title: Synthesis and binding characteristics of [(3)H]neuromedin N, a NTS2 receptor ligand.
abstract: Neurotensin (NT) and its analog neuromedin N (NN) are formed by the processing of a common precursor in mammalian brain tissue and intestines. The biological effects mediated by NT and NN (e.g. analgesia, hypothermia) result from the interaction with G protein-coupled receptors. The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [(3)H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454nM in rat brain and 425nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8±30.18nM, while the Bmax value corresponded to 3.8±0.2pmol/mg protein in rat brain membranes. The specific binding of [(3)H]NN was saturable, interacting with a single set of homogenous binding sites. In sodium sensitivity experiments, a very weak inhibitory effect of Na(+) ions was observed on the binding of [(3)H]NN, resulting in an IC50 of 150.6mM. In [(35)S]GTPγS binding experiments the Emax value was 112.3±1.4% in rat brain and 112.9±2.4% in rat spinal cord membranes and EC50 values of 0.7nM and 0.79nM were determined, respectively. NN showed moderate agonist activities in stimulating G proteins. The stimulatory effect of NN could be maximally inhibited via use of the NTS2 receptor antagonist levocabastine, but not by the opioid receptor specific antagonist naloxone, nor by the NTS1 antagonist SR48692. These observations allow us to conclude that [(3)H]NN labels NTS2 receptors in rat brain membranes.
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62, 6726 Szeged, Hungary.
authors.forename: Fanni
authors.initials: F
authors.lastname: Tóth
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62, 6726 Szeged, Hungary.
authors.forename: Jayapal Reddy
authors.initials: JR
authors.lastname: Mallareddy
authors.affiliation: Research Group of Organic Chemistry, Department of Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Dirk
authors.initials: D
authors.lastname: Tourwé
authors.affiliation: Department of Neuropeptides, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, 02106 Warsaw, Poland.
authors.forename: Andrzej W
authors.initials: AW
authors.lastname: Lipkowski
authors.affiliation: Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, 1B Banacha Str., 02-106 Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Bujalska-Zadrozny
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62, 6726 Szeged, Hungary.
authors.forename: Sándor
authors.initials: S
authors.lastname: Benyhe
authors.affiliation: Research Group of Organic Chemistry, Department of Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium.
authors.forename: Steven
authors.initials: S
authors.lastname: Ballet
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62, 6726 Szeged, Hungary.
authors.forename: Géza
authors.initials: G
authors.lastname: Tóth
authors.affiliation: Department of Neuropeptides, Mossakowski Medical Research Centre, Polish Academy of Sciences, 5 Pawinskiego Street, 02106 Warsaw, Poland; Department of Pharmacodynamics, Centre for Preclinical Research and Technology, Medical University of Warsaw, 1B Banacha Str., 02-106 Warsaw, Poland. Electronic address: hazufiel@wp.pl.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kleczkowska
mesh_terms: D000818:Animals; D001921:Brain / Q000000981:diagnostic imaging / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020128:Inhibitory Concentration 50; D008024:Ligands; D008297:Male; D009496:Neurotensin / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000493:pharmacokinetics*; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017208:Rats, Wistar; D018028:Receptors, Neurotensin / Q000378:metabolism*; D013116:Spinal Cord / Q000000981:diagnostic imaging / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000493:pharmacokinetics; D014316:Tritium / Q000493:pharmacokinetics
chemical_list: D008024:Ligands; C099727:Ntsr2 protein, rat; D010446:Peptide Fragments; D018028:Receptors, Neurotensin; D013462:Sulfur Radioisotopes; D014316:Tritium; C042090:neuromedin N; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009496:Neurotensin
keywords: NTS2; Neuromedin N; Rat; Receptor binding assays