Pharmacologic characterization of the novel ligand [4,5- 3H-Leu 9]Neurokinin-A binding to NK-2 receptors on hamster urinary bladder membranes

Abstract

We synthesized a novel ligand [4,5- 3H-Leu 9]-Neurokinin A ( 3H-NKA, S.A 117–144 Ci/mmol), and evaluated its binding to hamster urinary bladder membranes (HUBM). The ligand bound to HUBM in a highly-specific (94 ± 4%) and protein-dependent manner. Binding was rapid (k 1 = 0.037 nM −1∗min −1) and saturable (B max = 1210 ± 177 fmol/mg protein), to a single population of high-affinity sites (K D = 2.41 ± 0.15 nM, nH = 0.99 ± 0.02). Binding was inhibited by non-hydrolyzable GTP analogs. Competition experiments with HUBM demonstrated the following rank order of potency: NKA > Kassinin > [β-Ala8]-NKA(4–10) > [Nle10]-NKA(4–10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar 9-Met (O2) 11]-SP, (±) CP96,345 and Senktide respectively, did not inhibit binding at 10 μM, whereas, the selective NK-2 antagonists: (±) SR-48,968 ⪢ L-659,877 > R396 ⪢ MEN-10,207 > MEN-10,376, inhibited binding in a competitive manner. In contrast, the low specific binding (<30%) detected in guinea pig lung membranes, was not inhibited by selective NK-2 ligands. Over 30 ligands (0.1–10 μM) from other receptor classes, were not inhibitory. The data suggest that this new ligand binds with high-affinity and selectivity to homogeneous population of NK-2 receptors on HUBM but not on lung membranes, and is a suitable ligand to study NK-2 receptors.