The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.

Molecular evolution of GPCRs: 26Rfa/GPR103.

Neuropeptides possessing the Arg-Phe-NH2 (RFamide) motif at their C-termini (designated as RFamide peptides) have been characterized in a variety of animals. Among these, neuropeptide 26RFa (also termed QRFP) is the latest member of the RFamide peptide family to be discovered in the hypothalamus of vertebrates. The neuropeptide 26RFa/QRFP is a 26-amino acid residue peptide that was originally identified in the frog brain. It has been shown to exert orexigenic activity in mammals and to be a ligand for the previously identified orphan G protein-coupled receptor, GPR103 (QRFPR). The cDNAs encoding 26RFa/QRFP and QRFPR have now been characterized in representative species of mammals, birds, and fish. Functional studies have shown that, in mammals, the 26RFa/QRFP-QRFPR system may regulate various functions, including food intake, energy homeostasis, bone formation, pituitary hormone secretion, steroidogenesis, nociceptive transmission, and blood pressure. Several biological actions have also been reported in birds and fish. This review summarizes the current state of identification, localization, and understanding of the functions of 26RFaQRFP and its cognate receptor, QRFPR, in vertebrates.

Identification, localization and function of a novel neuropeptide, 26RFa, and its cognate receptor, GPR103, in the avian hypothalamus.

Several neuropeptides possessing the RFamide motif at their C-termini (designated RFamide peptides) have been characterized in the hypothalamus of a variety of vertebrates. 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, we have identified 26RFa in the avian brain by molecular cloning of the cDNA encoding the 26RFa precursor and mass spectrometry analysis of the mature peptide. 26RFa-producing neurons are exclusively located in the hypothalamus whereas GPR103 is widely distributed in the avian brain. Furthermore, avian 26RFa stimulates feeding behavior in broiler chicks. This 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.

Gonadotropin-releasing hormone stimulates the biosynthesis of pregnenolone sulfate and dehydroepiandrosterone sulfate in the hypothalamus.

The sulfated neurosteroids pregnenolone sulfate (Δ(5)PS) and dehydroepiandrosterone sulfate (DHEAS) are known to play a role in the control of reproductive behavior. In the frog Pelophylax ridibundus, the enzyme hydroxysteroid sulfotransferase (HST), responsible for the biosynthesis of Δ(5)PS and DHEAS, is expressed in the magnocellular nucleus and the anterior preoptic area, two hypothalamic regions that are richly innervated by GnRH1-containing fibers. This observation suggests that GnRH1 may regulate the formation of sulfated neurosteroids to control sexual activity. Double labeling of frog brain slices with HST and GnRH1 antibodies revealed that GnRH1-immunoreactive fibers are located in close vicinity of HST-positive neurons. The cDNAs encoding 3 GnRH receptors (designated riGnRHR-1, -2, and -3) were cloned from the frog brain. RT-PCR analyses revealed that riGnRHR-1 is strongly expressed in the hypothalamus and the pituitary whereas riGnRHR-2 and -3 are primarily expressed in the brain. In situ hybridization histochemistry indicated that GnRHR-1 and GnRHR-3 mRNAs are particularly abundant in preoptic area and magnocellular nucleus whereas the concentration of GnRHR-2 mRNA in these 2 nuclei is much lower. Pulse-chase experiments using tritiated Δ(5)P and DHEA as steroid precursors, and 3'-phosphoadenosine 5'-phosphosulfate as a sulfonate moiety donor, showed that GnRH1 stimulates, in a dose-dependent manner, the biosynthesis of Δ(5)PS and DHEAS in frog diencephalic explants. Because Δ(5)PS and DHEAS, like GnRH, stimulate sexual activity, our data strongly suggest that some of the behavioral effects of GnRH could be mediated via the modulation of sulfated neurosteroid production.

Gliotransmission and brain glucose sensing: critical role of endozepines.

Hypothalamic glucose sensing is involved in the control of feeding behavior and peripheral glucose homeostasis, and glial cells are suggested to play an important role in this process. Diazepam-binding inhibitor (DBI) and its processing product the octadecaneuropeptide (ODN), collectively named endozepines, are secreted by astroglia, and ODN is a potent anorexigenic factor. Therefore, we investigated the involvement of endozepines in brain glucose sensing. First, we showed that intracerebroventricular administration of glucose in rats increases DBI expression in hypothalamic glial-like tanycytes. We then demonstrated that glucose stimulates endozepine secretion from hypothalamic explants. Feeding experiments indicate that the anorexigenic effect of central administration of glucose was blunted by coinjection of an ODN antagonist. Conversely, the hyperphagic response elicited by central glucoprivation was suppressed by an ODN agonist. The anorexigenic effects of centrally injected glucose or ODN agonist were suppressed by blockade of the melanocortin-3/4 receptors, suggesting that glucose sensing involves endozepinergic control of the melanocortin pathway. Finally, we found that brain endozepines modulate blood glucose levels, suggesting their involvement in a feedback loop controlling whole-body glucose homeostasis. Collectively, these data indicate that endozepines are a critical relay in brain glucose sensing and potentially new targets in treatment of metabolic disorders.

Organization of two independent kisspeptin systems derived from evolutionary-ancient kiss genes in the brain of zebrafish.

Kisspeptins are new actors in the neuroendocrine regulation of reproduction. In vertebrates, the number of kiss genes varies from none to three. Zebrafish have two kiss genes, kiss1 and kiss2, and two kiss receptors (GPR54), kiss1r and kiss2r. To provide detailed information on the organization of the kiss systems in zebrafish, antibodies were raised against the C terminus of zebrafish preproKiss1 and preproKiss2. Immunohistochemistry fully confirmed in situ hybridization data, showing that kiss1-expressing neurons are only located in the habenular nucleus, while kiss2-expressing neurons are found in the dorsal and ventral hypothalamus. Kiss1-expressing cells project only to the interpeduncular and raphe nuclei and strongly expressed the kiss1r receptor. In contrast, kiss2-expressing cells are mostly present in the dorsal and ventral hypothalamus and project widely into the subpallium, the preoptic area, the thalamus, the ventral and caudal hypothalamus, and the mesencephalon. All these regions strongly expressed the kiss2r messengers. Kiss2 fibers profusely innervate the ventral forebrain and notably made close apposition with GnRH3 neurons. Estrogen treatment of juvenile fish with estradiol causes increase in kiss2 and kiss2r expression. In the pituitary gland, no proKiss2- positive fibers were detected, while positive cells were observed in the pars intermedia. In addition to proposing a successful strategy to develop antibodies to kisspeptins, these data indicate that the kiss2 systems of zebrafish are implicated in reproductive events, while the kiss1 gene would play other functions that remain to be established.

An indispensable role of secretin in mediating the osmoregulatory functions of angiotensin II.

Fluid balance is critical to life and hence is tightly controlled in the body. Angiotensin II (ANGII), one of the most important components of this regulatory system, is recognized as a dipsogenic hormone that stimulates vasopressin (VP) expression and release. However, detailed mechanisms regarding how ANGII brings about these changes are not fully understood. In the present study, we show initially that the osmoregulatory functions of secretin (SCT) in the brain are similar to those of ANGII in mice and, more important, we discovered the role of SCT as the link between ANGII and its downstream effects. This was substantiated by the use of two knockout mice, SCTR(-/-) and SCT(-/-), in which we show the absence of an intact SCT/secretin receptor (SCTR) axis resulted in an abolishment or much reduced ANGII osmoregulatory functions. By immunohistochemical staining and in situ hybridization, the proteins and transcripts of SCT and its receptor are found in the paraventricular nucleus (PVN) and lamina terminalis. We propose that SCT produced in the circumventricular organs is transported and released in the PVN to stimulate vasopressin expression and release. In summary, our findings identify SCT and SCTR as novel elements of the ANGII osmoregulatory pathway in maintaining fluid balance in the body.

Identification, localization, and function of a novel avian hypothalamic neuropeptide, 26RFa, and its cognate receptor, G protein-coupled receptor-103.

Several neuropeptides with the C-terminal RFamide sequence have been identified in the hypothalamus of a variety of vertebrates. Among the RFamide peptide groups, however, only LPXRFamide peptides, including gonadotropin-inhibitory hormone, have been characterized in the avian brain. In the present study, we sought for the presence of other RFamide peptides in the avian hypothalamus. We identified a cDNA encoding an RFamide peptide orthologous to 26RFa (also referred to as QRFP) in the hypothalamus of the Japanese quail. The deduced quail 26RFa precursor consisted of 120-amino-acid residues, encoding one RFamide peptide with 27 amino acids. This RFamide peptide was flanked at the N terminus by a dibasic amino acid cleavage site and at the C terminus by a glycine amidation signal. Quantitative RT-PCR analysis demonstrated specific expression of quail 26RFa mRNA in the diencephalon including the hypothalamus. Furthermore, mass spectrometry analysis revealed the presence of a peptide exhibiting the mass of mature 26RFa, indicating that the peptide is actually produced from the precursor in the diencephalon. 26RFa-producing cell bodies were localized in the anterior hypothalamic nucleus in the brain. Synthetic 26RFa increased intracellular Ca(2+) concentration in HEK293T cells transfected with the chicken G protein-coupled receptor GPR103. Intracerebroventricular injection of 26RFa in broiler chicks stimulated feeding behavior. These data provide the first evidence for the occurrence of the peptide 26RFa in the avian hypothalamus and indicate that this peptide exerts orexigenic activity.

Neuroanatomical distribution of MCH in the brain and pituitary of submammalian vertebrates.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that has been initially characterized from a salmon pituitary extract and subsequently identified in various species from all classes of vertebrates. The present review summarizes the current knowledge regarding the neuroanatomical distribution of MCH-immunoreactive neurons in submammalian vertebrates. In all species examined, MCH-immunoreactive perikarya are confined to the hypothalamus, with the exception of the cyclostome Lampetra fluvialis and the lungfish Protopterus annectens, in which additional populations of MCH-immunoreactive cell bodies occur in the telencephalon, and the frogs Rana ridibunda and Rana esculenta which exhibit MCH-positive perikarya in thalamic nuclei. In teleosts, in the frog R. ridibunda and in the L. fluvialis, MCH is present in the classical hypothalamic-neurohypophysial system indicating that the peptide may play the role of a neurohormone. In other groups, MCH-immunoreactive nerve fibers are widely distributed in various brain regions suggesting that, in these species, MCH in the central nervous system may act as a neurotransmitter or/and a neuromodulator rather than a neurohormone.

Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates.

Kisspeptin and its receptor GPR54 play important roles in mammalian reproduction and cancer metastasis. Because the KiSS and GPR54 genes have been identified in a limited number of vertebrate species, mainly in mammals, the evolutionary history of these genes is poorly understood. In the present study, we have cloned multiple forms of kisspeptin and GPR54 cDNAs from a variety of vertebrate species. We found that fish have two forms of kisspeptin genes, KiSS-1 and KiSS-2, whereas Xenopus possesses three forms of kisspeptin genes, KiSS-1a, KiSS-1b, and KiSS-2. The nonmammalian KiSS-1 gene was found to be the ortholog of the mammalian KiSS-1 gene, whereas the KiSS-2 gene is a novel form, encoding a C-terminally amidated dodecapeptide in the Xenopus brain. This study is the first to identify a mature form of KiSS-2 product in the brain of any vertebrate. Likewise, fish possess two receptors, GPR54-1 and GPR54-2, whereas Xenopus carry three receptors, GPR54-1a, GPR54-1b, and GPR54-2. Sequence identity and genome synteny analyses indicate that Xenopus GPR54-1a is a human GPR54 ortholog, whereas Xenopus GPR54-1b is a fish GPR54-1 ortholog. Both kisspeptins and GPR54s were abundantly expressed in the Xenopus brain, notably in the hypothalamus, suggesting that these ligand-receptor pairs have neuroendocrine and neuromodulatory roles. Synthetic KiSS-1 and KiSS-2 peptides activated GPR54s expressed in CV-1 cells with different potencies, indicating differential ligand selectivity. These data shed new light on the molecular evolution of the kisspeptin-GPR54 system in vertebrates.

Pituitary adenylate cyclase-activating polypeptide inhibits food intake in mice through activation of the hypothalamic melanocortin system.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and the proopiomelanocortin (POMC)-derived peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), exert anorexigenic activities. While alpha-MSH is known to inhibit food intake and stimulate catabolism via activation of the central melanocortin-receptor MC4-R, little is known regarding the mechanism by which PACAP inhibits food consumption. We have recently found that, in the arcuate nucleus of the hypothalamus, a high proportion of POMC neurons express PACAP receptors. This observation led us to investigate whether PACAP may inhibit food intake through a POMC-dependent mechanism. In mice deprived of food for 18 h, intracerebroventricular administration of PACAP significantly reduced food intake after 30 min, and this effect was reversed by the PACAP antagonist PACAP6-38. In contrast, vasoactive intestinal polypeptide did not affect feeding behavior. Pretreatment with the MC3-R/MC4-R antagonist SHU9119 significantly reduced the effect of PACAP on food consumption. Central administration of PACAP induced c-Fos mRNA expression and increased the proportion of POMC neuron-expressing c-Fos mRNA in the arcuate nucleus. Furthermore, PACAP provoked an increase in POMC and MC4-R mRNA expression in the hypothalamus, while MC3-R mRNA level was not affected. POMC mRNA level in the arcuate nucleus of PACAP-specific receptor (PAC1-R) knock-out mice was reduced as compared with wild-type animals. Finally, i.c.v. injection of PACAP provoked a significant increase in plasma glucose level. Altogether, these results indicate that PACAP, acting through PAC1-R, may inhibit food intake via a melanocortin-dependent pathway. These data also suggest a central action of PACAP in the control of glucose metabolism.

Novel stable PACAP analogs with potent activity towards the PAC1 receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala15, Ala20]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala15, Ala20]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.

Structure-activity relationships of a series of analogs of the endozepine octadecaneuropeptide (ODN(11)(-)(18)) on neurosteroid biosynthesis by hypothalamic explants.

We have previously shown that the endozepine octadecaneuropeptide (ODN) stimulates the biosynthesis of neurosteroids from frog hypothalamic explants. In the present study, we have investigated the structure-activity relationships of a series of analogs of the C-terminal octapeptide of ODN (OP) on neurosteroid formation. We found that OP and its cyclic analog cyclo1-8OP stimulate in a concentration-dependent manner the synthesis of various steroids including 17-hydroxypregnenolone, progesterone, 17-hydroxyprogesterone and dehydroepiandrosterone. Deletion or Ala-substitution of the Arg1 or Pro2 residues of OP did not affect the activity of the peptide. In contrast, deletion or replacement of any of the amino acids of the C-terminal hexapeptide fragment totally abolished the effect of OP on neurosteroid biosynthesis. The present study indicates that the C-terminal hexapeptide of ODN/OP is the minimal sequence retaining full biological activity on steroid-producing neurons.

Immunohistochemical distribution of the secretogranin II-derived peptide EM66 in the rat hypothalamus: a comparative study with jerboa.

EM66 is a 66-amino acid peptide derived from secretogranin II, a member of granin acidic secretory protein family, by proteolytic processing. EM66 has been previously characterized in the jerboa (Jaculus orientalis) hypothalamus and its potential implication in the neuroendocrine regulation of feeding behaviour has been demonstrated. In the present study, an immunohistochemical analysis of the localization of EM66 within hypothalamic structures of rat was performed and compared to the distribution of EM66 in the jerboa hypothalamus. In the rat hypothalamus, as in the jerboa, EM66 immunostaining was detected in the parvocellular paraventricular, preoptic and arcuate nuclei, as well as the lateral hypothalamus which displayed an important density of EM66-producing neurones. However, unlike jerboa, the suprachiasmatic and supraoptic nuclei of the rat hypothalamus were devoid of cellular EM66-immunolabeling. Thus, the novel peptide EM66 may exert common neuroendocrine activities in rat and jerboa, e.g. control of food intake, and species-specific roles in jerboa such as the regulation of biological rhythms and hydromineral homeostasis. These results suggest the existence of differences between jerboas and rats in neuroendocrine regulatory mechanisms involving EM66.

Pharmacological characterization of the receptor mediating the anorexigenic action of the octadecaneuropeptide: evidence for an endozepinergic tone regulating food intake.

Peptides of the endozepine family, including diazepam-binding inhibitor, the triakontatetraneuropeptide, and the octadecaneuropeptide (ODN), act through three types of receptors, that is, central-type benzodiazepine receptors (CBR), peripheral-type (mitochondrial) benzodiazepine receptors (PBR) and a metabotropic receptor positively coupled to phospholipase C via a pertussis toxin-sensitive G protein. We have previously reported that ODN exerts a potent anorexigenic effect in rat and we have found that the action of ODN is not affected by the mixed CBR/PBR agonist diazepam. In the present report, we have tested the possible involvement of the metabotropic receptor in the anorexigenic activity of ODN. Intracerebroventricular administration of the C-terminal octapeptide (OP) and its head-to-tail cyclic analog cyclo(1-8)OP (cOP) at a dose of 100 ng mimicked the inhibitory effect of ODN on food intake in food-deprived mice. The specific CBR antagonist flumazenil and the PBR antagonist PK11195 did not prevent the effect of ODN, OP, and cOP on food consumption. In contrast, the selective metabotropic endozepine receptor antagonist cyclo(1-8)[DLeu(5)]OP (100-1000 ng; cDLOP) suppressed the anorexigenic effect of ODN, OP, and cOP. At the highest concentration tested (1000 ng), cDLOP provoked by itself a significant increase in food intake. Taken together, the present results indicate that the anorexigenic effect of ODN and OP is mediated through activation of the metabotropic receptor recently characterized in astrocytes. The data also suggest that endogenous ODN, acting via this receptor, exerts an inhibitory tone on feeding behavior.

Anatomical distribution and biochemical characterization of the novel RFamide peptide 26RFa in the human hypothalamus and spinal cord.

26RFa is a novel RFamide peptide originally isolated in the amphibian brain. The 26RFa precursor has been subsequently characterized in various mammalian species but, until now, the anatomical distribution and the molecular forms of 26RFa produced in the CNS of mammals, in particular in human, are unknown. In the present study, we have investigated the localization and the biochemical characteristics of 26RFa-like immunoreactivity (LI) in two regions of the human CNS--the hypothalamus and the spinal cord. Immunohistochemical labeling using specific antibodies against human 26RFa and in situ hybridization histochemistry revealed that in the human hypothalamus 26RFa-expressing neurons are located in the paraventricular and ventromedial nuclei. In the spinal cord, 26RFa-expressing neurons were observed in the dorsal and lateral horns. Characterization of 26RFa-related peptides showed that two distinct molecular forms of 26RFa are present in the human hypothalamus and spinal cord, i.e. 26RFa and an N-terminally elongated form of 43 amino acids designated 43RFa. These data provide the first evidence that 26RFa and 43RFa are actually produced in the human CNS. The distribution of 26RF-LI suggests that 26RFa and/or 43RFa may modulate feeding, sexual behavior and transmission of nociceptive stimuli.

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.

Phenylpyrroles, a new chemolibrary virtual screening class of 5-HT7 receptor ligands.

Virtual screening studies have identified a series of phenylpyrroles as novel 5-HT7 receptor ligands. The synthesis and the affinity for the 5-HT7 receptor of these phenylpyrroles are described. Some of these compounds exhibited high affinity for the 5-HT7 receptors.

Identification of 26RFa from frog brain: a novel hypothalamic neuropeptide with orexigenic activity in mammals.

In the present study, we report the identification, in the frog brain, of a novel neuropeptide, termed 26RFa, that belongs to the RFamide peptide family. The cDNAs encoding the precursors for 26RFa have been characterized in human and rats. In rats, prepro-26RFa mRNA is expressed exclusively in two hypothalamic nuclei involved in the control of feeding behavior. Intracerebroventricular injection of 26RFa in mice induced a dose-dependent increase in food consumption. Taken together, these data indicate that 26RFa is a novel neuropeptide that may have important biological functions in vertebrates.

Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release.

Apelin, a recently isolated neuropeptide that is expressed in the supraoptic and the paraventricular nuclei, acts on specific receptors located on vasopressinergic neurons. The increased phasic pattern of these neurons facilitates sustained antidiuresis during dehydration or lactation. Here, we investigated whether apelin interacts with arginine vasopressin (AVP) to maintain body fluid homeostasis. We first characterized the predominant molecular forms of endogenous hypothalamic and plasma apelin as corresponding to apelin 13 and, to a lesser extent, to apelin 17. We then demonstrated that, in lactating rats, apelin was colocalized with AVP in supraoptic nucleus magnocellular neurons and given intracerebroventricularly inhibited the phasic electrical activity of AVP neurons. In lactating mice, intracerebroventricular administration of apelin 17 reduced plasma AVP levels and increased diuresis. Moreover, water deprivation, which increases systemic AVP release and causes depletion of hypothalamic AVP stores, decreased plasma apelin concentrations and induced hypothalamic accumulation of the peptide, indicating that AVP and apelin are conversely regulated to facilitate systemic AVP release and suppress diuresis. Opposite effects of AVP and apelin are likely to occur at the hypothalamic level through autocrine modulation of the phasic electrical activity of AVP neurons. Altogether, these data demonstrate that apelin acts as a potent diuretic neuropeptide counteracting AVP actions through inhibition of AVP neuron activity and AVP release. The coexistence of apelin and AVP in magnocellular neurons, their opposite biological effects, and regulation are likely to play a key role for maintaining body fluid homeostasis.