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

Receptor binding site(s) on the rat urinary bladder membranes were characterized using a biologically active analog of bombesin, [Tyr4,Leu14]bombesin, and a 50,000 x 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 x 10(9) M-1 min-1, and the rate constant of dissociation k-1 = 0.050 min-1, the apparent equilibrium dissociation constant Kd = 6.57 +/- 1.09 pM was determined. A linear Scatchard plot of the specific binding of 125I-[Tyr4,Leu14]bombesin to the membranes revealed that the radioligand bound with high affinity, Kd = 6.38 +/- 0.86 pM, to a single class of sites (Bmax = 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 binding sites. Both bombesin and neuromedin B displaced the radioligand dose dependently (IC50 = 0.3 nM). Neurokinin A and neurokinin B were less potent (IC50 = 20 and 110 nM, respectively). Substance P, or the specific bombesin receptor antagonists [D-Phe6]bombesin-(6-13) methyl ester, [D-F5Phe6,D-Ala11]bombesin-(6-11) methyl ester, [D-Phe6]bombesin-(6-13) propylamide, [D-Phe6,Leu13psi(CH2NH)Leu14]bombesin or [D-Cpa6,Phe14(psi13-14)]bombesin-(6-14) had an IC50 greater than 1 microM. 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.(ABSTRACT TRUNCATED AT 250 WORDS)