Characterization of the rat uterine neuromedin U receptor

Abstract

Neuromedin U (NmU) is a novel peptide that potently contracts smooth muscle, including rat uterus in vitro. No receptors have so far been demonstrated in any tissue. We have characterized the receptor for NmU in rat uterine membrane preparations. The binding of [125I] rat NmU was saturable, specific, reversible, and time, temperature, and pH dependent. The nonhydrolysable analog of GTP, GTP-gamma-S, reduced binding in a dose-dependent manner, suggesting that the binding site is coupled to a G-protein. Scatchard analysis of saturation binding studies showed a single class of binding site with a maximal binding capacity of 580 +/- 140 fmol/mg membrane protein and a dissociation constant of 0.35 +/- 0.09 nM (n = 3). Rat NmU and NmU-8 (the C-terminal region of the larger molecule) displaced [125I]rat NmU, displaying an IC50 of 1 x 10(-9) M and 6 x 10(-8) M, respectively. Chemical cross-linking studies demonstrated NmU binding sites on the rat uterus to have an M(r) of 48,500. This band was absent in the presence of 0.2 microM NmU. Thus the rat uterus contains specific NmU binding sites through their physiological role awaits identification.

Isolation, Structural Characterization, and Bioactivity of a Novel Neuromedin U Analog from the Defensive Skin Secretion of the Australasian Tree Frog, Litoria caerulea

“…The latter peptide produced similar EC 50 values to those reported previously (16 Ϯ 5 nM (22); 46 Ϯ 8 nM (38)). In addition, the IC 50 value obtained for inhibition of monoradioiodinated rat NmU binding to rat uterine membrane preparations (60 nM) was of a similar magnitude to that previously reported (35)). However, in the absence of suitable NmU receptor antagonists, it is not possible to state unequivocally whether both peptides activate or indeed bind to the same receptor.…”

“…Uterus membranes were prepared by differential centrifugation, as described previously (35). Membranes were resuspended in ice-cold HEPES (50 mM) buffer (pH 7.6) containing soybean trypsin inhibitor (10 g/ml), pepstatin (0.5 g/ml), leupeptin (0.5 g/ml), antipain (0.5 g/ml), benzamidine (0.1 g/ml), aprotinin (30 g/ml), and bacitracin (0.1 g/ml) to a final concentration of approximately 10 mg of protein/ml and stored at Ϫ80°C.…”

“…Synthetic rat-NmU-23 was iodinated by the chloramine T method as described previously (35). Peak fractions were assayed for binding to uterine membranes, and the average specific activity, as determined by a specific NmU radioimmunoassay (37), was 24 Bq/fmol.…”

“…Peak fractions were assayed for binding to uterine membranes, and the average specific activity, as determined by a specific NmU radioimmunoassay (37), was 24 Bq/fmol. The assay was performed as described by Nandha et al (35). Briefly, incubations were carried out at 4°C for 60 min in a final volume of 0.5 ml of binding buffer containing 50 mM Tris-HCl (pH 7.4) and aprotinin (30 g/ml).…”

Identification and Functional Characterization of a Novel Subtype of Neuromedin U Receptor

“…Our analyses for FM-3 mRNA expression levels using quantitative reverse transcription-polymerase chain reaction (RT-PCR) indicated that FM-3 mRNA was expressed highly in the small intestine and lung of rats, but only very low levels were detected in the uterus. Nonetheless neuromedin U has reportedly shown very strong contractile activity (1,2), and numerous binding sites have been detected in rat uterine tissue (11,12). We considered that this discrepancy might be due to the existence of an unknown receptor subtype for neuromedin U in the rat uterus.…”