title: Characterization of bradykinin receptors in human lung fibroblasts using the binding of 3[H][Des-Arg10,Leu9]kallidin and [3H]NPC17731.
abstract: Bradykinin (BK) receptors are involved in pain and inflammation. Two BK receptor subtypes, B1 and B2, have been defined based on their pharmacological properties. Both B1 and B2 receptors are G-protein coupled membrane receptors. B1 receptors are present in smooth muscle tissue, whereas B2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg10,Leu9]kallidin (DALKD) is a selective B1 receptor antagonist, and NPC17731 is a selective B2 receptor antagonist. To develop binding assays for the two known BK receptor subtypes, [3H]DALKD and [3H]NPC17731 were used as selective ligands for B1 and B2 receptors respectively. Both ligands bound to the CCD-16 human lung fibroblast membranes reaching equilibrium at 25 degrees C within 30 min. Binding was stable for at least 60 min. The Kd of [3H]DALKD was 0.33 nM and Bmax was 52 fmol/mg membrane protein. The Kd of [3H]NPC17731 was 0.39 nM and Bmax was 700 fmol/mg membrane protein. Competition for [3H]DALKD binding with BK receptor agonists was in the order: [des-Arg10]KD (DAKD) > KD >> [des-Arg9]BK (DABK) > BK, and competition for [3H]DALKD binding with BK receptor antagonists was in the order: DALKD > [des-Arg10]Hoe 140 (DAHoe 140) > [des-Arg9,Leu8]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [3H]DALKD bound selectively to B1 receptors. By contrast, competition for [3H]NPC17731 binding by BK agonists was in the order: BK > KD >> DAKD > DABK, and competition for [3H]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 >> DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [3H]NPC17731 labeled B2 receptors selectively. These results demonstrate that [3H]DALKD and [3H]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous evaluation of B1 and B2 BK receptor subtypes.
authors.affiliation: Drug Discovery, The R.W. Johnson Pharmaceutical Research Institute, Spring House, PA 19477, USA. spzhang@prius.jnj.com
authors.forename: S P
authors.initials: SP
authors.lastname: Zhang
authors.forename: E E
authors.initials: EE
authors.lastname: Codd
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D006801:Humans; D007609:Kallidin / Q000031:analogs & derivatives* / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D011869:Radioligand Assay; D018002:Receptors, Bradykinin / Q000032:analysis*; D012965:Sodium Chloride / Q000494:pharmacology; D014316:Tritium
chemical_list: D008565:Membrane Proteins; D009842:Oligopeptides; D018002:Receptors, Bradykinin; C082229:bradykinin, Lys-Leu(8)-desArg(9)-; D015636:Magnesium Chloride; D014316:Tritium; C071327:NPC 17731; D007609:Kallidin; D012965:Sodium Chloride