Identification of neuromedin U precursor-related peptide and its possible role in the regulation of prolactin release

Abstract

The discovery of neuropeptides provides insights into the regulation of physiological processes. The precursor for the neuropeptide neuromedin U contains multiple consensus sequences for proteolytic processing, suggesting that this precursor might generate additional peptides. We performed immunoaffinity chromatography of rat brain extracts and consequently identified such a product, which we designated neuromedin U precursor-related peptide (NURP). In rat brain, NURP was present as two mature peptides of 33 and 36 residues. Radioimmunoassays revealed NURP immunoreactivity in the pituitary, small intestine, and brain of rats, with the most intense reactivity in the pituitary. Intracerebroventricular administration of NURP to both male and female rats robustly increased plasma concentrations of prolactin but not of other anterior pituitary hormones. In contrast, NURP failed to stimulate prolactin release from dispersed anterior pituitary cells. Pretreatment of rats with bromocriptine, a dopamine receptor agonist, blocked the prolactin-releasing activity of NURP. In rats pretreated with the antagonist sulpiride, intracerebroventricular administration of NURP did not increase plasma prolactin concentrations more than administration of saline. These data suggest that NURP induces prolactin release by acting indirectly on the pituitary; dopamine from the hypothalamus, which inhibits prolactin release, may be involved in this activity of NURP.

Figure 1

Structure of NURP. (a) Comparison of amino acid sequences between the human NMU precursor, prepro-NMU (accession number NM_006681), and the human NMS precursor, prepro-NMS (NM_001011717). Identical residues are shown in red. The arrowheads indicate proteolytic cleavage sites conserved in both precursors. The sequences corresponding to putative mature NURP33 and NSRP34 are surrounded by dotted lines, and those for mature NURP36 and NSRP37 are boxed by solid lines. The sequences of NMU and NMS are indicated by solid underlines. (b) Structures of mature NURP and NSRP peptides. Amino acid identities between NURP and NSRP are shown in red. The residues conserved only within the NURP or NSRP are indicated in green and blue, respectively.

Figure 2

Purification of NURP from rat brain extracts. (a) RP-HPLC on a μ-Bondasphere C18 column of the materials eluted from the immunoaffinity column for rat NURP. The peaks containing NURP36 (B1) and NURP33 (B2) are indicated by arrows. (b) Final purification of rat NURP36 (upper; B1) and rat NURP33 (lower; B2) using a Chemcosorb 3ODS-H column. Purified peptides are indicated by arrows.

Figure 3

Representative RP-HPLC profiles of immunoreactivities for NURPs and NMU in rat tissues. (a–d) Peptide extracts from the whole brain (a), pituitary (b), and small intestine (c) of rat, and synthetic peptides (d) were applied to RP-HPLC under the same conditions. Samples of eluate were subjected to RIAs for rat NURP (upper, a–c) and for rat NMU (lower, a–c). Black bars indicate immunoreactivities (a–c). RP-HPLC profiles of immunoreactivities are each representative of three experiments. Elution positions of synthetic rat NURP36 and NMU are indicated by the arrowhead and arrow, respectively (d).

Figure 4

In vivo effects of NURP on prolactin release. (a) Effects of ICV-administered rat NURP36 on the plasma concentrations of anterior pituitary hormones and corticosterone in male rats. The plasma concentrations of the peptide-administered groups are shown as fold change relative to those of the saline-administered group. Data are presented as means ± SEM (n = 9 rats for the NURP36 and NSRP37 groups and n = 8 rats for the saline group). *P < 0.05; **P < 0.01 compared with the saline-administered group and ^† P < 0.01 compared with the NSRP37-administered group, one-way ANOVA followed by the Tukey–Kramer multiple-comparisons test. GH, growth hormone; LH, luteinizing hormone; PRL, prolactin; TSH, thyroid-stimulating hormone; FSH, follicle-stimulating hormone; CS, corticosterone. (b) Dose- and time-dependent effects of ICV-administered rat NURP36 on prolactin release in male rats. Rats were injected ICV with saline or NURP36 at the indicated doses. Data are presented as means ± SEM (n = 7 rats per group). *P < 0.05; **P < 0.01 compared with the saline-administered group at each time point, one-way ANOVA followed by the Tukey–Kramer multiple-comparisons test.

Figure 5

In vitro effect of NURP on prolactin release from anterior pituitary cells harvested from male rats. The cells were incubated for 30 min with increasing concentrations of rat NURP36 or TRH. Data are presented as means ± SEM (n = 3 wells per group). TRH, thyrotropin-releasing hormone.

Figure 6

Effects of a dopamine receptor agonist and antagonist on prolactin release induced by ICV-administered NURP. (a) Effect of bromocriptine. Saline or rat NURP36 (1 nmol) was administered ICV to rats that had received a preinjection of saline or bromocriptine. Data are presented as means ± SEM (n = 5 rats per group). (b) Effect of sulpiride. Saline or rat NURP36 (1 nmol) was administered ICV to rats preinjected with saline or sulpiride. Data are presented as means ± SEM (n = 5 rats per group). **P < 0.01, one-way ANOVA followed by the Tukey–Kramer multiple-comparisons test. The same saline-pretreated groups were used in both panels.

Figure 7

Pharmacological characterization of NURP by using CHO cells that stably express either rat NMUR1 or NMUR2. (a,b) Agonistic activities of rat NURP36, NSRP37, and NMU were evaluated by using the calcium-mobilization assay with CHO/rNMUR1-4 (a) and CHO/rNMUR2-11 (b) cells. Data points are presented as means ± SEM of triplicates for each experiment.