PHARMACOLOGICAL CHARACTERIZATION OF THE NOVEL LIGAND [4,5-H-3-LEU(9)]NEUROKININ-A BINDING TO NK-2 RECEPTORS ON HAMSTER URINARY-BLADDER MEMBRANES

We synthesized a novel ligand [4,5-H-3-Leu9]-Neurokinin A (H-3-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 (k1 = 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 > [beta-Ala8]-NKA(4-10) > [Nle10]-NKA(4-10) = Eledoisin = NKB > Physaelamin > Substance P. The selective NK-1 and NK-3 ligands, [Sar9-Met (O2)11]-SP, (+/-) CP96,345 and Senktide respectively, did not inhibit binding at 10 muM, 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 muM) 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.