Neuropeptide B and W: neurotransmitters in an emerging G-protein-coupled receptor system

Abstract

Deorphanised G-protein-coupled receptors represent new and expanding targets for drug development. Neuropeptide B (NPB) and W (NPW) have recently been identified as the cognate endogenous ligands for the orphan receptor GPR7, now designated as NPBW₁. NPB and NPW also bound to a second related orphan receptor, GPR8, now designated as NPBW₂ that is present in humans but not rats or mice. In humans, high levels of NPW mRNA have been visualised in the substantia nigra, whereas moderate expression levels have been detected in the amygdala and hippocampus. In peripheral tissues, expression of NPW mRNA has been confirmed in the progenital system, comprising the kidney, testis, uterus, ovary and placenta, and also in stomach homogenates. Immunocytochemical, molecular biological and autoradiography techniques have revealed a discrete CNS distribution for NPBW₁ in human, mouse and rat. Highest expression of NPBW₁ mRNA and protein was identified in the amygdala and hypothalamic nuclei known to regulate feeding behaviour. [¹²⁵I]-NPW bound with a single high affinity to rat amygdala, K_D=0.44 nM and 150 fmol mg⁻¹ protein. Physiological studies demonstrate that intracerebroventricular infusion of NPBW₁ ligands modulates feeding behaviour, regulates the release of corticosterone, prolactin and growth hormone while also manipulating pain pathway. Mouse knockout models of the gene encoding either NPB or NPBW₁ have a gender-specific phenotype, with moderate obesity evident in males but not females. Further investigation is required to elucidate the precise physiological role of NPB and NPW as neurotransmitters.

Introduction

G-protein-coupled receptors (GPCRs) represent the largest family of transmembrane spanning proteins. Members of Class 1 GPCRs (Family A) are the targets for a significant proportion of currently prescribed drugs (Wilson & Bergsma, 2000; Maguire & Davenport, 2005). Following the completion of 99% of the human genome (Lander et al., 2001), bioinformatics has been applied to identify most of, if not all, the human genes that could potentially encode a GPCR (Foord et al., 2005). Recently, about 85 ‘orphan' Class 1 GPCRs have been identified, so-called because the endogenous ligand(s) activating the receptor is as yet unknown. During the last 10 years, over 50 orphan GPCRs have been deorphanised (paired with their cognate ligands), using reverse pharmacology, with the majority turning out to be peptides (Davenport, 2003). The novel neuropeptide B (NPB) and neuropeptide W (NPW), paired with the previously designated orphan receptors GPR7 and GPR8, have received growing attention as they have been shown to modulate the release of pituitary-derived hormones, regulate feeding behaviour and manipulate pain pathways. Therefore, understanding the role of the NPB/NPW system may provide new insights into human physiology and may offer novel targets for therapeutic drug development. The purpose of this review is therefore to summarise the physiology and pharmacology of the NPB/NPW transmitter system.

Identification of NPB and NPW as cognate ligands for orphan receptors GPR7 and GPR8

O'Dowd et al. (1995) originally identified two genes, GPR7 and GPR8, predicted to encode two GPCRs (designated as orphan receptors). Following pairing of these receptors with NPB and NPW, they were reclassified by IUPHAR as NPBW₁ (GPR7) and NPBW₂ (GPR8) (see Davenport & Singh, 2005a, 2005b). NPBW₁ and NPBW₂ (Table 1) are predicted to comprise 328 and 333 amino acids, respectively, with 64% sequence homology. Among other families of GPCRs, NPBW₁ and NPBW₂ are most closely related to opioid receptors.

Table 1.

Structural information on NPBW₁ and NPBW₂

Receptor	Species	Amino acid	Accession number	Chromosomal location	Gene name
NPBW1	Human	328	P48145	8q11.23	GPR7
NPBW1	Rat	329	Q56UD9	5q12	Gpr7
NPBW1	Mouse	146	XP_136404	1 (A1–A2)	Gpr7
NPBW2	Human	333	P48146	20q13.3	GPR8

Amino-acid analysis of NPBW₁ orthologues in rat and mouse has revealed high degree of conservation throughout evolution (Lee et al., 1999). In contrast, the gene-encoding NPBW₂ has not been detected in rat or mouse (Lee et al., 1999) and the receptor may not be present in these species. However, the gene encoding NPBW₂ has been discovered in other mammalian species such as rabbit (Lee et al., 1999).

Anatomical localisation of mRNA encoding NPBW₁ and NPBW₂ receptors

The distribution of messenger RNA (mRNA) encoding NPBW₁ and NPBW₂, determined by RT–PCR or in situ hybridisation in a panel of human, rat and mouse tissues, is summarised in Table 2. Although RT–PCR requires homogenisation of tissue resulting in the loss of anatomical structure, it provides crucial evidence that a particular tissue is capable of expressing mRNA encoding one or both receptors. In some cases, results have been confirmed by in situ hybridisation that, by retaining the anatomical structure, has given some initial information about the possible physiological function of these receptors.

Table 2.

Distribution of NPBW₁ and its homologue in human, mouse and rat

	Human		Mouse	Rat
	NPBW1	NPBW2	NPBW1	NPBW1
CNS tissues
Cerebellum	−a	+a	−b,c	−a,b,c
Cerebral cortex	ND	ND	−b,c	+a,b,c
Parietal cortex	−a	++a	−b,c	−c
Hippocampus	+++a	+++a	+b,c	+a,b,c
Amygdala	+++a	+++1	+++b,c	+++a,b,c,d
Hypothalamus	−a	−a	+++b,c	+++a,b,c,d
Thalamus	+a	++a	+b,c	++a,b,c
Caudate nucleus	−a	++a	−b,c	−c
Substantia nigra	+a	+a	−b,c	−c
Corpus callosum	+a	+a	−b,c	−c
Bed nucleus of the stria terminalis	ND	ND	++b,c	+++c
Dorsal vagal complex	ND	ND	−b,c	++c
Periaqueductal grey	ND	ND	−b,c	++c
Superficial grey layer of superior colliculus	ND	ND	−b,c	++c
Medulla oblongata	ND	ND	ND	++a
Spinal cord	−a	−a	ND	++a
Peripheral tissues
Pituitary gland	++a,b	++a	ND	+a
Adrenal gland	++a	++a	ND	−a
Testis	+a	+1	ND	+a
Uterus	−a	−a	ND	+++a

Expression of receptors NPBW₁ or NPBW₂: ND, not determined; –, not detected; +, low levels; ++, moderate levels; +++, high levels, as determined by

^aRT–PCR.

^bIn situ hybridisation.

^cIn vitro autoradiography or

^dImmunocytochemistry. These data are from Brezillon et al. (2003), Fujii et al. (2002), Lee et al. (1999), O'Dowd et al. (1995), Singh et al. (2004) and Tanaka et al. (2003).

RT–PCR analysis of a panel of human tissues revealed a predominant central nervous system (CNS) distribution for NPBW₁ mRNA (Brezillon et al., 2003). Highest expression of NPBW₁ mRNA was detected in the hippocampus and amygdala. NPBW₂ mRNA was also detected at high levels within these regions of the brain with moderate expression further detected in the thalamus (Brezillon et al., 2003). However, the precise anatomical distribution of both NPBW₁ and NPBW₂ in the human brain is currently unknown and warrants further investigation.

In peripheral tissues, moderate expression levels of NPBW₁ and NPBW₂ mRNA were reported in the pituitary gland, whereas mRNA for only the latter receptor was detected in the adrenal gland (Brezillon et al., 2003). In contrast, Mazzocchi et al. (2005) were able to confirm mRNA expression of both NPBW₁ and NPBW₂ to cultured human adrenal zona glomerulosa, zona fasciculata and reticularis cells. Within the pituitary gland, the distribution of NPBW₁ was further refined to the lateral wings of the anterior pituitary using in situ hybridisation (O'Dowd et al., 1995).

A similar distribution of NPBW₁ mRNA was detected in rat tissues using RT–PCR, with almost exclusive expression to the CNS (Fujii et al., 2002). Moderate to high levels were present in peripheral tissues such as the pituitary gland and progenital system (Fujii et al., 2002). In situ hybridisation revealed high levels of NPBW₁ mRNA expression in discrete regions of rat brain, including various hypothalamic and amygdaloid nuclei (Lee et al., 1999). In particular, NPBW₁ mRNA was detected in the ventromedial hypothalamic nuclei (VMH), dorsal medial hypothalamic nucleus (DMH) and paraventricular hypothalamic nucleus (PVN), all of which are strongly associated with the modulation of feeding behaviour and metabolic disorders (Williams et al., 2001). NPBW₁ mRNA was also found in the arcuate (Arc) and supraoptic nucleus (SO). Interestingly, the former nucleus consists of neurones that contain neuropeptide Y (NPY), a peptide that has pronounced effects on feeding (Woods et al., 1998), or contain other neurotransmitters regulating the release of anterior pituitary-derived hormones. In contrast, the SO contains neurones that extend into the posterior pituitary, releasing vasopressin to have peripheral effects (Johnston, 1985). High NPBW₁ mRNA levels were also present in the suprachiasmatic nucleus (SCh), which regulates body functions over 24 h in response to the light and dark cycle (Hastings, 1997). Strongest expression of NPBW₁ mRNA was in the amygdala, particularly within the medial amygdaloid nucleus, whereas moderate levels were also detected in the posteromedial amygdaloid nucleus. These nuclei represent part of the limbic system, which including areas such as the amygdala, hypothalamus and hippocampus, is responsible for the emotional state of mammals (Sah et al., 2003).

Anatomical localisation and characterisation of [¹²⁵I]-NPW binding by quantitative autoradiography and image analysis

mRNA levels do not necessarily correlate with protein expression (Curtis et al., 2000; Greenbaum et al., 2003; Neumann et al., 2004). Radioligand binding assays using tissue sections followed by image analysis of the resulting autoradiographs provide direct evidence of receptors within discrete anatomical regions and have the advantage of being quantitative. Using [¹²⁵I]-NPW, highest receptor densities (∼150 fmol mg⁻¹ protein) (Figure 1) were measured in the amygdala, and this distribution was confirmed to be the receptor NPBW₁ using immunocytochemistry (Singh et al., 2004). NPBW₁ receptors were also localised in a range of hypothalamic nuclei such as the VMH, DMH and SCh, demonstrating high correlation with NPBW₁ mRNA expression. Interestingly, one mismatch between receptor protein and mRNA was observed in the hippocampus, where NPBW₁ expression was reported (Lee et al., 1999), but protein was not detected by ligand binding (Singh et al., 2004). Comparison of the distribution of NPBW₁ mRNA and protein in mouse brain revealed a limited distribution of receptors when compared to rats (O'Dowd et al., 1995; Tanaka et al., 2003; Singh et al., 2004). However, the pattern of NPBW₁ mRNA in mouse was similar to that observed in rats and humans, with the expression of NPBW₁ predominantly in the amygdala and hypothalamus (O'Dowd et al., 1995; Tanaka et al., 2003; Singh et al., 2004).

Figure 1.

Localisation of NPBW₁ in coronal sections of rat brain using [¹²⁵I]-NPW receptor autoradiography. [¹²⁵I]-NPW binding was detected to the (a) DVC, dorsal vagal complex, (b) PAG, periaqueductal grey, (c) SuG, superficial grey layer of superior colliculus, (d) MeA, medial amygdaloid nuclei, (e) SFO, subfornical organ, BST, bed nucleus of the stria terminalis, MPA, medial preoptic area and the SCh of rat brain (n=4). Specific binding was not detected in the presence of unlabelled NPW (f). Scale bar=1 cm. Reproduced with permission from Brain Research (Singh et al., 2004).

Identification of NPB and NPW as cognate ligands for orphan receptors by reverse pharmacology

To identify the cognate endogenous ligand for GPR7 (NPBW₁) and GPR8 (NPBW₂), Shimomura et al. (2002) artificially expressed these receptors in Chinese hamster ovary cells, exposing them to porcine hypothalamus extracts while monitoring alterations in cAMP levels. Hypothalamic extracts were assayed for the endogenous ligand as the receptor NPBW₁ mRNA was highly expressed in various hypothalamic nuclei, and in accordance with classical neurotransmitters, the ligands should be in close proximity to their receptors. When these cell lines expressing either NPBW₁ or NPBW₂ were incubated with the hypothalamic extracts, specific inhibition of forskolin-induced cAMP production was detected. This also suggested that both NPBW₁ and NPBW₂ are coupled to the hetrotrimeric G_i protein. Purification, isolation and further analysis of the ligand responsible for this inhibition of cAMP production revealed the discovery of the novel peptide, NPW. In a similar manner, Fujii et al. (2002), as well as two other independent groups (Brezillon et al., 2003; Tanaka et al., 2003), identified NPB as an additional endogenous ligand for NPBW₁ and NPBW₂.

NPB and NPW belong to a distinct family of peptides that do not display any significant sequence similarity to other known peptides while sharing a high degree of sequence similarity with each other (Fujii et al., 2002; Shimomura et al., 2002; Brezillon et al., 2003; Tanaka et al., 2003). The amino-acid sequence of the human forms of NPB and NPW are aligned with their respective orthologues in other mammalian species in Figure 2. NPB has a post-translational modification, bromination of the N-terminus tryptophan (Fujii et al., 2002) that is presently unique among the mammalian bioactive peptides. Whether this modification is essential for the peptide to function in tissues or stability in vivo remains to be determined. However, it has been demonstrated that des-Br-NPB is equipotent in in vitro cAMP inhibition assays to brominated NPB (Tanaka et al., 2003).

Figure 2.

Bovine, human, mouse, porcine and rat amino-acid sequences of NPB and NPW. Sequence similarities between NPB and NPW orthologues are shown in black. Further identical amino acids within the NPB or NPW orthologues are shown in grey.

In common with other peptides, NPB and NPW are derived from larger precursor proteins with signal peptides of 24 and 32 amino acids, respectively. These peptides, of separate gene products, are then cleaved at basic pairs of amino acids to yield shorter forms of the peptides, both consisting of 23 amino acids (NPW-23 and NPB-23) (sharing 61% amino-acid sequence similarity), or C-terminally extended forms of 29 amino acids (NPB-29) and 30 amino acids (NPW-30) (sharing 66% amino-acid sequence similarity) (Fujii et al., 2002; Brezillon et al., 2003). The rank order of potency has been determined in artificial cell lines expressing two different receptors. The four peptides bound with high potency to both receptors but NPBW₁ have slightly higher affinity for both forms of NPB, whereas NPBW₂ have higher affinity for the short and C-terminal extended forms of NPW (Fujii et al., 2002; Shimomura et al., 2002; Brezillon et al., 2003; Tanaka et al., 2003). Consistent with the sequence, NMR conformational analysis of the shorter forms of NPB and NPW (Lucyk et al., 2005) reveled that they displayed similar C-terminal secondary structures, whereas the N-terminal secondary structures differed, which may contribute to the slight differences in potencies reported for the peptides. However, these differences are small and it is unlikely that the two receptors can be distinguished pharmacologically using the rank order of these agonists. At present, no antagonists have been developed that are selective for either receptor.

A radiolabelled analogue of NPW-23 ([¹²⁵I]-NPW), which binds saturably, specifically and with high affinity to NPBW₁ in rat amygdala (K_D of 0.44 nM) has been synthesised, thus providing a potentially useful pharmacological tool to quantify receptor density (Singh et al., 2004). Fixed concentrations of NPB-23 were found to compete with [¹²⁵I]-NPW binding at levels comparable to that seen for nonspecific binding (Singh et al., 2004). As NPBW₂ is not expressed in rat, the results represent binding to NPBW₁ and provide pharmacological evidence that NPB and NPW, at least in rat brain, share one common receptor. Similarly, NPBW₂ is also not detected in mice (Lee et al., 1999). C-terminally truncated forms of NPB or depletion of Trp¹ in either peptide resulted in the loss of potency, suggesting that the C- and N-terminus comprise regions consisting of critical amino acids for the efficacy and binding of the peptides to their receptors (Brezillon et al., 2003; Tanaka et al., 2003).

Anatomical localisation of the endogenous peptide ligands, NPB and NPW

In humans, high levels of NPW mRNA were visualised in the substantia nigra, whereas moderate expression levels were detected in the amygdala and hippocampus (Fujii et al., 2002). In peripheral tissues, expression of NPW mRNA was confirmed in the progenital system, comprising the kidney, testis, uterus, ovary and placenta, and also in stomach homogenates (Fujii et al., 2002). The distribution of NPB mRNA was similar to NPW mRNA, but with higher expression within the CNS. Unlike NPW, NPB mRNA was detected in the thalamus and hypothalamus (Table 3).

Table 3.

CNS distribution of NPB and NPW in human, mouse and rat

	Human		Mouse		Rat
	NPB	NPW	NPB	NPW	NPB	NPW
Cerebellum	++a	++a	−b	−b	+a	ND
Parietal cortex	+a	++a	−b	−b	ND	ND
Hippocampus	++a	++a	++b	−b	++a,b	ND
Amygdala	++a	++a	−b	−b	ND	++c
Hypothalamus	++a	−a	+++b	−b	++a,b	++c
Thalamus	+a	−a	+b	−b	+a	ND
Caudate nucleus	+a	−a	−b	−b	ND	ND
Substantia nigra	+++a	+++a	−b	−b	++c	ND
Corpus callosum	++a	−a	−b	−b	ND	ND
Chorid plexus	+a	++a	−b	−b	ND	ND
Periaqueductal grey	ND	ND	−b	++b	ND	ND
Ventral tegmental area	ND	ND	−b	+b	ND	++c
Dorsal raphe nuclei	ND	ND	−b	+++b	++c	+++c
Edinger Westphal nuclei	ND	ND	+b	++b	++c	ND
Spinal cord	+++a	−a	ND	ND	+a	ND

Expression of NPB or NPW: ND, not determined; −, not detected; +, low levels; ++, moderate levels; +++, high levels, as determined by

^aRT–PCR.

^bIn situ hybridisation, or

^cImmunocytochemistry. These data are from Brezillon et al. (2003), Dun et al. (2005), Dun et al. (2003), Fujii et al. (2002), Singh et al. (2004) and Tanaka et al. (2003).

Using immunocytochemical techniques, NPW-like immunoreactivity (IR) has been detected in the dorsal raphe nuclei (DRN) and ventral tegmental area (VTA) (Singh et al., 2004) as well as various hypothalamic nuclei such as the PVN, SO, dorsal hypothalamic areas, Arc and accessory neurosecretory nuclei of rat brain (Dun et al., 2003). NPW-IR was also detected in the cell processes of the basomedial and central nuclei of the amygdala (Dun et al., 2003). Although the method of immunocytochemistry employed was not quantitative, it was observed that NPW-IR was more abundant in male hypothalamus compared to their female counterparts (Dun et al., 2003). IR to NPW was also detected in the pituitary gland, both the anterior and posterior parts with dense network of NPW-IR fibres visualised within the median eminence (Dun et al., 2003). A similar distribution for NPB-IR was reported in rat brain (Dun et al., 2005).

In situ hybridisation techniques compared the distribution of NPB and NPW mRNA in mouse brain. NPW mRNA was detected in the periaqueductal grey, VTA and the DRN (Tanaka et al., 2003). In contrast, NPB mRNA was more abundant and detected in the Edinger Westphal nuclei as well as the sensory and motor root of the trigeminal nerve. The former nuclei of the trigeminal nerve is known to provide preganglionic parasympathetic inputs to the eye, which results in the control of pupil formation in order to accommodate the lens, whereas the latter nerve is responsible for receiving sensory information from the face and providing motor connections to the muscles involved in mastication (Trimarchi, 1992; Lund et al., 1998). NPB mRNA was also detected in the hypothalamus, particularly the medial parvicellular region of the PVN (Tanaka et al., 2003).

Physiological role of NPBW₁

Feeding behaviour, metabolic rate and obesity

From the distribution of NPBW₁, it was hypothesised that NPBW₁ may modulate feeding behaviour (Shimomura et al., 2002). The distribution of the receptor and the ligands were also comparable with the distribution of substance P, corticotrophin-releasing factor (CRF) and NPY, all of which are well-established regulators of food consumption (Holmes et al., 2003). The first physiological study on the action of NPW reported acute hyperphagia in male rats when NPW was administered intracerebroventricular (i.c.v.) (Shimomura et al., 2002). These initial observations were confirmed by other groups who studied the actions of NPBW₁ activation over 24 h by i.c.v. application of NPB or NPW (Baker et al., 2003; Samson et al., 2004; Levine et al., 2005). In agreement and further exploring the site of NPW action, Levine et al. (2005) demonstrated that administration of NPW into the PVN and lateral hypothalamus (LH) resulted in hyperphagia. However, Tanaka et al. (2003) showed that, although the acute effects of NPB administration were to induce hyperphagia, the long-term effect was to significantly attenuate feeding. Significant hypophagia was also reported by Mondal et al. (2003), with the effect of high doses lasting for up to 48 h. Interestingly, a recent study identified a source of endogenous NPW from rat stomach antral cells and reported decreased levels of NPW in fasted animals in which, when re-fed, levels of NPW increase (Mondal et al., 2006), consistent with the notion that NPW may act as a suppressant to feeding.

In all studies, animals had free access to food and water and unless stated were injected i.c.v.; therefore, availability of food and route of administration cannot account for the observed differences. Furthermore, administration of different doses of NPB/NPW did not result in different physiological responses. The variations in response to NPB/NPW administration could in part be attributed to circadian rhythm, as NPBW₁ receptors have been localised to the SCh (Lee et al., 1999; Singh et al., 2004). Thus, the effect of NPB/NPW treatment may depend on the stage of light/dark cycle. However, there is little evidence suggesting that feeding behaviour in male rats administered NPB/NPW differs during the light/dark cycle and requires further investigation (Tanaka et al., 2003).

The chronic effects of NPBW₁ receptor modulation had not been evaluated until Ishii et al. (2003) generated mice with targeted disruption of the gene encoding NPBW₁ (NPBW₁−/−). Intriguingly, female NPBW₁−/− mice remained similar to wild type in all phenotypes examined, whereas moderate adult onset obesity was evident in NPBW₁−/− male mice, greatly exaggerated when animals were fed with a high-fat diet (Ishii et al., 2003). This was further emphasised by the generation of a strain of NPB−/− mice, in which adult onset obesity was specifically observed in males but not females (Kelly et al., 2005). The disruption to the receptor or peptide did not cause fatalities or attenuation of growth in either sex. The mechanism of obesity was further investigated and importantly shown to be independent of the leptin and melanocortin signalling pathway (Ishii et al., 2003). Furthermore, mRNA levels of NPY were reduced, whereas proopiomelanocortin (POMC) was increased compared to wild-type male mice, indicative of a lean rather than obese state. Importantly, POMC is the precursor to opioid peptides and melanocortin (Low, 2004). Although melanocortin is thought to be an anti-orexigenic peptide (O'Rahilly et al., 2003), evidence suggests it may also act as an orexigen (Appleyard et al., 2003).

In NPBW₁ −/− male mice, spontaneous locomotor activity was decreased as were both resting O₂ consumption and CO₂ production (Ishii et al., 2003). Although the genes for NPB, NPW and NPBW₁ have not been manipulated in rats, male rats administered NPB show significantly increased locomotor activity (Baker et al., 2003), which correlates with the data from the mouse NPBW₁ knockout studies (Ishii et al., 2003). Interestingly, i.c.v. administration of NPW in rats increased c-Fos IR in neurones expressing hypocretins in the LH (Levine et al., 2005). The hypocretins are peptides, which are associated with wakefulness (Berridge & Espana, 2005), and it can be speculated that the hypocretins may be involved in NPB/NPW-induced increases in spontaneous locomotor activity, which may in turn cause acute hyperphagia (Williams et al., 2001). Hence, this is consistent with the development of obesity if the NPBW₁ receptor is blocked or expression is disrupted, as chronic calorie intake would exceed energy expenditure. However, Mondal et al. (2003) were not able to detect enhanced locomotor activity when NPW was administered to rats, but they did report increased O₂ consumption and increased CO₂ production while also demonstrating an increase in body temperature when compared with rats administered vehicle alone. This latter effect, independent of locomotor activity, may result from protein and triacylglycerol catabolism induced by corticosterone.

A second significant phenotypic characteristic of NPBW₁−/− male mice is that they become progressively hyperglycaemic and hyperinsulinaemic (Ishii et al., 2003). As indicated previously, these effects were specific to male mice, as female NPBW₁ −/− mice did not differ from their wild type (Ishii et al., 2003). The reason for these sexual dimorphic effects could, in part, be explained by the reduced localisation of NPW-like IR in female rats when compared to male rats (Dun et al., 2003). Although immunocytochemistry is not quantitative, it allows speculation that NPW (and NPB) may be more abundant in male mice in relation to their female counterparts. However, the sexual dimorphic results demonstrated for NPBW₁ are not unique. Female wild-type rats with lesions to the amygdala complex have been shown to gain significantly more weight owing to hyperphagia compared to male rats (King et al., 1999). It is possible that NPBW₁ may be a sex-specific receptor, regulating a novel-independent arm in the complex process of energy homeostasis. It should be emphasised that these results are from only a limited number of papers and further studies are required to support these hypotheses.

Hormone release

The distribution of NPBW₁ in the PVN, SO, Arc and SCh (Lee et al., 1999) combined with the detection of NPBW₁ in the pituitary gland (O'Dowd et al., 1995; Fujii et al., 2002; Brezillon et al., 2003) strongly suggest the NPW transmitter system to regulate the release of pituitary gland-derived hormones (O'Dowd et al., 1995; Shimomura et al., 2002). NPB/NPW infusion into rats has since been demonstrated to induce secretion of corticosterone and prolactin while inhibiting growth hormone release dose dependently (Baker et al., 2003; Samson et al., 2004). NPB or NPW did not affect the release of other anterior pituitary-derived hormones. These endocrine effects of NPB/NPW administration were not mediated by direct binding of ligand to NPBW₁ on the pituitary but indirectly, possibly by regulating the release of CRF, dopamine and GHRH/somatostatin (Baker et al., 2003; Samson et al., 2004). One mechanism of NPB-mediated release of corticosterone was attributed to the effects of the release of CRF (which normally induces adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary, which in turn promotes corticosterone secretion from the adrenal cortex) as neutralisation using anti-CRF serum resulted in significant attenuation of NPB-mediated release of corticosterone (Samson et al., 2004). This is supported by the observation that PVN cells depolarise in response to NPW application, resulting in increased plasma corticosterone (Taylor et al., 2005). This effect is attenuated when rats are pretreated with a CRF antagonist (Taylor et al., 2005). Thus, there is strong evidence suggesting that NPW induced secretion of corticosterone is, in part, mediated through NPBW₁ receptors in the PVN, which induce CRF release into the median eminence; CRF then acts on the endocrine cells of the anterior pituitary to release ACTH, which in turn results in the secretion of corticosterone from the adrenal gland (Figure 3). This latter hormone then has effects on various organs of the body, most prominently to induce gluconeogenesis among other important effects such as modulating the body's response to stress and regulating the immune system (Dallman et al., 2004).

Figure 3.

Mechanism of corticosterone secretion induced by NPB/NPW. NPB/NPW released from afferents terminating at the PVN cause depolarisation, resulting in the release of CRF from the PVN into the hypothalamopituitary portal vessels. Once arriving at the anterior pituitary, CRF acts on the cells to induce secretion of ACTH, which is then released in the venous circulation. On arriving to the adrenal cortex, ACTH acts to promote the secretion of corticosterone, which then has its effects on glucose production, protein and triacylglycerol catabolism as well as stimulating appetite (Dallman et al., 2004; la Fleur et al., 2004).

Pain

This novel transmitter system has also been implicated in modulating responses to acute inflammatory pain. Originally, Tanaka et al. (2003) demonstrated an analgesic effect of i.c.v. injection of NPB in rats. This initial work has been progressed and intrathecal (i.t.) injection of either NPB or NPW at the level of the lumbar enlargement has been shown to induce an analgesic effect on pain caused by subcutaneous application of formalin (Yamamoto et al., 2005). Interestingly, i.t. injection of these peptides at the mid-thoracic level did not show any significant effects when compared to vehicle controls (Yamamoto et al., 2005). Similarly, NPB and NPW exhibited a significant anti-allodynia effect on rats challenged with carrageenan (Yamamoto et al., 2005). These effects were not attenuated by the opioid receptor antagonist naloxone, suggesting an independent analgesic and anti-allodynia pathway compared to the opioid peptides (Yamamoto et al., 2005). On the contrary, both NPB and NPW were ineffective in reducing the pain response to thermal hyperalgesia or mechanical nociceptive tests. Mouse models in which the mRNA encoding NPB has been knocked out show a typical phenotype expected from the above-described physiology (Tanaka et al., 2003; Kelly et al., 2005; Yamamoto et al., 2005). These mice demonstrated significant hyperalgesia to acute inflammatory stimulus, induced by intraperitoneal application of acetic acid, when compared with wild-type mice (Kelly et al., 2005). In agreement with results obtained in rats, these NPB knockout mice did not demonstrate hyperalgesia to chemical or thermal pain (Kelly et al., 2005). Therefore, activation of NPBW₁ at the spinal level has been shown to produce an analgesic and anti-allodynia effect in response to acute inflammatory pain (Yamamoto et al., 2005, 2006), consistent with the reported effect of upregulation of NPBW₁ in nerves of the peripheral nervous system during the pathogenesis of inflammatory neuropathies (Zaratin et al., 2005).

Future directions

Two components of the NPB/NPW system have been disrupted using knockout mice: the ligand NPB and the receptor NPBW₁. Further studies on NPBW₁−/− mice will advance the understanding of the role of this receptor. As both peptides are able to bind this receptor, interpreting the results of deleting the NPB gene is more difficult because the receptor may be activated by NPW. Future studies are required in which NPW is deleted. Interestingly, preliminary studies suggest that NPW knockouts (Kelly et al., 2005) may have a different phenotype to that of NPB. This suggests that the two peptides might be present in distinct compartments and have different physiological roles. Crosses between the two phenotypes may elucidate these functions. Development of an antagonist for NPBW₁ is crucial in clarifying the physiological function of this novel transmitter system. Furthermore, the role of NPBW₂ also requires investigation, as this is one of a small number of GPCRs that have no rat or mouse orthologue.

Orphan GPCRs from Class 1, recently paired with their cognate ligands, are emerging as important targets for the development of novel drugs (Wilson et al., 1998; Lin & Civelli, 2004). NPBW₁ has been demonstrated to modulate feeding, metabolism and to be involved in obesity, which intriguingly appears to be specific to male mice. Whether this is also true in humans remains to be discovered. Interestingly, in humans, abdominal obesity is more prevalent in males and is an independent risk factor for coronary heart disease (Lakka et al., 2002). Thus far, activating or disrupting the NPBW₁ transmitter system (Ishii et al., 2003; Kelly et al., 2005), at least in mice, did not have pronounced effects on growth and mortality, which is an important consideration for initiating a drug discovery programme based on this emerging transmitter system whether as a potential target for treatment of inflammatory mediated pain or for modulating feeding behaviour.

Abbreviations

ACTH

adrenocorticotrophin hormone

Arc

arcuate nucleus

BST

bed nucleus of the stria terminalis

CRF

corticotrophin-releasing factor

DMH

dorsal medial hypothalamic nucleus

DRN

dorsal raphe nuclei

DVC

dorsal vagal complex

GPCR

G-protein-coupled receptor

i.c.v.

intracerebroventricular

i.t.

intrathecal

IR

immunoreactivity

LH

lateral hypothalamus

MeA

medial amygdaloid nuclei

MPA

medial preoptic area

NPB

neuropeptide B

NPBW₁

(NC-IUPHAR Nomenclature) neuropeptide B/W 1 receptor

NPBW₂

(NC-IUPHAR Nomenclature) neuropeptide B/W 2 receptor

NPW

neuropeptide W

NPY

neuropeptide Y

PAG

periaqueductal grey

POMC

proopiomelanocortin

PVN

paraventricular nucleus

SCh

suprachiasmatic nucleus

SFO

subfornical organ

SO

supraoptic nucleus

SuG

superficial grey layer of the superior colliculus

VMH

ventromedial hypothalamic nucleus

VTA

ventral tegmental area