Identification and initial characterization of a putative neuromedin B-type receptor from rat urinary bladder membranes

Abstract

Receptor binding site(s) on the rat urinary bladder membranes were characterized using a biologically active analog of bombesin, [Tyr 4, Leu 14]bombesin, and a 50 000 × g total particulate preparation. The binding was specific, reversible, saturable, time- and concentration-dependent. A dissociation curve showed that both bombesin and neuromedin B equally displaced the radioligand in the first 10 min after saturation. From the rate constant of association k +1 = 7.60 × 10 9 M −1 min −1, and the rate constant of dissociation k −1 = 0.050 min −1, the apparent equilibrium dissociation constant K d = 6.57 ± 1.09 pH was determined. A linear Scatchard plot of the specific binding of 125I-[Tyr 4, Leu 14]bombesin to the membranes revealed that the radioligand bound with high affinity, K d = 6.38 ± 0.86 pM, to a single class of sites (B max = 2.3 fmol/mg protein). The Hill coefficient of the same binding data was 1.05 ± 0.21, indicating that the radioligand was binding to a single population of noninteracting bindings sites. Both bombesin and neuromedin B displaced the radioligand dose dependently (IC 50 = 0.3 nM). Neurokinin A and neurokinin B were less potent (IC 50 = 20 and 110 nM, respectively). Substance P, or the specific bombesin receptor antagonists [D-Phe 6]bombesin-(6–13) methyl ester, [D-F 5Phe 6,D-Ala 11]bombesin-(6–11) methyl ester, [D-Phe 6]bombesin-(6–13) propylamide, [D-Phe 6,Leu 13psi(CH 2NH)Leu 14]bombesin or [D-Cpa 6,Phe 14(psi13–14)]bombesin-(6–14) had an IC 50 > 1 μM. The results presented suggest the presence of neuromedin B receptor sites on the rat urinary bladder membranes that can be occupied also by some other peptides, notably bombesin, neurokinin A and neurokinin B. The presence of a non-specific bombesin receptor subtype cannot be excluded.