Recent advances in mammalian RFamide peptides: the discovery and functional analyses of PrRP, RFRPs and QRFP

Since the first discovery of a peptide with RFamide structure at its C-terminus (i.e., an RFamide peptide) from an invertebrate in 1977, numerous studies on RFamide peptides have been conducted, and a variety have been identified in various phyla throughout the animal kingdom. The first reported mammalian RFamide peptides were neuropeptide FF (NPFF) and neuropeptide AF (NPAF) in 1985. However, for many years after this, no new novel RFamide peptides were identified in mammals. A breakthrough in discovering mammalian RFamide peptides was made possible by reverse pharmacology on the basis of orphan G protein-coupled receptor (GPCR) research. The first report of an RFamide peptide identified from orphan GPCR research was prolactin (PRL)-releasing peptide (PrRP) in 1998. To date, a total of five RFamide peptide genes have been discovered in mammals. Orphan GPCR research has contributed considerably to the identification of these peptides and their receptor genes. This paper examines these mammalian RFamide peptides focusing especially on PrRP, RFamide-related peptides (RFRPs) and, the most recently identified, pyroglutamylated RFamide peptide (QRFP), the discovery of all of which the authors were at least partly involved in. We review here the strategies employed for the identification of these peptides and examine their characteristics, tissue distribution, receptors and functions.     

Nicotine stimulates prolactin-releasing peptide (PrRP) cells and non-PrRP cells in the solitary nucleus

Nicotine has been reported to regulate food intake and body weight. But the mechanisms underlying these roles have not been fully elucidated. In the present study, we showed that acute administration of nicotine (0.5 mg/kg s.c.) could activate prolactin-releasing peptide (PrRP)-bearing neurons in the A2 area of the NTS of rats, suggesting that PrRP may be associated with nicotine-induced effects in the central nervous system (CNS). We next treated rats with nicotine chronically (4 mg/kg/day for 7 days i.p.), and the results showed that the body weight was strongly reduced and food intake was greatly suppressed compared to the vehicle control group (p<0.01). Immunocytochemical studies revealed that PrRP-bearing neurons in the NTS were evidently activated after chronic administration of nicotine, suggesting that PrRP was involved in the regulation of nicotine-mediated body weight loss and food intake suppression in rats. We also found that acute/chronic administration of nicotine activated PrRP-negative neurons in the NTS, and the majority of these neurons were shown to be TH-negative, suggesting that noncatecholaminergic, PrRP-negative neurons in the NTS are associated with the roles of nicotine. Nicotine has also been shown to stimulate the secretion of ACTH, a stress responsive hormone. In the present study, rats received nicotine (0.5 mg/kg s.c.) or saline followed by restraint stress for 30 min. The immunocytochemical results showed that nicotine/stress and saline/stress both activated the majority of the PrRP neurons in the NTS, there being no significant difference between the two treatments (p>0.05). Nicotine/stress also greatly activated PrRP/TH-negative neurons in the NTS. Saline/stress, however, caused much lower effect on the activation of PrRP/TH-negative neurons. In addition, the activation effect of nicotine/stress on PrRP/TH-negative neurons was much stronger than that of nicotine alone (p<0.01). These results indicated that PrRP was associated with stress responses, but it had little effect on nicotine-mediated stress responses. On the other hand, nicotine and restraint stress may synergistically activate PrRP/TH-negative neurons in the NTS. Taken together, our data show that PrRP is involved in the nicotine-induced regulation of body weight and food intake, but may not be involved in the mediation of nicotine on stress responses. PrRP/TH-negative neurons in the NTS are also associated with the roles of nicotine in the CNS.     

Physiological roles of prolactin-releasing peptide

Prolactin-releasing peptide (PrRP) was first isolated from bovine hypothalamus as an orphan G-protein-coupled receptor using the strategy of reverse pharmacology. The initial studies showed that PrRP was a potent and specific prolactin-releasing factor. Morphological and physiological studies, however, indicated that PrRP may play a wide range of roles in neuroendocrinology other than prolactin release, i.e., metabolic homeostasis, stress responses, cardiovascular regulation, gonadotropin secretion, GH secretion and sleep regulation. This review will provide the current knowledge of PrRP, especially its roles in energy metabolism and stress responses.     

Driving reproduction: RFamide peptides behind the wheel

The availability of tools for probing the genome and proteome more efficiently has allowed for the rapid discovery of novel genes and peptides that play important, previously uncharacterized roles in neuroendocrine regulation. In this review, the role of a class of neuropeptides containing the C-terminal Arg-Phe-NH(2) (RFamide) in regulating the reproductive axis will be highlighted. Neuropeptides containing the C-terminal Phe-Met-Arg-Phe-NH(2) (FMRFamide) were first identified as cardioregulatory elements in the bi-valve mollusk Macrocallista nimbosa. During the past two decades, numerous studies have shown the presence of structurally similar peptides sharing the RFamide motif across taxa. In vertebrates, RFamide peptides have pronounced influences on opiatergic regulation and neuroendocrine function. Two key peptides in this family are emerging as important regulators of the reproductive axis, kisspeptin and gonadotropin-inhibitory hormone (GnIH). Kisspeptin acts as the accelerator, directly driving gonadotropin-releasing hormone (GnRH) neurons, whereas GnIH acts as the restraint. Recent evidence suggests that both peptides play a role in mediating the negative feedback effects of sex steroids. This review presents the hypothesis that these peptides share complementary roles by responding to internal and external stimuli with opposing actions to precisely regulate the reproductive axis.     

Structure and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 26RFa in vertebrates

A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.     

Evolutionary origin and divergence of PQRFamide peptides and LPXRFamide peptides in the RFamide peptide family. Insights from novel lamprey RFamide peptides

Among the RFamide peptide groups, PQRFamide peptides, such as neuropeptide FF (NPFF) and neuropeptide AF (NPAF), share a common C-terminal Pro-Gln-Arg-Phe-NH(2) motif. LPXRFamide (X = L or Q) peptides, such as gonadotropin-inhibitory hormone (GnIH), frog growth hormone-releasing peptide (fGRP), goldfish LPXRFamide peptide and mammalian RFamide-related peptides (RFRPs), also share a C-terminal Leu-Pro-Leu/Gln-Arg-Phe-NH(2) motif. Such a similar C-terminal structure suggests that these two groups may have diverged from a common ancestral gene. In this study, we sought to clarify the evolutionary origin and divergence of these two groups, by identifying novel RFamide peptides from the brain of sea lamprey, one of only two extant groups of the oldest lineage of vertebrates, Agnatha. A novel lamprey RFamide peptide was identified by immunoaffinity purification using the antiserum against LPXRFamide peptide. The lamprey RFamide peptide did not contain a C-terminal LPXRFamide motif, but had the sequence SWGAPAEKFWMRAMPQRFamide (lamprey PQRFa). A cDNA of the precursor encoded one lamprey PQRFa and two related peptides. These related peptides, which also had the C-terminal PQRFamide motif, were further identified as mature endogenous ligands. Phylogenetic analysis revealed that lamprey PQRFamide peptide precursor belongs to the PQRFamide peptide group. In situ hybridization demonstrated that lamprey PQRFamide peptide mRNA is expressed in the regions predicted to be involved in neuroendocrine and behavioral functions. This is the first demonstration of the presence of RFamide peptides in the agnathan brain. Lamprey PQRFamide peptides are considered to have retained the most ancestral features of PQRFamide peptides.     

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.     

Molecular evolution and functional characterization of the orexigenic peptide 26RFa and its receptor in vertebrates

Several neuropeptides possessing the RFamide motif at their C-termini (designated RFamide peptides) have been characterized in the hypothalamus of a variety of vertebrates. To date, five groups of the RFamide peptide family have been shown to exert several important neuroendocrine, behavioral, sensory, and autonomic functions. Since the discovery of the 26-amino acid RFamide peptide (termed 26RFa) from the frog brain, 26RFa has been shown to exert orexigenic activity in mammals and to be a ligand of the previously identified orphan G-protein-coupled receptor GPR103. Recently, 26RFa and its cognate receptor GPR103 have been identified in the brain of birds. This mini-review summarizes the advances in the identification, localization, and functions of 26RFa and its cognate receptor GPR103 in vertebrates and highlights recent progress made in birds.     

Possible inhibitory role of prolactin-releasing peptide for ACTH release associated with running stress

Exercise around the lactate threshold induces a stress response, defined as "running stress." We have previously demonstrated that running stress is associated with activation of certain regions of the brain, e.g., the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus, that are hypothesized to play an integral role in regulating stress-related responses, including ACTH release during running. Thus we investigated the role of prolactin-releasing peptide (PrRP), found in the ventrolateral medulla and the nucleus of the solitary tract, which is known to project to the PVN during running-induced ACTH release. Accumulation of c-Fos in PrRP neurons correlated with running speeds, reaching maximal levels under running stress. Intracerebroventricular injection of neutralizing anti-PrRP antibodies led to increased plasma ACTH level and blood lactate accumulation during running stress, but not during restraint stress. Exogenous intracerebroventricular administration of low doses of PrRP had the opposite effects. Therefore, our results suggest that, during running stress, PrRP-containing neurons are activated in an exercise intensity-dependent manner, and likewise the produced endogenous PrRP attenuates ACTH release and blood lactate accumulation during running stress. Here we provide a novel perspective on understanding of PrRP in the endocrine-metabolic response associated with running stress.     

Neuronal effects of orexins: relevant to sympathetic and cardiovascular functions

Orexin A and B, also called hypocretin 1 and 2, were recently discovered in the hypothalamus. This organ, in which a number of neuropeptides have been demonstrated to stimulate or suppress food intake, is considered important for the regulation of appetite and energy homeostasis. Orexins were initially reported as a regulator of food intake. More recent reports suggest their possible important roles in the multiple functions of neuronal systems, such as narcolepsy, a sleep disorder. Orexins and their receptors are distributed in neural tissue and brain regions involved in the autonomic and neuroendocrine control. Functional studies have shown that these peptides evoke changes in cardiovascular and sympathetic responses. The data from our in vivo and in vitro studies suggest that the peptide acting on neurons in the hypothalamic paraventricular nucleus increases the cardiovascular responses. This review will focus on the neural effects of orexins and how these peptides may participate in the regulation of cardiovascular and sympathetic functions.     

RFamide Peptides in the Leech, Hirudo medicinalis

RFamide peptides have been localized to a number of neuronsof the CNS of the leech, Hirudo medicinalis, using immunocytochemicaltechniques. The majority of this immunoreactivity appears tobe due to the peptide FMRFamide. Most of the identified RFamideimmunoreactive cells are cholinergic motor neurons, though someare interneurons. Superfused FMRFamide is active on the targetsof these identified neurons; in a few well studied cases, ithas been possible to show that FMRFamide mimics a specific physiologicalaction of an identified neuron on its target. In the leech as in other phyla where they occur, RFamide peptidesare widely distributed in neurons, and are neuromodulators withdiverse physiological effects.     

Evolutionary Origin of GnIH and NPFF in Chordates: Insights from Novel Amphioxus RFamide Peptides

Gonadotropin-inhibitory hormone (GnIH) is a newly identified hypothalamic neuropeptide that inhibits pituitary hormone secretion in vertebrates. GnIH has an LPXRFamide (X = L or Q) motif at the C-terminal in representative species of gnathostomes. On the other hand, neuropeptide FF (NPFF), a neuropeptide characterized as a pain-modulatory neuropeptide, in vertebrates has a PQRFamide motif similar to the C-terminal of GnIH, suggesting that GnIH and NPFF have diverged from a common ancestor. Because GnIH and NPFF belong to the RFamide peptide family in vertebrates, protochordate RFamide peptides may provide important insights into the evolutionary origin of GnIH and NPFF. In this study, we identified a novel gene encoding RFamide peptides and two genes of their putative receptors in the amphioxus Branchiostoma japonicum. Molecular phylogenetic analysis and synteny analysis indicated that these genes are closely related to the genes of GnIH and NPFF and their receptors of vertebrates. We further identified mature RFamide peptides and their receptors in protochordates. The identified amphioxus RFamide peptides inhibited forskolin induced cAMP signaling in the COS-7 cells with one of the identified amphioxus RFamide peptide receptors expressed. These results indicate that the identified protochordate RFamide peptide gene is a common ancestral form of GnIH and NPFF genes, suggesting that the origin of GnIH and NPFF may date back to the time of the emergence of early chordates. GnIH gene and NPFF gene may have diverged by whole-genome duplication in the course of vertebrate evolution.     

A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry

Recently, cDNAs encoding novel RFamide-related peptides (RFRPs) have been reported in the mammalian brains by a gene database search and the deduced RFRPs have been suggested to participate in neuroendocrine and pain mechanisms in the rat. Two peptides have been predicted to be encoded in the cDNA of rodent RFRPs. To assess precise functions of rodent RFRPs in the brain, in the present study we identified a naturally occurring RFRP in the rat hypothalamus by immunoaffinity purification combined with mass spectrometry (MS). The affinity chromatography showed that the rat hypothalamus contained RFRP-like immunoreactivity. The immunoreactive material was analyzed by a nanoflow electrospray ionization time-of-flight MS followed by tandem MS analysis. The mass peak corresponding to octadecapeptide was detected at 1010.54 m/z ([M+2H](2+)) and its sequence, ANMEAGTMSHFPSLPQRF-NH(2), was revealed by the fragmentation, showing a mature form encoded in the cDNA sequence of RFRPs. The identified endogenous RFRP will aid not only in defining its physiological roles but also facilitate the development of its agonists and antagonists in the rodent brain.     

The action of RFamide neuropeptides on molluscs, with special reference to the gastropods Buccinum undatum and Busycon canaliculatum

The ever-growing RFamide neuropeptide superfamily has members in all animal phyla. Their effects in molluscs, on both smooth and cardiac muscle as well as on neurons, has been studied in detail. These neuropeptides exert a variety of functions: excitatory, inhibitory or even biphasic. Firstly, the literature on the excitatory effect of the RFamide neuropeptides on molluscan muscle and neurons has been reviewed, with greater emphasis and examples from the gastropods Buccinum undatum and Busycon canaliculatum. The peptides seem to be potent activators of contraction, sometimes generating slow tonic force and other times twitch activity. Secondly, the literature on the inhibitory effect of the superfamily has been reviewed. These peptides can exert an inhibitory effect, hyperpolarizing the cells rather than depolarizing them. Thirdly, the neuropeptides may play a variety of other roles, such as contributing to the regulation or maturation process of the animals. There have been cases recorded of RFamide neuropeptides acting as potent venoms in members of the Conus sp. The pathway of action of these multiple roles, their interaction with the parent neurotransmitters acetylcholine and serotonin, as well as the calcium dependency of the RFamide neuropeptides has been discussed, again with special reference to the above mentioned gastropods. A better understanding of the mode of action, the effects, and the importance of the RFamide neuropeptides on molluscan physiology and pharmacology has been attempted by reviewing the existing literature, recognizing the importance of the RFamide neuropeptide actions on molluscs.     

Disrupted organization of RFamide pathways in the hypothalamus is associated with advanced puberty in female rats neonatally exposed to bisphenol A

Hypothalamic neurons, which produce the kisspeptin family of peptide hormones (Kp), are critical for initiating puberty and maintaining estrous cyclicity by stimulating gonadotropin-releasing hormone (GnRH) release. Conversely, RFamide-related peptide-3 (RFRP3) neurons inhibit GnRH activity. It has previously been shown that neonatal exposure to bisphenol A (BPA) can alter the timing of female pubertal onset and induce irregular estrous cycles or premature anestrus. Here we tested the hypothesis that disrupted ontogeny of RFamide signaling pathways may be a mechanism underlying advanced puberty. To test this, we used a transgenic strain of Wistar rats whose GnRH neurons express enhanced green fluorescent protein. Pups were exposed by daily subcutaneous injection to vehicle, 17beta-estradiol (E2), 50 μg/kg BPA, or 50 mg/kg BPA, from Postnatal Day (PND) 0 through PND 3, and then cohorts were euthanized on PNDs 17, 21, 24, 28, and 33 (5-8 animals per age per exposure; males were collected on PNDs 21 and 33). Vaginal opening was advanced by E2 and 50 μg/kg BPA. On PND 28, females exposed to E2 and 50 μg/kg BPA had decreased RFRP-3 fiber density and contacts on GnRH neurons. RFRP3 perikarya were also decreased in females exposed to 50 μg/kg BPA. Data suggest that BPA-induced premature puberty results from decreased inhibition of GnRH neurons.     

Identification of the prolactin-releasing peptide-producing cell in the rat adrenal gland

Prolactin-releasing peptide (PrRP) is a novel peptide found in bovine hypothalamus as an endogenous ligand of an orphan G-protein-coupled receptor (hGR3). It is known that PrRP is widely distributed and plays roles in the central nervous system (CNS). In particular, PrRP acts as a neurotransmitter that mediates stress and activates the hypothalamo-pituitary-adrenal axis. On the other hand, only a few studies have so far been performed on PrRP in peripheral tissues. Among peripheral tissues, appreciable levels of PrRP are found only in the adrenal gland; however, the PrRP-producing cells in the adrenal gland have not been identified. In this study, we detected PrRP mRNA in the rat adrenal medulla. So, we tried to identify the PrRP-producing cells in primary culture cells of the adrenal medulla. We found immunopositive PrRP cells among the cultured cells from the adrenal gland, but not in the adrenal gland tissue, by means of immunocytochemistry. The PrRP immunopositive cells were double positive for tyrosine hydroxylase (TH) and for phenylethanolamine N-methyltransferase (PNMT), which indicates that PrRP may be produced in a part of the adrenaline cells in the adrenal gland. This is the first report that PrRP is produced in the adrenaline-containing cells of the adrenal gland.     

食欲素神经肽在应激过程中的作用

食欲素因其在调节能量代谢、睡眠和唤醒等生理功能中的作用而备受关注.近年来研究逐渐发现,食欲素参与应激和奖赏过程的调节,特别是其在药物成瘾过程中的作用是目前的研究热点.主要介绍食欲素系统与应激相关系统之间的神经联系,阐述了其在应激相关的生理、神经内分泌与行为反应中的作用.并进一步介绍了食欲素系统在应激诱发药物成瘾复吸过程中的作用.食欲素对应激反应的调控作用具有相对特异性,受应激的种类、其他应激相关神经递质系统及食欲素神经元的投射通路等多种因素影响.     

Biological properties of prolactin-releasing peptide analogs with a modified aromatic ring of a C-terminal phenylalanine amide

Prolactin-releasing peptide (PrRP)-induced secretion of prolactin is not currently considered a primary function of PrRP, but the development of late-onset obesity in both PrRP and PrRP receptor knock-out mice indicates the unique anorexigenic properties of PrRP. In our recent study, we showed comparable potencies of peptides PrRP31 and PrRP20 in binding, intracellular signaling and prolactin release in pituitary RC-4B/C cells, and anorexigenic effect after central administration in fasted mice. In the present study, eight analogs of PrRP20 with C-terminal Phe amide modified with a bulky side chain or a halogenated aromatic ring revealed high binding potency, activation of mitogen-activated protein kinase/extracellular-regulated kinase (MAPK/ERK1/2) and cAMP response element-binding protein (CREB) and prolactin release in RC-4B/C cells. In particular, [PheNO₂³¹]PrRP20, [1-Nal³¹]PrRP20, [2-Nal³¹]PrRP20 and [Tyr³¹]PrRP20 showed not only in vitro effects comparable or higher than those of PrRP20, but also a very significant and long-lasting anorexigenic effect after central administration in fasted mice. The design of potent and long-lasting PrRP analogs with selective anorexigenic properties promises to contribute to the study of food intake disorders.     

Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons

Abstract: Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI-MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI- MS study of direct peptide fingerprinting in the single neuron these experients demonstrate that MALDI-MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single-cell studies in vertebrates, including humans.     

Repeated administration of the anorectic factor prolactin-releasing peptide leads to tolerance to its effects on energy homeostasis

Central administration of a single dose of prolactin-releasing peptide (PrRP) causes a reduction in both fast-induced and nocturnal food intake and body weight gain. The aim of this study was to examine the effect of repeated administration of PrRP on energy homeostasis, including a measure of the expression of the mitochondrial uncoupling protein-1 (UCP-1) in brown adipose tissue. Conscious, free-feeding animals received central injections of PrRP (4 nmol icv) or vehicle. A single injection at 1000 caused a sustained hyperthermia over the 4-h test period and an increase in the expression of UCP-1 mRNA. Repeated, twice daily injection caused a reduction in body weight gain greater than that seen in pair-fed animals for the first 48-72 h. After 72 h, the animals became refractory to the actions of PrRP. The pair-fed group showed a reduction in UCP-1 mRNA expression at 48 h, which was reversed by PrRP treatment. This study indicates that PrRP exerts its effects on energy homeostasis in the short-medium term by reducing food intake and increasing energy expenditure.