title: Binding of the novel ligand [4,5-3H-Leu10]substance P to high-affinity NK-1 receptors on guinea pig lung membranes: modulation by GTP analogs and sulfhydryl modifying agents.
abstract: A novel ligand, [4,5-3H-Leu10]substance P ([3H]SP), with high specific activity (137 Ci/mmole) was utilized to investigate the properties of NK (neurokinin)-1 receptors on guinea pig lung membranes (GPLM) and compared them to NK-1 receptors on rat submaxillary glands (RSGM). In the presence of a neutral endopeptidase inhibitor, thiorphan (100 microM), [3H]SP bound with high specificity (greater than 95%), rapidly (k1 = 0.116 nM-1 x min-1) and in a reversible (k-1 = 0.012 min-1) manner to a single class of high-affinity (Kd = 0.16 nM) and saturable (Bmax = 256 fmol/mg protein) receptors. High specific binding with higher density (5-fold) was also detected in RSGM, albeit with a lower affinity (Kd = 1.36 nM). Guanyl-5'-yl-imidodiphosphate and guanosine-5'-O-3-thiotriphosphate inhibited binding to GPLM (and RSGM) in a concentration-related manner. In GPLM, this effect was mediated by a reduction in affinity, mainly via enhancement of ligand dissociation rates and appearance of a lower affinity state (Kd = 3.4 nM). Preincubation of GPLM with sulfhydryl modifying agents (p-chloromercuriphenyl sulfonic acid and N-ethylmaleimide) reduced receptor density and affinity in a time- and concentration-dependent manner. Competition experiments with tachykinins and analogs illustrated a rank order of potency of: SP greater than or equal to [Sar9,Met(O2)11]SP greater than SP-methyl ester greater than or equal to physalaemin greater than SP(6-11) much greater than kassinin greater than neurokinin A = eledoisin much greater than neurokinin B greater than Nle10-NKA(4-10), clearly demonstrating that these receptors are of NK-1 type. Moreover, analysis of over 30 peptide and non-peptide hormones and antagonists demonstrated exquisite selectivity (greater than 10,000-fold) towards NK-1-selective agonists (vs. other ligands. A highly significant (P less than .005) linear correlation (r = 0.924) exists between agonist affinities in GPLM and RSGM. Combined, the data suggest that [3H]SP labels a nearly homogeneous population of high-affinity, G-protein coupled NK-1 receptors on GPLM and RSGM, with very high degree of selectivity.
authors.affiliation: Department of Pharmacology, ICI Pharmaceuticals Group, Wilmington, Delaware.
authors.forename: D
authors.initials: D
authors.lastname: Aharony
authors.forename: C A
authors.initials: CA
authors.lastname: Catanese
authors.forename: D P
authors.initials: DP
authors.lastname: Woodhouse
mesh_terms: D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D008024:Ligands; D008168:Lung / Q000378:metabolism*; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013373:Substance P / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D015320:Tachykinins / Q000494:pharmacology; D014316:Tritium
chemical_list: D002413:Cations, Divalent; D008024:Ligands; D018041:Receptors, Neurokinin-2; D017981:Receptors, Neurotransmitter; D015320:Tachykinins; D014316:Tritium; D013373:Substance P; D006160:Guanosine Triphosphate; D004229:Dithiothreitol