Discovery of gonadotropin-inhibitory hormone (GnIH), progress in GnIH research on reproductive physiology and behavior and perspective of GnIH research on neuroendocrine regulation of reproduction.

Based on extensive studies on gonadotropin-releasing hormone (GnRH) it was assumed that GnRH is the only hypothalamic neurohormone regulating gonadotropin release in vertebrates. In 2000, however, Tsutsui's group discovered gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide that inhibits gonadotropin release, in quail. Subsequent studies by Tsutsui's group demonstrated that GnIH is conserved among vertebrates, acting as a new key neurohormone regulating reproduction. GnIH inhibits gonadotropin synthesis and release through actions on gonadotropes and GnRH neurons via GnIH receptor, GPR147. Thus, GnRH is not the sole hypothalamic neurohormone controlling vertebrate reproduction. The following studies by Tsutsui's group have further demonstrated that GnIH has several important functions in addition to the control of reproduction. Accordingly, GnIH has drastically changed our understanding about reproductive neuroendocrinology. This review summarizes the discovery of GnIH, progress in GnIH research on reproductive physiology and behavior and perspective of GnIH research on neuroendocrine regulation of reproduction.

Phoenixin-14: detection and novel physiological implications in cardiac modulation and cardioprotection.

Phoenixin-14 (PNX) is a newly identified peptide co-expressed in the hypothalamus with the anorexic and cardioactive Nesfatin-1. Like Nesfatin-1, PNX is able to cross the blood-brain barrier and this suggests a role in peripheral modulation. Preliminary mass spectrography data indicate that, in addition to the hypothalamus, PNX is present in the mammalian heart. This study aimed to quantify PNX expression in the rat heart, and to evaluate whether the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). By ELISA the presence of PNX was detected in both hypothalamus and heart. In plasma of normal, but not of obese rats, the peptide concentrations increased after meal. Exposure of the isolated and Langendorff perfused rat heart to exogenous PNX induces a reduction of contractility and relaxation, without effects on coronary pressure and heart rate. As revealed by immunoblotting, these effects were accompanied by an increase of Erk1/2, Akt and eNOS phosphorylation. PNX (EC50 dose), administered after ischemia, induced post-conditioning-like cardioprotection. This was revealed by a smaller infarct size and a better systolic recovery with respect to those detected on hearts exposed to I/R alone. The peptide also activates the cardioprotective RISK and SAFE cascades and inhibits apoptosis. These effects were also observed in the heart of obese rats. Our data provide a first evidence on the peripheral activity of PNX and on its direct cardiomodulatory and cardioprotective role under both normal conditions and in the presence of metabolic disorders.

A novel reproductive peptide, phoenixin.

Normal anterior pituitary function is essential for fertility. Release from the gland of the reproductive hormones luteinising hormone and follicle-stimulating hormone is regulated primarily by hypothalamically-derived gonadotrophin-releasing hormone (GnRH), although other releasing factors (RF) have been postulated to exist. Using a bioinformatic approach, we have identified a novel peptide, phoenixin, that regulates pituitary gonadotrophin secretion by modulating the expression of the GnRH receptor, an action with physiologically relevant consequences. Compromise of phoenixin in vivo using small interfering RNA resulted in the delayed appearance of oestrus and a reduction in GnRH receptor expression in the pituitary. Phoenixin may represent a new class of hypothalamically-derived pituitary priming factors that sensitise the pituitary to the action of other RFs, rather than directly stimulating the fusion of secretary vesicles to pituitary membranes.

A new key neurohormone controlling reproduction, gonadotrophin-inhibitory hormone in birds: discovery, progress and prospects.

In vertebrates, the neuropeptide control of gonadotrophin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotrophin-releasing hormone (GnRH). Gonadal sex steroids and inhibin inhibit gonadotrophin secretion via feedback from the gonads, but a hypothalamic neuropeptide inhibiting gonadotrophin secretion was, until recently, unknown in vertebrates. In 2000, we discovered a novel hypothalamic dodecapeptide that directly inhibits gonadotrophin release in quail and termed it gonadotrophin-inhibitory hormone (GnIH). GnIH acts on the pituitary and GnRH neurones in the hypothalamus via a novel G-protein-coupled receptor for GnIH to inhibit gonadal development and maintenance by decreasing gonadotrophin release and synthesis. The pineal hormone melatonin is a key factor controlling GnIH neural function. GnIH occurs in the hypothalamus of several avian species and is considered to be a new key neurohormone inhibiting avian reproduction. Thus, the discovery of GnIH provides novel directions to investigate neuropeptide regulation of reproduction. This review summarises the discovery, progress and prospects of GnIH, a new key neurohormone controlling reproduction.

Orphan seven transmembrane receptor screening.

Drug discovery has successfully exploited the superfamily of seven transmembrane receptors (7TMR), with over 35% of clinically marketed drugs targeting them. However, it is clear that there remains an undefined potential within this protein family for successful drugs of the future. The human genome sequencing project identified approximately 720 genes that belong to the 7TMR superfamily. Around half of these genes encode sensory receptors, while the other half are potential drug targets. Natural ligands have been identified for approximately 215 of these, leaving 155 receptors classified as orphan 7TMRs having no known ligand. Deorphanisation of these receptors by identification of natural ligands has been the traditional method enabling target validation by use of these ligands as tools to define biological relevance and disease association. Such ligands have been paired with their cognate receptor experimentally by screening of small molecule and peptide ligands, reverse pharmacology and the use of bioinformatics to predict candidate ligands. In this manuscript, we review the methodologies developed for the identification of ligands at orphan 7TMRs and exemplify these with case studies.

A novel amphibian hypothalamic neuropeptide: isolation, localization, and biological activity.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.

A novel avian hypothalamic peptide inhibiting gonadotropin release.

The neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), which was originally isolated from mammals and subsequently from non-mammals. To date, however, an inhibitory peptide of gonadotropin release is unknown in vertebrates. Here we show, in a bird, that the hypothalamus also contains a novel peptide which inhibits gonadotropin release. Acetic acid extracts of quail brains were passed through C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). The peptide was isolated from avian brain and shown to have the sequence Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH(2). Cell bodies and terminals containing this peptide were localized immunohistochemically in the paraventricular nucleus and median eminence, respectively. This peptide inhibited, in a dose-related way, gonadotropin release from cultured quail anterior pituitaries. This is the first hypothalamic peptide inhibiting gonadotropin release reported in a vertebrate. We therefore term it gonadotropin-inhibitory hormone (GnIH).

Multiple prolactin-releasing activity in the bovine hypothalamic extract.

The prolactin (PRL)-releasing activity (PRA) in the bovine hypothalamic extract (BHE) was compared to that of known substances with PRA and further characterized by gel filtration and reversed-phase high performance liquid chromatography (RP-HPLC). Crude BHE produced marked dose-dependent stimulation of PRL secretion from the cultured rat adenohypophysial cells. Among the synthetic substances examined, vasoactive intestinal peptide (VIP), thyrotropin-releasing hormone (TRH) and beta-endorphin (END) showed significant PRA. However, the flatter dose-response slope for TRH compared with BHE or the small amounts of VIP and END in BHE suggested that these peptides could not account for the major active elements of BHE. Oxytocin and interleukin-1beta were also tested, but they exhibited no PRA in our assay system. Gel filtration of BHE on the Sephadex G-100 column yielded two peaks of PRA distinct from TRH, VIP and END. One eluted in the void and the other in more retarded fractions. The latter fractions were pooled and subjected to the two-step RP-HPLC. The PRA was separated into three peaks designated peaks I, II and III in the first RP-HPLC experiment. Furthermore, the second RP-HPLCs with finer resolution revealed that peak II as well as peak III consisted of three peaks, while peak I eluted as a single peak. Most of these seven PRA peaks exhibited different RP-HPLC profiles from those of the newly characterized PRL-releasing peptides. These findings again provide confirmatory evidence that BHE contained unique factors different from the above known substances.

The quest for novel bioactive peptides utilizing orphan seven-transmembrane-domain receptors.

Various sorts of bioactive molecules including hormones, neurotransmitters, and chemokines transmit signals into cells by binding to so-called seven-transmembrane-domain receptors (7TMRs). The recent progress in cDNA and genome DNA analyses has brought the discovery of numerous genes encoding ligand-unknown "orphan" 7TMRs. We have developed a strategy to identify the ligands of orphan 7TMRs by monitoring specific signal transductions induced in cells expressing orphan 7TMRs. Employing this method, we succeeded in identifying the natural ligands of the orphan 7TMRs, hGR3, and APJ. The ligand peptide identified for hGR3 was found to show a specific prolactin release promoting activity in rat anterior pituitary cells in in vitro culture and was therefore named "prolactin-releasing peptide." We named another novel bioactive peptide "apelin," for "APJ endogenous ligand." Although the biological functions of apelin are still under investigation, APJ reportedly acts as a coreceptor in the process of human immunodeficiency virus infection. We believe that the identification of orphan 7TMR ligands will provide clues to reveal the unknown regulatory mechanisms of various physiological phenomena and opportunities for novel drug discovery in the future.

Identification of melanin concentrating hormone (MCH) as the natural ligand for the orphan somatostatin-like receptor 1 (SLC-1).

To identify possible ligands of the orphan somatostatin-like receptor 1 (SLC-1), rat brain extracts were analyzed by using the functional expression system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G protein-gated inwardly rectifying potassium channels (GIRK). A strong inward current was observed with crude rat brain extracts which upon further purification by cation exchange chromatography and high performance liquid chromatography (HPLC) yielded two peptides with a high agonist activity. Mass spectrometry and partial peptide sequencing revealed that one peptide is identical with the neuropeptide melanin concentrating hormone (MCH), the other represents a truncated version of MCH lacking the three N-terminal amino acid residues. Xenopus oocytes expressing the MCH receptor responded to nM concentrations of synthetic MCH not only by the activation of GIRK-mediated currents but also by the induction of Ca(2+) dependent chloride currents mediated by phospholipase C. This indicates that the MCH receptor can couple either to the G(i)- or G(q)-mediated signal transduction pathway, suggesting that MCH may serve for a number of distinct brain functions including food uptake behavior.

Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor.

Melanin-concentrating hormone (MCH), which is an orexigenic peptide, was isolated and identified as the endogenous ligand of the SLC-1 receptor. We established a CHO cell line expressing the rat SLC-1 receptor to search for its endogenous ligand. The extract of rat whole brain showed inhibition of intracellular forskolin-induced cAMP accumulation in rat SLC-1-expressing CHO cells and was purified. Using HPLC purification, we isolated and identified MCH as the endogenous ligand of the SLC-1 receptor. The authentic MCH demonstrated a dose-dependent inhibitory effect on cAMP accumulation in forskolin-stimulated rat and human SLC-1-expressing CHO cells with an EC(50) value of 0.2 nM for both the rat and human SLC-1 receptors. This is the first description of the functional receptor for MCH.

Synthesis of new peptides with prolactin-releasing activity by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction.

A newly isolated peptide from bovine hypothalamus with prolactin-releasing activity (prolactin-releasing peptide; PrRP) was synthesized by a combination of recombinant DNA technology and a cysteine-specific cyanylation reaction, together with rat and human homologs. The peptides were expressed in the form of fusion proteins with basic fibroblast growth factor mutein, which were purified by heparin-affinity chromatography. The fusion proteins were cleaved at the cysteine residues of the junction site by cyanylation, followed by treatment with ammonia for C-terminal amidation. Purification of the resulting crude peptides was performed using chromatography on a gel-filtration column, a cation-exchange column, and a reversed-phase column. As an example, about 90 mg of bovine PrRP (bPrRP) was obtained from 201 of culture bloth. The purified b PrRP showed full biological activities in binding to its receptor expressed on CHO cells and releasing arachidonic acid metabolite from the same cells, while the C-terminal acid form of bPrRP had little of these activities. These results indicate that the C-terminal amide structure is very important for expressing biological activity. The peptides obtained here might be very useful for studies on their biological significance and roles in vivo.

A prolactin-releasing peptide in the brain.

Hypothalamic peptide hormones regulate the secretion of most of the anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin. These peptides do not regulate the secretion of prolactin, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an 'orphan' receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.

Neuro-humoral control of insulin secretion.

Hypothalamic extract from normal rats was shown to contain a principle able to stimulate insulin secretion both in vivo and in vitro. This substance, whose peptidic nature was demonstrated by enzymatic digestion, has a molecular weight of about 1000 Daltons. An active release of this peptide was obtained when incubating hypothalamic fragments under appropriate stimulatory conditions (i.e. KCl 50 mmol l-1 in the incubation medium) (i.e. molecular weight and insulin secretion stimulating activity) similar to the hypothalamic peptide was evidentiated in the plasma of normal rats. Since manipulations of hte hypothalamus (i.e. electrical lesions or stimulations) could respectively decrease or enhance the plasma concentration of this substance, the hypothalamic origin of the insulin secretion stimulating principle present in the plasma was strongly suggested. The possible participation of this hypothalamic peptide to the neural control of insulin secretion is suggested.

Biological and chemical evidence for the existence of a porcine hypothalamic parotid hormone-releasing factor.

Methodology has been developed to achieve partial purification of a parotid hormone-releasing peptide from porcine hypothalamus-thalamus tissue using an in vivo parotid hormone stimulation test in pigs and a dentinal fluid transport stimulation test in rats. The purification steps include: acetone-water extraction of the tissue at pH 5.2, ultrafiltration through Amicon PM10 membrane, size exclusion chromatography on Bio-Gel P2, and open-column reversed-phase chromatography on Lichroprep RP8. A 100-fold increase in specific activity was attained. Intravenous infusion of porcine hypothalamus-thalamus extract stimulates a dentinal fluid transport mechanism in teeth of intact anesthetized rats, and the release of plasma immunoreactive parotid hormone in conscious catheterized pigs. Parotidectomy in both species suppresses the response, suggesting that the expression of parotid hormone-releasing activity requires the integrity of a putative hypothalamus-parotid gland endocrine axis.

Partial purification of a hypothalamic factor that inhibits gonadotropin-releasing hormone-stimulated luteinizing hormone release.

A protein that suppresses GnRH-stimulated LH release from dispersed rat anterior pituitary cells was purified from rat hypothalami by chromatography on Sephadex G-25, carboxymethyl cellulose, and high performance gel permeation. The final increase in inhibitory activity was 214-fold. The inhibitor is a glycoprotein with an apparent mol wt of 12,252 +/- 638, as determined by gel permeation on HPLC and electrophoreses as a monomer after treatment with 8 M urea. The inhibitor has a Stokes radius of 16 A, an S value of 2.14 +/- 0.08, an isoelectric point value near 4.1, and a frictional ratio of 1.05. A preliminary amino acid composition is presented.

Purification of a glucagon releasing factor from the rat hypothalamus.

The purification of a peptide from the rat hypothalamus which is capable of stimulating the release of glucagon from the endocrine pancreas and therefore named glucagon releasing factor (GlRF), is described. Compositional analysis of the product obtained using well established techniques for peptide isolation revealed that GlRF appeared to consist of 30-31 amino acids. The significance of GlRF in the physiological regulation of glucagon secretion is yet to be determined but the existence of this very potent factor supports the concept of a hypothalamic-pancreatic neurohormonal link.

[Bovine epiphyseal lactotropin, biologically and immunochemically related to pituitary prolactin]. / Laktotropin iz épifiza byka, biologicheski i immunokhimicheski rodstvennyki prolaktinu gipofiza.

A substance biologically and immunologically related to the hypophysis lactogenic hormone (HLH) was detected in a purified preparation obtained by extracting the bovine pineal glands (epiphyses) with acidified acetonic saline fractionation, precipitation in isoelectric point and gel filtration. Bioactivity of the pineal gland lactogen was evaluated according to its ability for stimulating the pigeon thyroid gland proliferation. Equal doses of the pineal gland hormone and HLH produced the same biological effect. The lactogen immunochemical properties were studied by gel precipitation in agar gel, immunoelectrophoresis and radioimmunoconcurrent technique. Absolute identity of the hormone immunochemical properties was revealed. Molecular weights of both lactogens, determined by chromatography on Sefadex C-75, were equal. The average HLH content in the organ, determined by radioimmunochemistry, was 4.7 X 10(5) micrograms for the hypophysis and 9.6 X 10(2) micrograms for the pineal gland. HLH content, calculated per 1 mg protein, was 3 X 10(3) micrograms for the hypophysis and 24 micrograms for the pineal gland. The hormone content in the epiphysis was 1000 times higher than HLH concentration in the blood serum.

Isolation and structure of several peptides from porcine hypothalami.

Ten peptides were isolated from porcine hypothalami and structurally elucidated. These included four dipeptides Arg-Phe, Phe-Tyr, Val-Trp, and Tyr-Phe; a tripeptide Lys-Phe-Tyr; two tetrapeptides Gly-Lys-Val-Asn and Phe-Glu-His-Glu, a nonapeptide Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe; a decapeptide Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe and a hexadecapeptide Phe-Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe-Asn-Leu. The tetrapeptide Gly-Lys-Val-Asn, the nonapeptide, the decapeptide and the hexadecapeptide most probably represent artifact fragments of alpha- and beta-chains of porcine hemoglobin. The natural or synthetic Gly-Lys-Val-Asn and Phe-Glu-His-Glu had some growth hormone releasing activity while Val-Trp, Tyr-Phe and Lys-Phe-Tyr had slight prolactin releasing activity. The biological activities of other peptides have not been determined yet.