Neuropeptide FF receptor antagonist, RF9, attenuates the fever induced by central injection of LPS in mice

The endogenous opioid system has been found to be involved in the fever caused by lipopolysaccharide (LPS). Neuropeptide FF (NPFF, FLFQPQRF-NH₂) is an endogenous peptide known to modulate opioid activity, mainly in the central nervous system. Therefore, those data suggested a link between LPS-induced fever and NPFF systems. Using a model of acute neuroinflammation, we sought to determine the effects of NPFF systems on the fever induced by i.c.v. injection of LPS. Coinjected with different doses of NPFF (10 and 30 nmol), the fever of LPS (125 ng) was not modified. Interestingly, the selective NPFF receptors antagonist RF9 (30 nmol) injected into the third ventricle failed to induce significant effect, but it decreased the fever of LPS (125 ng) after cerebral administration in mice. These results suggest that NPFF receptors activation is required for LPS to produce fever. This interaction is the first evidence that NPFF systems participate in the control of acute neuroinflammation in conscious animals.     

神经肽FF的生物活性研究进展

神经肽FF(neuropeptide FF,NPFF)最初于1985年从牛脑中分离得到,是一种哺乳动物体内普遍存在的八肽。NPFF最早因具有调节阿片镇痛活性而引起关注,而后陆续发现NPFF具有多种生理功能,包括调节体温、心血管、神经内分泌、胃肠运动、摄食、抗炎、免疫调节、以及神经保护等。现在认为NPFF是一种具有激素样活性的神经递质,因此对其生理功能的深入研究有助于理解NPFF在多种生理系统中的作用机制及其潜在多肽药物的研发。本文综述了近年来NPFF生物活性的最新研究进展,结合本实验室已有研究基础,重点介绍了NPFF在神经内分泌、免疫调节、抗炎、神经保护、及信号通路方面的进展,并展望了NPFF类多肽药物今后的发展方向。     

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe(1)]NC(1-13)NH(2) (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.     

Regulation of endogenous human NPFF2 receptor by neuropeptide FF in SK-N-MC neuroblastoma cell line

Neuropeptide FF has many functions both in the CNS and periphery. Two G protein-coupled receptors (NPFF1 and NPFF2 receptors) have been identified for neuropeptide FF. The expression analysis of the peptide and receptors, together with pharmacological and physiological data, imply that NPFF2 receptor would be the primary receptor for neuropeptide FF. Here, we report for the first time a cell line endogenously expressing hNPFF2 receptor. These SK-N-MC neuroblastoma cells also express neuropeptide FF. We used the cells to investigate the hNPFF2 receptor function. The pertussis toxin-sensitive inhibition of adenylate cyclase activity upon receptor activation indicated coupling to Gi/o proteins. Upon agonist exposure, the receptors were internalized and the mitogen-activated protein kinase cascade was activated. Upon neuropeptide FF treatment, the actin cytoskeleton was reorganized in the cells. The expression of hNPFF2 receptor mRNA was up-regulated by neuropeptide FF. Concomitant with the receptor mRNA, the receptor protein expression was increased. The homologous regulation of hNPFF2 receptor correlates with our previous results in vivo showing that during inflammation, the up-regulation of neuropeptide FF mRNA precedes that of NPFF2 receptor. The regulation of hNPFF2 receptor by NPFF could also be important in the periphery where neuropeptide FF has been suggested to function as a hormone.     

Neuropeptide FF (NPFF) reduces the expression of cocaine-induced conditioned place preference and cocaine-induced sensitization in animals

The endogenous brain opioid system is believed to play an important role in mediating reward mechanisms. Opioid innervation is high in many limbic regions and reinforcing actions of many drugs of abuse, including cocaine, are thought to be mediated via endogenous opioid system. The aim of the present study was to indicate whether the anti-opioid peptide, neuropeptide FF (NPFF; FLFQPQRF-NH₂) was able to modify the rewarding effect of cocaine (5 mg/kg) measured in the expression of conditioned place preference (CPP) test in rats and the expression of sensitization to hyperlocomotor effect of cocaine (10 mg/kg) in mice. Our results indicate that NPFF (5, 10, and 20 nmol) given intracerebroventricularly (i.c.v.) inhibited the expression of cocaine-induced CPP at the dose of 10 nmol (P < 0.01) and 20 nmol (P < 0.001). Moreover, NPFF inhibited the expression of cocaine-induced sensitization to its hyperlocomotor effect at the dose of 20 nmol (P < 0.05) and acute hyperlocomotor effect of cocaine at doses of 5 nmol (P < 0.01), 10 nmol (P < 0.01), and 20 nmol (P < 0.05). Our study suggests that NPFF may participate in a rewarding effect of cocaine measured in the CPP paradigm. On the other hand, our experiments indicate that NPFF is involved in the mechanism of expression of sensitization to cocaine hyperlocomotion but this effect seems to be non-specific because NPFF also inhibited the acute hyperlocomotor effect of cocaine.     

Identification and Functional Characterization of the Phosphorylation Sites of the Neuropeptide FF2 Receptor

The neuropeptide FF₂ (NPFF₂) receptor belongs to the rhodopsin family of G protein-coupled receptors and mediates the effects of several related RFamide neuropeptides. One of the main pharmacological interests of this system resides in its ability to regulate endogenous opioid systems, making it a potential target to reduce the negative effects of chronic opioid use. Phosphorylation of intracellular residues is the most extensively studied post-translational modification regulating G protein-coupled receptor activity. However, until now, no information concerning NPFF₂ receptor phosphorylation is available. In this study, we combined mass spectrometric analysis and site-directed mutagenesis to analyze for the first time the phosphorylation pattern of the NPFF₂ receptor and the role of the various phosphorylation sites in receptor signaling, desensitization, and trafficking in a SH-SY5Y model cell line. We identified the major, likely GRK-dependent, phosphorylation cluster responsible for acute desensitization, ⁴¹²TNST⁴¹⁵ at the end of the C terminus of the receptor, and additional sites involved in desensitization (³⁷²TS³⁷³) and internalization (Ser³⁹⁵). We thus demonstrate the key role played by phosphorylation in the regulation of NPFF₂ receptor activity and trafficking. Our data also provide additional evidence supporting the concept that desensitization and internalization are partially independent processes relying on distinct phosphorylation patterns.     

Crypteins derived from the mouse neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice

NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests.     

Pancreatic polypeptide, microinjected into the dorsal vagal complex, potentiates glucose-stimulated insulin secretion in the rat

Specific binding sites for circulating pancreatic polypeptide (PP) have been found within the dorsal vagal complex (DVC) in the caudal medulla oblongata. Therefore, the effects of rat PP on pancreatic hormone secretion upon its microinjection into the DVC in halothane-anesthetized rats at doses of 0.4–40 pmol were investigated. At this range of doses, the changes in plasma concentrations of insulin, glucagon and glucose over basal levels did not differ from those after vehicle microinjection. In a separate series of experiments, vehicle and PP at doses of 0.4 and 4 pmol were microinjected into the right DVC 40 min after the continuous infusion of -glucose had been started. In animals receiving continuous infusion of -glucose, PP microinjected into the DVC (4 pmol), resulted in markedly higher insulin levels at corresponding time points compared to those with vehicle microinjected into the DVC. These data indicate, for the first time, that microinjection of PP into the DVC may potentiate glucose-stimulated insulin secretion in halothane-anesthetized rats.     

Assessing activation of the human neuropeptide FF2 receptor with a non-radioactive GTP binding assay

We have evaluated a novel, time-resolved fluorometric GTP binding assay for its suitability for functional screening of neuropeptide FF (NPFF) receptor ligands. Our results suggest that this assay, which relies on the use of a europium-labeled GTP analogue, Eu-GTP, provides a powerful alternative to the [³⁵S]guanosine-5′-O-(3-thio)triphosphate binding assay for assessing the functional properties of NPFF analogs. Further, we demonstrate that the tetrapeptide PMRF-NH₂ exhibited high agonist potency at the NPFF2 receptor, and that the efficacies of this peptide and another shortened NPFF analog were greater than that of NPFF.     

Functional properties of Pfr(Tic)amide and BIBP3226 at human neuropeptide FF2 receptors

The functional characteristics of two putative neuropeptide FF (NPFF) antagonists, BIBP3226 and PFR(Tic)amide, on the human neuropeptide FF receptor subtype 2 (hNPFF2) were investigated. Surprisingly, PFR(Tic)amide was shown to exhibit agonist properties in the [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding assay. The efficacy of PFR(Tic)amide was significantly greater than that of (1DMe)Y8Fa, a stable analog of NPFF, and PFR(Tic)amide can therefore be classified as a ‘super-agonist’. BIBP3226 did act as a reversible competitive antagonist on the hNPFF2 receptor. However, high concentrations of BIBP3226 also non-specifically increased [³⁵S]GTPγS binding. The usefulness of BIBP3226 as an antagonist tool on the NPFF receptor is thus limited.     

迷走神经背核对胃机能调控的研究进展

神经解剖学和生理学的研究证明,迷走神经背核（dorsal motor nucleus of the vagus,DMV）是调控胃机能的重要副交感初级中枢。支配胃的迷走神经纤维主要发自于延髓的DMV。就DMV的细胞构筑和突触联系、DMV对胃的神经支配、电刺激DMV对胃机能的影响以及DMV内的神经递质和受体对胃机能的调控进行综述。     

Release of Neuropeptide FF, an Anti-Opioid Peptide, in Rat Spinal Cord Slices Is Voltage- and Ca2+-Sensitive: Possible Involvement of P-Type Ca2+ Channels

Abstract: Neuropeptide FF (NPFF), an FMRFamide-like peptide with antiopioid properties, inhibits morphine-induced analgesia but also produces hyperalgesia. In the present study, the mechanisms of NPFF release were investigated in an in vitro superfusion system with rat spinal cord slices. The opening of voltage-sensitive Na⁺ channels with veratridine (20 µ M ) induced calcium-dependent NPFF release, which was abolished by tetrodotoxin (1 µ M ), suggesting that NPFF release depends on nerve impulse activity. We also showed that NPFF release was a function of the extent of depolarization and was calcium dependent. The 30 m M K⁺-induced release was blocked by Co²⁺ or Ni²⁺ (2.5 m M ) but was unaffected by Ca²⁺ channel blockers of the L type—Cd²⁺ (100 µ M ), nifedipine or nimodipine (10 µ M ), diltiazem (20 µ M ), or verapamil (50 µ M )—or the N type—ω-conotoxin GVIA (1 µ M ). In contrast, ω-agatoxin IVA (1 µ M ) led to a 65% reduction in NPFF release, suggesting that P-type Ca²⁺ channels play a prominent role. The 35% remaining release resulted from activation of an unknown subtype. The NPFF-like material in superfusates recognized spinal NPFF receptors, suggesting that NPFF release in the spinal cord has a physiological role.     

Localization of the CB1 cannabinoid receptor in the rat brain. An immunohistochemical study

The presence of central cannabinoid receptor (CB1), involving the N-terminal 14 amino acid peptide, was demonstrated in the rat brain by immunohistochemistry. Intensely stained neurons were observed in the principal neurons of the hippocampus, striatum, substantia nigra, cerebellar cortex, including the Purkinje cells. Moderate CB1-IR cell bodies and fibers were present in the olfactory bulb, cingulate, entorhinal and piriform cortical areas, amygdala and nucleus accumbens. The perivascular glial fibers have shown moderate to high density CB1-IR in olfactory and limbic structures. Low density was detected in the thalamus and hypothalamus and area postrema. The CB1 receptor was widely distributed in the forebrain and sparsely in the hindbrain. These new data support the view that the endogenous cannabinoids play an important role in different neuronal functions as neuromodulators or neurotransmitters.     

NPY and NPY Y1 receptor effects on noradrenaline overflow from the rat brain in vitro

Neurotransmitters and neuropeptides play important roles in the regulation of various neuroendocrine functions particularly feeding. The aim of this study was to investigate whether a functional interaction occurs among neuropeptide Y (NPY) at NPY Y₁ receptors and noradrenaline overflow, as this may contribute to the regulation of appetite. The release of endogenous noradrenaline and its metabolite 3,4-dihydroxyphenylglycol (DHPG) were examined from hypothalamic and medullary prisms using the technique of in vitro superfusion and high performance liquid chromatography (HPLC) with coulometric detection. Noradrenaline and DHPG overflow was investigated at rest, in response to NPY (0.1 μM) and in response to the NPY Y₁ receptor agonist, [Leu³¹,Pro³⁴]NPY (0.1 μM). Perfusion with NPY and [Leu³¹,Pro³⁴]NPY significantly reduced noradrenaline overflow from the hypothalamus and medulla. Perfusion with NPY and [Leu³¹,Pro³⁴]NPY was without significant effect on hypothalamic DHPG overflow, while medullary DHPG overflow was significantly reduced by NPY and [Leu³¹,Pro³⁴]NPY. Results from this study provide evidence of NPY Y₁ receptor-mediated inhibition of noradrenaline release in the hypothalamus and medulla, further illustrating a complex interaction between neurotransmitters and neuropeptides within the rat brain.     

Progesterone receptor mRNA expression and distribution in the female rabbit brain

Progesterone regulates diverse functions in the rabbit brain through the interaction with its nuclear receptor (PR). Although PR protein has been detected in some regions of the rabbit forebrain, PR mRNA expression and distribution in the rabbit brain are unknown. Hence, we investigated these issues by in situ hybridization. New Zealand adult female rabbits were ovariectomized and treated with vehicle or estradiol (5 μg/(kg day)) for 3 days. The results show an extended distribution of PR mRNA expression in the rabbit brain. The highest expression was detected in preoptic area and hypothalamic anterior nuclei such as paraventricular, periventricular and arcuate nuclei. A high expression was also detected in thalamic and telencephalic areas, including hippocampus and cerebral cortex. Estradiol treatment induced an increase in PR mRNA expression in many brain areas, particularly in the hippocampus and the hypothalamic and preoptic area regions. The wide distribution of PR mRNA in the rabbit brain suggests that progesterone through PR activation is involved in several functions apart from reproductive behavior in rabbits, and that PR expression is up-regulated by estradiol in the rabbit brain.     

An immunohistochemical study of the distribution of ABH antigens in human submandibular glands at the light and electron microscopic levels.

The distribution of blood group antigens ABH in submandibular glands was studied at light and electron microscopy levels by applying ImmunoGold Silver Staining (IGSS) and post-embedding ImmunoGold (IGS) methods, respectively. In IGSS treated samples, a cytoplasmic and a surface form of antigen localization were discernible in the glandular parenchyma. The former was restricted to most mucous cells and to scattered serous cells: A and B antigens were demonstrated in mucous cells of A and B type glands, while H antigen appeared in most mucous and occasional serous elements regardless of the blood type of donors. The latter appeared as a strong H reactivity on cell surfaces of serous acini and ducts regardless of the patient blood type. The IGS method was applied both on non-osmicated samples embedded in LR White resin and on osmicated, Epon embedded samples. In non-osmicated tissues, antigen labelling was revealed in secretory granules and cell surfaces. Positive secretory granules were found in most mucous cells and occasional serous, intercalated, and striated duct cells. A and B antigens weakly reacted in mucous cells of A and B type glands, respectively, while strong H reactivity was seen in mucous, serous, intercalated and striated duct cells of glands of all types. Surfaces labelled with H antigen were found on both lumenal and basolateral membranes of striated ducts in glands of all types. IGS method applied on osmicated, Epon embedded samples, selectively revealed blood group antigens in secretory granules of serous cells but not in the apical vesicles of striated ductal cells. Cell surfaces were completely unreactive.     

Suppressor of cytokine signaling 3 in the human hypothalamus

In rodents, the mediobasal hypothalamus and the hypothalamic paraventricular nucleus (PVN) are implicated in leptin signaling. Surprisingly little data is available on the human hypothalamus. We set out to study the expression of suppressor-of-cytokine-signaling 3 (SOCS3), α-melanocyte stimulating hormone (αMSH) and agouti-related protein (AgRP) in the infundibular nucleus (IFN) and to investigate the relationship between these neuropeptide expressions and serum leptin concentrations in a blood sample taken within 24h before death. We studied post-mortem human brain material by means of quantitative immunocytochemistry. We found that SOCS3 immunoreactivity was widely distributed throughout the hypothalamus, and most prominent in the PVN, whereas expression levels in the IFN were low. Surprisingly, SOCS3 expression in the PVN was inversely related to serum leptin. A significant positive correlation was observed between AgRP and NPY expression in the IFN. The inverse correlation between SOCS3 expression in the PVN and serum leptin was unexpected and may be related to the hypothalamic adaptation to fatal illness rather than to nutritional status, or may represent an interspecies difference.     

Cytochrome P450 aromatase (CYP19) in the non-human primate brain: distribution, regulation, and functional significance

In adult male primates, estrogens play a role in both gonadotropin feedback and sexual behavior. Inhibition of aromatization in intact male monkeys acutely elevates serum levels of luteinizing hormone, an effect mediated, at least partially, within the brain. High levels of aromatase (CYP19) are present in the monkey brain and regulated by androgens in regions thought to be involved in the central regulation of reproduction. Androgens regulate aromatase pretranslationally and androgen receptor activation is correlated with the induction of aromatase activity. Aromatase and androgen receptor mRNAs display both unique and overlapping distributions within the hypothalamus and limbic system suggesting that androgens and androgen-derived estrogens regulate complimentary and interacting genes within many neural networks. Long-term castrated monkeys, like men, exhibit an estrogen-dependent neural deficit that could be an underlying cause of the insensitivity to testosterone that develops in states of chronic androgen deficiency. Future studies of in situ estrogen formation in brain in the primate model are important for understanding the importance of aromatase not only for reproduction, but also for neural functions such as memory and cognition that appear to be modulated by estrogens.