Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5904079 | Neuropeptides | 2016 | 6 Pages |
Abstract
The goal of this study consisted of the synthesis and radiolabeling of NN, as well as the determination of the binding characteristics of [3H]NN and G protein activation by the cold ligand. In homologous displacement studies a weak affinity was determined for NN, with IC50 values of 454 nM in rat brain and 425 nM in rat spinal cord membranes. In saturation binding experiments the Kd value proved to be 264.8 ± 30.18 nM, while the Bmax value corresponded to 3.8 ± 0.2 pmol/mg protein in rat brain membranes. The specific binding of [3H]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 [3H]NN, resulting in an IC50 of 150.6 mM. In [35S]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 0.7 nM and 0.79 nM 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 [3H]NN labels NTS2 receptors in rat brain membranes.
Keywords
SPPSPEIHOBtBmaxEC50TFAEmaxIC50TBTUPseudo-first-order rate constantSR 48692NTS1Receptor binding assaysDMFN,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate1-hydroxybenzotriazoleBSANeuromedin NN,N-dimethylformamideN,N-diisopropylethylamine[35S]GTPγSbovine serum albuminTrifluoroacetic acidtriethylamineequilibrium dissociation constantGDPPCsSolid phase peptide synthesislevocabastineRatDIPEAneurotensinhalf-maximal inhibitory concentrationhalf maximal effective concentrationProprotein convertasespolyethyleneimineTEAhigh performance liquid chromatographyHPLCkobsguanosine 5′-diphosphateneurotensin receptor 1
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Authors
Fanni Tóth, Jayapal Reddy Mallareddy, Dirk Tourwé, Andrzej W. Lipkowski, Magdalena Bujalska-Zadrozny, Sándor Benyhe, Steven Ballet, Géza Tóth, Patrycja Kleczkowska,