chem 36:2519–2525

    CAS  PubMed  Google Scholar 

  • Jerman JC, Brough SJ, Gager T, Wood M, Coldwell MC, Smart D, Middlemiss DN (2001) Pharmacological characterisation of human 5-HT2 receptor subtypes. Eur J Pharmacol 414:23–30

    Article  CAS  PubMed  Google Scholar 

  • Kennett GA (1993) 5-HT1C receptors and their therapeutic relevance. Curr Opin Invest Drugs 2:317–362

    Google Scholar 

  • Kennett GA, Curzon G (1988) Evidence that hypophagia induced by mCPP and TFMPP requires 5-HT1C and 5-HT1B receptors; hypophagia induced by RU 24969 only requires 5-HT1B receptors. Psychopharmacology 96:93–100

    Google Scholar 

  • Kennett GA, Bailey F, Piper DC, Blackburn TP (1995) Effect of SB 200646A, a 5-HT2C/5-HT2B receptor antagonist in two conflict models of anxiety. Psychopharmacology 118:178–182

    Google Scholar 

  • Kennett GA, Wood MD, Bright F, Cilia J, Piper DC, Gager T, Thomas D, Baxter GS, Forbes IT, Ham P, Blackburn TP (1996) In vitro and in vivo profile of SB-206553, a potent 5-HT2C/5-HT2B receptor antagonist with anxiolytic-like properties. Br J Pharmacol 117:427–434

    CAS  PubMed  Google Scholar 

  • Kennett GA, Wood MD, Bright F, Trail B, Riley G, Holland V, Avenell KY, Stean T, Upton N, Bromidge S, Forbes IT, Brown AM, Middlemiss DN, Blackburn TP (1997a) SB 242084, a selective and brain penetrant 5-HT2C receptor antagonist. Neuropharmacology 36:609–620

    Article  CAS  PubMed  Google Scholar 

  • Kennett GA, Ainsworth K, Trail B, Blackburn TP (1997b) BW723C86, a 5-HT2B receptor agonist causes hyperphagia and reduced feeding in rats. Neuropharmacology 36:233–239

    Article  CAS  PubMed  Google Scholar 

  • Kursar JD, Nelson DL, Wainscott DB, Cohen ML, Baez M (1994) Molecular cloning, functional expression and mRNA tissue distribution of the human 5-hydroxytryptamine2B receptor. Mol Pharmacol 46:227–234

    CAS  PubMed  Google Scholar 

  • Launay J-M, Herve P, Peoc’h K, Tournois C, Callebert J, Nebigil CG, Etienne N, Drouet L, Humbert M, Simonneau G, Maroteaux L (2002) Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension. Nat Med 8:1129–1135

    Article  CAS  PubMed  Google Scholar 

  • Leonhardt S, Gorospe E, Hoffman BJ, Teitler M (1992) Molecular pharmacological differences in the interaction of serotonin with 5-hydroxytryptamine1C and 5-hydroxytryptamine2 receptors. Mol Pharmacol 42:328–335

    CAS  PubMed  Google Scholar 

  • Leysen D, Kelder J (1998) Ligands for the 5-HT2C receptor as potential antidepressants and anxiolytics. In: van Der Groot H (ed) Pharmacochemistry library, trends in drug research. Elsevier, Amsterdam, pp 49–61

    Google Scholar 

  • Liu Y-L, Heal DJ, Stock MJ (2002) Mechanism of the thermogenic effect of metabolite 2 (BTS 54 505), a major pharmacologically active metabolite of the novel anti-obesity drug, sibutramine. Int J Obes 26:1245–1253

    Article  CAS  Google Scholar 

  • Lopez-Gimenez JF, Villazon M, Brea J, Loza I, Palacios JM, Mengod G, Vilaro MT (2001) Multiple conformations of native and recombinant human 5-hydroxytrptamine2A receptors are labeled by agonists and discriminated by antagonists. Mol Pharmacol 60:690–699

    CAS  PubMed  Google Scholar 

  • Lucki I, Ward HR, Frazer A (1989) Effect of 1-(m-chlorophenyl)piperazine and 1-(m-trifluoromethylphenyl)piperazine on locomotor activity. J Pharmacol Exp Ther 249:155–164

    CAS  PubMed  Google Scholar 

  • Marek GJ, Aghajanian GK (1996) LSD and the phenylethylamine hallucinogen DOI are potent partial agonists at 5-HT2A receptors on interneurons in rat piriform cortex. J Pharmacol Exp Ther 278:1373–1382

    CAS  PubMed  Google Scholar 

  • Martin GR, Humphrey PPA (1994) Receptors for 5-hydroxytryptamine: current perspectives on classification and nomenclature. Neuropharmacology 33:261–273

    Article  CAS  PubMed  Google Scholar 

  • Nelson DL, Lucaites VL, Wainscott DB, Glennon RA (1999) Comparisons of hallucinogenic phenylisopropylamine binding affinities at cloned human 5-HT2A, 5-HT2B and 5-HT2C receptors. Naunyn-Schmiedebergs Arch Pharmacol 359:1–6

    CAS  PubMed  Google Scholar 

  • Nozulak J, Kalkmann HO, Floersheim P, Hoyer D, Schoeffter P, Buerki HR (1994) (+-cis-4,5,6,7a,8,9,10,11a-Octahydro-7H-10-methylindolo[1,7-bc][2,6]-aphthyridine: a 5-HT2C/2B receptor antagonist with low 5-HT2A receptor affinity. J Med Chem 38:28–33

    Google Scholar 

  • Peroutka SJ, Snyder SH (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol. Mol Pharmacol 16:687–699

    CAS  PubMed  Google Scholar 

  • Pompeiano M, Palacios JM, Mengod G (1994) Distribution of the serotonin 5-HT2 receptor family mRNAs: comparison between 5-HT2A and 5-HT2C receptors. Mol Brain Res 23:163–178

    Article  CAS  PubMed  Google Scholar 

  • Porter RHP, Benwell KR, Lamb H, Malcolm CS, Allen NH, Revell DF, Adams DR, Sheardown MJ (1999) Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells. Br J Pharmacol 128:13–20

    CAS  PubMed  Google Scholar 

  • Quirk K, Lawrence A, Jones J, Misra A, Harvey V, Lamb H, Revell D, Porter RHP, Knight AR (2001) Characterisation of agonist binding on human 5-HT2C receptor isoforms. Eur J Pharmacol 419:107–112

    Article  CAS  PubMed  Google Scholar 

  • Roth BL (1994) Multiple serotonin receptors: clinical and experimental aspects. Ann Clin Psychiatry 6:67–78

    CAS  PubMed  Google Scholar 

  • Roth BL, Willins DL, Kristiansen K, Kroeze WK (1998) 5-hydroxytryptamine2-family receptors (5-hydroxytryptamine2A, 5-hydroxytryptamine2B, 5-hydroxytryptamine2C): where structure meets function. Pharmacol Ther 79:231–257

    Article  CAS  PubMed  Google Scholar 

  • Rothman RB, Baumann MH, Savage JE, Rauser L, McBride A, Hufeisen SJ, Roth BL (2000) Evidence for possible involvement of 5-HT2B receptors in the cardiac valvulopathy associated with fenfluramine and other serotonergic medications. Circulation 102:2836–2841

    CAS  PubMed  Google Scholar 

  • Sadzot B, Baraban JM, Glennon RA, Lyon RA, Leonhardt S, Jan CR, Titeler RA (1989) Hallucinogenic drug interactions at human brain 5-HT2 receptors: implications for treating LSD-induced hallucinogenesis. Psychopharmacology 98:495–499

    Google Scholar 

  • Setola V, Hufeisen SJ, Grande-Allen KJ, Vesely I, Glennon RA, Blough B, Rothman RB, Roth BL (2003) 3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”) induces fenfluramine-like proliferative actions on human cardiac valvular interstitial cells in vitro. Mol Pharmacol 63:1223–1229

    Article  CAS  PubMed  Google Scholar 

  • Sleight AJ, Stam NJ, Mutel V, Vanderheyden PML (1996) Radiolabelling of the human 5-HT2A receptor with an agonist, a partial agonist and an antagonist: effects on apparent agonist affinities. Biochem Pharmacol 51:71–76

    Article  CAS  PubMed  Google Scholar 

  • Titeler RA, Lyon RA, Glennon RA (1988) Radioligand binding evidence implicates the brain 5-HT2 receptor as a site of action for LSD and phenylisoproprylamine hallucinogens. Psychopharmacology 94:213–216

    CAS  PubMed  Google Scholar 

  • Vickers SP, Clifton PG, Dourish CT, Tecott LH (1999) Reduced satiating effect of d-fenfluramine in serotonin 5-HT2C receptor mutant mice. Psychopharmacology 143:309–314

    Article  CAS  PubMed  Google Scholar 

  • Vickers SP, Dourish CT, Kennett GA (2001) Evidence that hypophagia induced by d-fenfluramine and d-norfenfluramine in the rat is mediated by 5-HT2C receptors. Neuropharmacology 41:200–209

    Article  CAS  PubMed  Google Scholar 

  • Wainscott DB, Lucaites VL, Kursar JD, Baez M, Nelson DL (1996) Pharmacologic characterization of the human 5-hydroxytryptamine 2B receptor: evidence for species differences. J Pharmacol Exp Ther 276:720–727

    CAS  PubMed  Google Scholar 

  • Wainscott DB, Sasso DA, Kursar JD, Baez M, Lucaites VL, Nelson DL (1998) [3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2B (5-HT2B) receptor. Naunyn-Schmiedebergs Arch Pharmacol 357:17–24

    CAS  PubMed  Google Scholar 

  • Wilkinson LO, Dourish CT (1991) Serotonin and animal behaviour. Serotonin receptor subtypes: basic and clinical aspects. Recept Biochem Methodol 1:147–210

    Google Scholar 

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Acknowledgements

The authors would like to acknowledge the assistance of Ms Sandra Binnie and the Vernalis Department of Tissue Culture for providing the cultured cells used in this study.

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Correspondence to Antony R. Knight.

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Knight, A.R., Misra, A., Quirk, K. et al. Pharmacological characterisation of the agonist radioligand binding site of 5-HT2A, 5-HT2B and 5-HT2C receptors. Naunyn-Schmiedeberg's Arch Pharmacol 370, 114–123 (2004). https://doi.org/10.1007/s00210-004-0951-4

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