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Binding of the novel radioligand [3H]UFP-101 to recombinant human and native rat nociceptin/orphanin FQ receptors

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Abstract

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-3H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [3H]labeled NOP antagonist, [Nphe1,4’-3H-Phe4,Arg14,Lys15]N/OFQ-NH2 ([3H]UFP-101). We have characterized [3H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHOhNOP) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [3H]UFP-101 (antagonist) was compared with [3H]N/OFQ (agonist). The effects of GTPγS (10 µM) and Na+ were investigated alone and in combination in competition experiments with both radioligands. In CHOhNOP, B max, and pK D, values were 561 and 580 fmol mg protein−1 and 9.97 and 10.19 for [3H]UFP-101 and [leucyl-3H]N/OFQ, respectively. In rat cerebrocortex B max and pK D, values were 65 and 88 fmol mg protein−1 and 10.12 and 10.34 for [3H]UFP-101 and [leucyl-3H]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation (r 2 0.92 in Rat and 0.97 in CHOhNOP). Naloxone was inactive. The binding of both radioligands was Na+-dependent with [3H]UFP-101 being more sensitive (IC50 ~20 mM). Unlike the agonist [leucyl-3H]N/OFQ, the antagonist [3H]UFP-101 was unaffected by GTPγS. [3H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-3H]N/OFQ in competition experiments. In addition, the binding of [3H]UFP-101 is unaffected by GTPγS. This radioligand will be useful to further characterize NOP in a range of binding paradigms.

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Acknowledgements

The authors would like to thank the International Association for the Study of Pain for provision of a collaborative travel grant between Universities of Leicester (UK) and Ferrara (Italy) and RET 08/2004, and OTKA T 46514 from National Bureau of Research and Technology (G.T.)

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Authors and Affiliations

  1. Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, LE1 5WW, UK

    Massimo Ibba, Masato Kitayama, John McDonald & David G. Lambert

  2. Department of Experimental and Clinical Medicine, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, via Fossato di Mortara, 17, 44100, Ferrara, Italy

    Massimo Ibba & Girolamo Calo

  3. Department of Pharmaceutical Sciences and Biotechnology Center, University of Ferrara, via Fossato di Mortara, 17, 44100, Ferrara, Italy

    Remo Guerrini

  4. Institute of Biochemistry, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary

    Judit Farkas & Geza Toth

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  1. Massimo Ibba
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Correspondence to David G. Lambert.

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Ibba, M., Kitayama, M., McDonald, J. et al. Binding of the novel radioligand [3H]UFP-101 to recombinant human and native rat nociceptin/orphanin FQ receptors. Naunyn-Schmied Arch Pharmacol 378, 553–561 (2008). https://doi.org/10.1007/s00210-008-0350-3

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