Amino acid sequences of NPB and NPW. (A) Amino acid sequences of mature NPB and NPW peptides. An asterisk indicates the posttranslational bromination site of the native bovine NPB. Peptide sequences of other species are deduced from cDNA sequences. Amino acid identities between NPB and NPW are shown in black. Shaded residues are conserved only within the NPB or NPW. (B) Deduced amino acid sequences of prepro-NPB and NPW precursor polypeptides. Mature peptides are marked by equal signs. Question marks indicate undetermined sequence of bovine prepro-NPB. Human and mouse prepro-NPW cDNA do not have a translation initiator ATG codon; putative translation initiation sites are indicated by a pound sign. An arrow indicates a possible additional processing site for NPW. Identical amino acids within the orthologues are shown in black. Lightly shaded residues are conserved in more than four of six (NPB) or two of three (NPW) species.

In vitro pharmacology of NPB and NPW assessed by melanophore pigment aggregation assay (A and B) and [Ca²⁺]i transient assay (C) in mammalian cells. (A and B) X. laevis melanophores were transiently transfected with human GPR7 (A) or GPR8 (B) cDNA, and challenged with various forms of synthetic human NPB and NPW peptides. Brominated NPB and nonbrominated NPB have approximately equal potencies on GPR7 (A), whereas both of them have much weaker potencies on GPR8 (B). Brominated NPW and nonbrominated NPW both have similar potencies on GPR7 and GPR8. des[l-Trp-1]NPB and des[l-Trp-1]NPW are both much less potent on GPR7 and GPR8. Data are the mean of eight separate experiments. (C) Ltk− cells were stably cotransfected with the human GPR7 cDNA and Gqi chimera cDNA, and challenged with nonbrominated synthetic human NPB and NPW. NPB has a modestly higher potency than NPW. When Gqi cDNA is not cotransfected, neither NPB nor NPW could induce the calcium mobilization (open and filled circles are all overlapping). Peak [Ca²⁺]i values are represented as percentages of the maximum response. Data are presented as the mean ± SEM of three experiments performed in duplicate.

In situ hybridization of NPB (A–H) and NPW (I–L) mRNA on mouse brain sections. (Scale bars are 300 μm.) (A–H) NPB mRNA in mouse brain. CA1–3, CA1–3 field of hippocampus; LHb, lateral habenular nucleus; PaMP, paraventricular hypothalamic nucleus medial parvicellular part; EW, EW nucleus; m5, motor root of the trigeminal nerve; s5, sensory root of the trigeminal nerve; LPBI, lateral parabrachial nucleus internal part; Me5, mesencephalic trigeminal nucleus; Sub CA, subcoeruleus nucleus α part; A5, noradrenergic cell group A5; LC, locus coeruleus; IOB, inferior olive subnucleus B. (I–L) NPW mRNA in mouse brain. PAG, periaqueductal gray matter; EW, EW nucleus; VTA, ventral tegmental area; DR, dorsal raphe nucleus; DRD, dorsal raphe nucleus dorsal part.

In vivo pharmacological effects of i.c.v.-injected NPB on food intake (A and B), locomotor activity (C), and nociception (D and E). (A) Vehicle or 3 or 10 nmol of synthetic rat NPB was i.c.v. injected in bolus into freely fed mice, and food consumption was measured. Injections were performed at 20:00 (beginning of dark phase) and food intake was measured at 22:00, 00:00, and 08:00 the next morning (end of dark phase). The data represent food intake between the indicated times (mean ± SEM, n = 4–5 per group). Asterisks indicate significant difference (P < 0.05, one-way ANOVA, Fisher's post hoc analysis). (B) Anorexic effect of NPB is enhanced by pretreatment with CRF. CRF (0.3 nmol) was i.c.v. injected 15 min before the injection of 3 nmol of synthetic rat NPB in mice. NPB was i.c.v. injected at 20:00 (beginning of dark phase), and food intake was measured at 22:00 and 00:00. n.d., no food intake detected (bars invisible). The data represent food intake between designated times in dark phase (mean ± SEM, n = 7 per group). Asterisks indicate the significant difference (P < 0.05, one-way ANOVA, Fisher's post hoc analysis). (C) NPB-induced hyperlocomotion in both dark and light phases. Three nanomols of synthetic rat NPB or vehicle was i.c.v. injected into rats placed in an open field apparatus. The data represent the distance traveled (meters) per 2 h (mean ± SEM, n = 6–8 per group). Asterisks indicate significant difference (P < 0.05, one-way ANOVA, Fisher's post hoc analysis). (D) Paw flick tests were performed 20 min after i.c.v. injection of vehicle or 3 nmol of synthetic rat NPB in rats. Tests were performed without (Car−) and with (Car+) the chemical inflammation induced by carrageenan. Two milligrams of carrageenan was injected into a hindpaw 3 h before the paw flick test. The data represent the latency (seconds) to flick the paw out of the path of the heat-producing light beam (mean ± SEM, n = 6–8 per Car− group and n = 4–5 per Car+ group). (E) Formalin tests were performed 10 min after injection of vehicle, 3 nmol of nonbrominated synthetic rat NPB, or 3 nmol of brominated NPB in rats. The data represent the licking duration (seconds per 5-min bin) at indicated minutes after injection of 50 μl of 5% formalin into a hindpaw (mean ± SEM, n = 4–5 per group). Asterisks indicate significant difference from vehicle treatment (P < 0.05, one-way ANOVA, Fisher's post hoc analysis).