CHARACTERIZATION OF BRADYKININ RECEPTORS IN HUMAN LUNG FIBROBLASTS USING THE BINDING OF [H-3][DES-ARG(10),LEU(9)]KALLIDIN AND [H-3]NPC17731

Bradykinin (BK) receptors are involved in pain and inflammation. Two B K receptor subtypes, B-1 and B-2, have been defined based on their pha rmacological properties. Both B-1 and B-2 receptors are G-protein coup led membrane receptors. B-1 receptors are present in smooth muscle tis sue, whereas B-2 receptors are found in both smooth muscle tissue and neurons. [Des-Arg(10),Leu(9)]kallidin (DALKD) is a selective B-1 recep tor antagonist, and NPC17731 is a selective B-2 receptor antagonist. T o develop binding assays for the two known BK receptor subtypes, [H-3] DALKD and [H-3]NPC17731 were used as selective ligands for B-1 and B-2 receptors respectively. Both ligands bound to the CCD-16 human lung f ibroblast membranes reaching equilibrium at 25 degrees C within 30 min . Binding was stable for at least 60 min. The K-d of [H-3]DALKD was 0. 33 nM and B-max was 52 fmol/mg membrane protein. The K-d of [H-3]NPC17 731 was 0.39 nM and B-max was 700 fmol/mg membrane protein. Competitio n for [H-3]DALKD binding with BK receptor agonists was in the order: [ des-Arg(10)]KD (DAKD) > KD much greater than [des-Arg(9)]BK (DABK) > B K, and competition for [H-3]DALKD binding with BK receptor antagonists was in the order: DALKD > [desArg(10)]Hoe 140 (DAHoe 140) > [des-Arg( 9),Leu(8)]BK (DALBK) > NPC17731 > Hoe 140 > DNMFBK, suggesting that [H -3]DALKD bound selectively to B-1 receptors. By contrast, competition for [H-3]NPC17731 binding by BK agonists was in the order: BK > KD muc h greater than DAKD > DABK, and competition for [H-3]NPC17731 binding by BK antagonists was in the order: NPC17731 = Hoe 140 much greater th an DNMFBK > DAHoe 140 > DALBK > DALKD, indicating that [H-3]NPC17731 l abeled B-2 receptors selectively. These results demonstrate that [H-3] DALKD and [H-3]NPC17731 can be used with CCD-16 human lung fibroblast membranes to provide a pair of binding assays for the simultaneous eva luation of B-1 and B-2 BK receptor subtypes.