General

The Neuropeptide FF receptor family is a member of the G protein-coupled receptor superfamily containing two subtypes, NPFFR1 and NPFFR2, which exhibit high affinities for Neuropeptide FF (NPFF) and Rfamide related peptides (RFRP). NPFFR1 is broadly distributed in the central nervous system with the highest levels found in the limbic system and the hypothalamus. NPFFR2 is present in high density, particularly in mammals in the superficial layers of the spinal cord where it is involved in nociception and modulation of opioid functions [3,25,30,43].

NPFF peptides

Neuropeptide FF (NPFF, FLFQPQRFamide) is representative of a family of RFamide peptides that regulate pain, cardiovascular functions, appetite, thirst, and body temperature. NPFF is an amidated neuropeptide first isolated from bovine brain [57]. Genes encoding two precursors, NPFF_A [37,55] and NPFF_B [14,25] have been cloned in mammals. NPFF_B products are also referred as RFamide-related peptides (RFRP) or gonadotropin-inhibitory hormone (GnIH) and regulate pain modulation, stress response and hypothalamic-pituitary-gonadal axis [22,25,53]; see [50] for review]. See table 1 for peptides originating from these precursors in humans.



The NPFF_A precursor could maturate peptides with a C-terminal PQRF-NH₂ sequence and an N-terminal sequence that differs in different species. Several peptides contained in the NPFF_A precursor have been biochemically identified in various species (see table 2 and [58] for review).



The NPFF_B precursor is a precursor for RFRP or GnIH [14,50]. It matures peptides with a C-terminal LPXRF-NH₂ (X= L or Q) sequence. Several peptides contained in the NPFF_B precursor have been biochemically identified in various mammalian species (see Table 3 and [50] for review).

Name	Peptide	Source
Human RFRP-1	MPHSFANLPLRF-NH2	Human brain [53]
Human RFRP-3	VPNLPQRF-NH2	Human brain [53]
Macaque RFRP-3	SGRNMEVSLVRQVLNLPQRF-NH2	Macaque brain [52]
Bovine RFRP-1	SLTFEEVKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH2	Bovine brain [9]
Bovine RFRP-3	AMAHLPLRLGKNREDSLSRWVPNLPQRF-NH2	Bovine brain [59]
Rat RFRP-3	ANMEAGTMSHFPSLPQRF-NH2	Rat brain [54]
Hamster RFRP-1	SPAPANKVPHSAANLPLRF-NH2	Hamster brain [51]
Hamster RFRP-3	TLSRVPSLPQRF-NH2	Hamster brain [51]

Table 3. Peptides contained in the NPFF_B precursor and their sources.

Receptor family

Two NPFF receptors have been identified; NPPF2 (also referred to as HLWAR77, NPGPR and GPR74) [3,8] and NPFF1 (also known as OT7TO22) [3,14].

Receptor structure

NPFF1 and NPFF2 receptors belong to the G protein-coupled receptor family. They are about 50% identical and are most similar to neuropeptide Y and orexin receptors (30-35% homology). Variants of the NPFF2 receptor in N- and/or C- terminals have been reported [60]. Comparative studies of the pharmacological profiles of both receptors indicate that NPFF1 receptors are not very discriminative towards the endogenous peptides from both precursors NPFF_A and NPFF_B. However, peptides from the NPFF_A precursor display a higher affinity for the NPFF2 receptor and, conversely, peptides from the NPFF_B precursor slightly prefer the NPFF1 receptor [25,28]. NPFF receptor activity in recombinant cells, especially NPFF1 activity, is reversed by BIBP3226, a selective antagonist for neuropeptide Y, Y1 receptors [27-28]. In vitro and in vivo pharmacological activities of NPFF receptors are reversed by the potent antagonist RF9 [16,45].

Selective agonists with high affinity for each NPFF receptor are available as well as selective radioiodinated ligands [28]. Moreover, the frog pancreatic polypeptide exhibits nanomolar affinity for NPFF2 receptors.

Signal transduction

NPFF receptors are coupled to G proteins and regulate adenylyl cyclase in recombinant cell lines (CHO, HEK 293, SH-SY5Y) [3,25,28-29]. NPFF receptors are also coupled to voltage-gated N-type Ca²⁺ channels [20]. At this time, the endogenous intracellular signalling pathways activated by NPFF receptors in neurones are still unknown.

Much data indicate that, at the cellular level, stimulation of NPFF receptors inactivates opioid receptors by a molecular mechanism which is still not understood. In acutely dissociated neurons, NPFF2 receptors specifically counteract N-type Ca²⁺ channel inhibition by opioids [40,42]. In SH-SY5Y neuroblastoma cells stably expressing human NPFF receptors, NPFF agonists also reduce the inhibitory effect of μ and δ-opioid receptor activation on an N-type Ca²⁺ channel [20,29]. These regulations could be due in part to receptor heteromerisation since NPFF2 receptors have been shown to physically interact with mu-opioid receptors [41] and induce their trans-phosphorylations [32].

The NPFF analog 1DMe blocks presynaptic δ-opioid autoreceptors in the spinal cord, leading to an increase in K⁺-evoked met-enkephalin release [2], that could explain the analgesic effect of an intrathecal injection of 1DMe [13]. 1DMe also inhibits the reduction induced by morphine, but not by noradrenaline, of the electrically evoked acetylcholine release in the guinea pig myenteric plexus [49]. Furthermore, on isolated neurons from rat periventricular and dorsal raphe nuclei that co-express nociceptin receptors and NPFF1 or NPFF2 receptors, respectively, NPFF analogs inhibit the nociceptin-induced reduction of N-type Ca²⁺ channel conductance [40,42].

NPFF causes an increase in miniature inhibitory postsynaptic currents in magnocellular neurosecretory cells of the paraventricular nucleus indicating a presynaptic locus of action [15].

NPFF1 couples with G_αi to inhibit adenylyl cyclase (AC) [14], and reduces the activities of cAMP-dependent protein kinase (PKA) and mitogen-activated protein kinase (MAPK) signaling cascade. The inhibitory action of RFRPs on gonadotropin gene expression was suggested to be mediated by an inhibition of AC/cAMP/PKA-dependent extracellular signal-regulated kinase (ERK) pathway [47]. In the sheep, RFRP-3 can inhibit both gonadotropin-releasing hormone (GnRH)-induced intracellular Ca²⁺ increase and ERK phosphorylation, impacting GnRH-induced gonadotropin release and synthesis [44].

Tissue distribution

NPFF receptors [10,12] and mRNA [3] are localised in brain regions known to contain opioid receptors and to be involved in nociception control. A comparative autoradiographic study [12], using two radioligands, [¹²⁵I]YVP and [¹²⁵I]EYF selective for NPFF1 and NPFF2 receptors, respectively, has revealed that NPFF2 receptors are mainly expressed in the rat central nervous system (especially in the dorsal horn of the spinal cord, the parafascicular and laterodorsal nuclei of the thalamus and some hypothalamus nuclei), indicating that most of the pharmacological effects of NPFF in this species are due to the NPFF2 receptor.

In situ hybridization, autoradiography, and immunohistochemistry in the rat brain have shown that NPFF1 is expressed in various brain regions including the limbic system of telencephalon, diencephalon, mesencephalon as well as the pituitary [3,25].

Comparison of NPFF1 and NPFF2 receptor distribution in rat, mouse, octodon, rabbit, guinea-pig and monkey reveals important species differences [11] and shows that the pattern of NPFF2 receptors is consistent with a potential role of NPFF in the modulation of sensory inputs while NPFF1 could participate in neuroendocrine functions.

Functions

NPFF is involved in several physiological functions such as hormonal modulation, cardiovascular and thermal regulation and food intake [30]. Furthermore, many pharmacological data suggest that NPFF plays an important role in pain modulation and acts mainly through the regulation of the opioid system [33,36,43]. A close relationship between NPFF and opioid systems exists in the central nervous system, since NPFF analogs increase or decrease opioid effects, depending on dose and site of administration [43]. In general, intracerebroventricular injected analogues exert pronociceptive activity while intrathecal injection induces analgesia [58]. The NPFF system is therefore considered to be an opioid-modulating system involved in homeostasis that counteracts the action of opioids and thus plays a role in opiate tolerance [36,43]. Furthermore, NPFF injected during conditioned place preference, influences the development of rewarding effects of morphine [26]. The NPFF antagonist RF9 prevents the development of analgesia tolerance and decreases the withdrawal syndrome following chronic morphine treatment [7]. The stimulation of NPFF receptors in the ventral tegmental area blocks morphine-induced release of dopamine in the rat [21] while i.c.v injections of an NPFF analog inhibits morphine-induced expression of c-fos in the same brain region in mice [31].

NPFF also displays potent effects in the periphery. Intravenous injection of NPFF causes a transient rise in arterial blood pressure and heart rate in rats [1,39].

The ICV administration of NPFF reduces food intake and the combination of NPFF and naloxone shows no additivity in the anorexigenic effect, suggesting that NPFF and naloxone reduced food intake by a common mechanism. This suggests that NPFF may function as an endogenous anorexigenic peptide with antiopioid function [23,35,48]. One of the emerging themes from recent research is that NPFF endogenous peptides are involved in control of feeding behaviour both in invertebrates and in vertebrates [5].

Abundant RFRP-immunoreactive (ir) neuronal fibers are observed in the median eminence (ME) of sheep [4], macaque [52], and humans [53]. RFRP-3, a mammalian GnIH ortholog, inhibits gonadotropin synthesis and/or release from cultured pituitaries in sheep [44] and cattle [18]. Peripheral administration of RFRP-3 also inhibits gonadotropin release in sheep [4] and cattle [18]. It was further shown that NPFF1 mRNA is expressed in gonadotrophs in the human pituitary [53]. Recent study has also shown that RFRP-3 is secreted in the portal blood in a pulsatile manner in sheep [46]. Taken together, it is likely that RFRP-3 directly acts on the pituitary to inhibit gonadotropin secretion from the pituitary at least in these mammalian species. On the contrary, it is thought that RFRP-3 does not act directly on the pituitary in rodents, because there are relatively few or no RFRP-ir fibers in the ME [24,51]. However, there are also several studies indicating that RFRP-3 can directly inhibit gonadotropin release from the pituitary [34] (see [50] for review).

Immunohistochemical studies using light and confocal microscopy indicate that neuronal axon terminals containing RFRP-3 are in probable contact with GnRH neurons in mammals [24,51-53]. NPFF1 also is expressed in GnRH neurons in hamsters [51]. Together with the observation that central administration of RFRP-3 inhibits the release of gonadotropin in hamsters [24] [51] and rats [17], RFRP-3 may inhibit the secretion of gonadotropins by decreasing GnRH neuronal activity in addition to regulating the gonadotroph directly. Direct application of RFRP-3, a mammalian GnIH ortholog, to GnRH cells in cultured mouse brain slices decreases firing rate in a subpopulation of cells, further indicating a direct action of RFRP-3 on GnRH neurons. In addition, RFRP-3 inhibits firing of kisspeptin-activated vGluT2 (vesicular glutamate transporter 2)-GnRH neurons as well as of kisspeptin-insensitive GnRH neurons in mice [6,56]. Central administration of RF9, an antagonist of NPFF receptors, to rats and mice led to marked increases in gonadotropin concentrations, providing a pronounced role of RFRP-3 as a key regulator of the reproductive axis in mammals [38].

Central administrations of RFRP-3 inhibit reproductive behavior of male rats [17]. On the other hand, central administrations of RFRP-3 stimulate feeding behavior in rats [17,34]. There is also a report that central administration of RFRP-3 can stimulate adrenocorticotropic hormone and oxytocin release in rats [19]. It was shown that RFRP-ir fibers project to various neurons in the brain, such as dopamine and/or proopiomelanocortin neurons in the rat, sheep and macaque [52], indicating multiple functions of GnIH and its orthologs in the brain of mammals.

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