title: Pharmacologic characterization of the novel ligand [4,5-3H-Leu9]neurokinin-A binding to NK-2 receptors on hamster urinary bladder membranes.
abstract: We synthesized a novel ligand [4,5-3H-Leu9]-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 (k1 = 0.037 nM-1*min-1) and saturable (Bmax = 1210 +/- 177 fmol/mg protein), to a single population of high-affinity sites (KD = 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 microM, 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 microM) 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.
authors.affiliation: Pulmonary Sct., ICI Americas Inc., Wilmington, Delaware.
authors.forename: D
authors.initials: D
authors.lastname: Aharony
authors.forename: G E
authors.initials: GE
authors.lastname: Conner
authors.forename: D P
authors.initials: DP
authors.lastname: Woodhouse
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008168:Lung / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D001743:Urinary Bladder / Q000378:metabolism*
chemical_list: D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; C078560:neurokinin A, Leu(9)-; D015288:Neurokinin A