Different distribution of neuromedin S and its mRNA in the rat brain: NMS peptide is present not only in the hypothalamus as the mRNA, but also in the brainstem

Abstract

Neuromedin S (NMS) is a neuropeptide identified as another endogenous ligand for two orphan G protein-coupled receptors, FM-3/GPR66 and FM-4/TGR-1, which have also been identified as types 1 and 2 receptors for neuromedin U structurally related to NMS. Although expression of NMS mRNA is found mainly in the brain, spleen, and testis, the distribution of its peptide has not yet been investigated. Using a newly prepared antiserum, we developed a highly sensitive radioimmunoassay for rat NMS. NMS peptide was clearly detected in the rat brain at a concentration of 68.3 ± 3.4 fmol/g wet weight, but it was hardly detected in the spleen and testis. A high content of NMS peptide was found in the hypothalamus, midbrain, and pons-medulla oblongata, whereas abundant expression of NMS mRNA was detected only in the hypothalamus. These differing distributions of the mRNA and peptide suggest that nerve fibers originating from hypothalamic NMS neurons project into the midbrain, pons, or medulla oblongata. In addition, abundant expression of type 2 receptor mRNA was detected not only in the hypothalamus, but also in the midbrain and pons-medulla oblongata. These results suggest novel, unknown physiological roles of NMS within the brainstem.

Keywords: brain; brainstem; neuromedin S; neuropeptide; peptide distribution; radioimmunoassay.

FIGURE 1

Characterization of antisera for rat neuromedin S (NMS). (A) Structural comparison of rat NMS and rat neuromedin U (NMU). Structure conserved in the two peptides is shaded. The box indicates the sequence used as antigen peptide to obtain antisera specific for rat NMS. (B) Specific binding curves of antisera. [¹²⁵I-Tyr²¹]-rat NMS[1-20] was incubated with serially diluted OB1321-1 (closed circles) and OB1321-2 (open circles) antisera, and then co-precipitated with antibody in a concentration-dependent manner. (C) Standard curve of radioimmunoassay for rat NMS using antiserum OB1321-2. [¹²⁵I-Tyr²¹]-rat NMS[1-20] binding to antibody was displaced by increasing concentrations of rat NMS (open circles) but not rat NMU (closed circles). B/B0 means tracer bound/tracer bound in zero standards. Inset shows the inhibition of tracer ligand binding to antibody by diluted rat brain extract (open squares).

FIGURE 2

Representative reversed-phase high performance liquid chromatography (RP-HPLC) profile of neuromedin S (NMS) immunoreactivity in rat brain. Peptide extract (equivalent to 1.2 g wet weight) from rat whole brain (A) and synthetic rat NMS (B) were applied to RP-HPLC under the same conditions. Black bars indicate immunoreactive NMS detected by radioimmunoassay (A). Elution position of rat NMS is indicated by the arrow (B).

FIGURE 3

Regional distribution of immunoreactive neuromedin S (ir-NMS) in rat brain. Concentrations of ir-NMS in various brain areas were quantified by radioimmunoassay. The brains were sampled during the light period. Data represent means ± SEM of three rats.

FigurE 4

Expression of neuromedin S (NMS) mRNA in rat brain. Rat NMS mRNA was quantified by quantitative reverse transcription–polymerase chain reaction analysis. The brains were sampled during the light period. Data represent means ± SEM of three rats.

FIGURE 5

Expression of neuromedin U receptors types 1 (NMUR1) and 2 (NMUR2) mRNA in rat brain. Rat NMUR1 (closed bars) and NMUR2 (open bars) mRNA were quantified by quantitative reverse transcription–polymerase chain reaction analysis. Data represent means ± SEM of three rats.

FIGURE 6

Levels of immunoreactive neuromedin S (ir-NMS) in the brain of rats maintained under light/dark cycling, and of fasted rats. (A–C) Concentrations of ir-NMS were quantified in the hypothalamus (A), midbrain (B), and pons–medulla oblongata (C) of rats under the light at ZT6 and dark at ZT18. (D–F) Concentrations of ir-NMS were quantified in the hypothalamus (D), midbrain (E), and pons–medulla oblongata (F) of free-fed and 48 h-fasted rats. Data represent means ± SEM of three rats. Data were analyzed statistically by Student’s t-test.