tBu-D-Gly5]NPS, a pure and potent antagonist of the neuropeptide S receptor: in vitro and in vivo studies

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, the NPSR ligand [(t)Bu-D-Gly(5)]NPS was generated and in vitro characterized as a pure antagonist at the mouse NPSR. In the present study the pharmacological profile of [(t)Bu-D-Gly(5)]NPS has been investigated. [(t)Bu-D-Gly(5)]NPS activity was evaluated in vitro in the calcium mobilization assay at the rat NPSR and in vivo in the locomotor activity and righting reflex tests in mice and in the elevated plus maze and defensive burying assays in rats. In vitro, [(t)Bu-D-Gly(5)]NPS was inactive per se while it inhibited the calcium mobilization induced by 30 nM NPS (pK(B) 7.42). In Schild analysis experiments [(t)Bu-D-Gly(5)]NPS (0.1-10 μM) produced a concentration-dependent rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 7.17. In mouse locomotor activity experiments, supraspinal injection of [(t)Bu-D-Gly(5)]NPS (1-10 nmol) dose dependently counteracted NPS (0.1 nmol) stimulant effects. In the mouse righting reflex assay [(t)Bu-D-Gly(5)]NPS (0.1-10 nmol) fully prevented the arousal-promoting action of the natural peptide (0.1 nmol). Finally, [(t)Bu-D-Gly(5)]NPS (3-30 nmol) was able to completely block NPS (1 nmol) anxiolytic-like actions in rat elevated plus maze and defensive burying assays. Collectively, the present results demonstrated that [(t)Bu-D-Gly(5)]NPS behaves both in vitro and in vivo as a pure and potent NPSR antagonist. This compound represents a novel and useful tool for investigating the pharmacology and neurobiology of the NPS/NPSR system.     

Effect of neuropeptide S receptor antagonists and partial agonists on palatable food consumption in the rat

Neuropeptide S (NPS) is the endogenous ligand for the previously orphan G-protein-coupled-receptor, now termed NPS receptor (NPSR). NPS has both anxiolytic and pro-arousal properties and decreases food intake. In this work we use a rat model of palatable food intake to test in vivo different analogs of human NPS developed in our laboratories and characterized in previous in vitro experiments as partial agonists ([Ala³]NPS and [Aib⁵]NPS), or antagonists ([d-Cys(^tBu)⁵]NPS and [^tBu-d-Gly⁵]NPS). Our results confirmed that intracerebroventricular (ICV) injection of NPS (1 nmol) decreases standard chow intake in food restricted rats as well as in freely feeding animals fed with standard or palatable food diets. [Aib⁵]NPS (30 and 60 nmol), like NPS, reduced palatable food intake, thus confirming in vivo its ability to activate NPSR. [Ala³]NPS (60 nmol) did not affect palatable food intake per se but blocked the anorectic effect of NPS, thus suggesting its ability to function as an antagonist in this model. Finally, [d-Cys(^tBu)⁵]NPS (20-60 nmol) and [^tBu-d-Gly⁵]NPS (10-30 nmol), described in previous in vitro studies as pure NPSR antagonists, did not affect palatable food intake when given alone, but fully blocked the anorectic effect of NPS. These results provide an important characterization of the pharmacological properties of these NPS analogs in vivo. Of particular relevance are the data showing that [d-Cys(^tBu)⁵]NPS and [^tBu-d-Gly⁵]NPS behave as pure antagonists at NPSR regulating food intake, indicating that these molecules are suitable tools for further investigation of the physiopharmacology of the NPS/NPSR system.     

Neuropeptide S Facilitates Mice Olfactory Function through Activation of Cognate Receptor-Expressing Neurons in the Olfactory Cortex

Neuropeptide S (NPS) is a newly identified neuromodulator located in the brainstem and regulates various biological functions by selectively activating the NPS receptors (NPSR). High level expression of NPSR mRNA in the olfactory cortex suggests that NPS-NPSR system might be involved in the regulation of olfactory function. The present study was undertaken to investigate the effects of intracerebroventricular (i.c.v.) injection of NPS or co-injection of NPSR antagonist on the olfactory behaviors, food intake, and c-Fos expression in olfactory cortex in mice. In addition, dual-immunofluorescence was employed to identify NPS-induced Fos immunereactive (-ir) neurons that also bear NPSR. NPS (0.1–1 nmol) i.c.v. injection significantly reduced the latency to find the buried food, and increased olfactory differentiation of different odors and the total sniffing time spent in olfactory habituation/dishabituation tasks. NPS facilitated olfactory ability most at the dose of 0.5 nmol, which could be blocked by co-injection of 40 nmol NPSR antagonist [D-Val⁵]NPS. NPS administration dose-dependently inhibited food intake in fasted mice. Ex-vivo c-Fos and NPSR immunohistochemistry in the olfactory cortex revealed that, as compared with vehicle-treated mice, NPS markedly enhanced c-Fos expression in the anterior olfactory nucleus (AON), piriform cortex (Pir), ventral tenia tecta (VTT), the anterior cortical amygdaloid nucleus (ACo) and lateral entorhinal cortex (LEnt). The percentage of Fos-ir neurons that also express NPSR were 88.5% and 98.1% in the AON and Pir, respectively. The present findings demonstrated that NPS, via selective activation of the neurons bearing NPSR in the olfactory cortex, facilitates olfactory function in mice.     

Neuropeptide S inhibits release of 5-HT and glycine in mouse amygdala and frontal/prefrontal cortex through activation of the neuropeptide S receptor

Neuropeptide S (NPS) is a neurotransmitter/neuromodulator that has been identified as the natural ligand of G protein-coupled receptors termed NPS receptors (NPSRs). The NPS-NPSR system is involved in the control of numerous centrally-mediated behaviours, including anxiety. As several classical transmitters play a role in fear/anxiety, we here investigated the regulation by NPS of the exocytotic release of 5-hydroxytryptamine (5-HT) and glycine in nerve terminals isolated from mouse frontal/prefrontal cortex and amygdala. Synaptosomes, prelabelled with the tritiated neurotransmitters, were depolarized in superfusion with 12–15 mM KCl and exposed to varying concentrations of NPS. The evoked release of [³H]5-HT in frontal/prefrontal cortex was potently inhibited by NPS (maximal effect about 25% at 0.1 nM). Differently, the neuropeptide exhibited higher efficacy but much lower potency in amygdala (maximal effect about 40% at 1 μM). NPS was an extremely potent inhibitor of the K⁺-evoked release of [³H]glycine in frontal/prefrontal nerve endings (maximal effect about 25% at 1 pM). All the inhibitory effects observed were counteracted by the NPSR antagonist SHA 68, indicating that the neuropeptide acted at NPSRs. In conclusion, NPS can inhibit the exocytosis of 5-HT and of glycine through the activation of presynaptic NPSRs situated on serotonergic and glycinergic terminals in areas involved in fear/anxiety behaviours. The possibility exists that the NPSRs in frontal/prefrontal cortex are high-affinity receptors involved in non-synaptic transmission, whereas the NPSRs on amygdala serotonergic terminals are low-affinity receptors involved in axo-axonic synaptic communication.     

In vitro and in vivo pharmacological characterization of the novel neuropeptide S receptor ligands QA1 and PI1

The pharmacological activity of the novel neuropeptide S (NPS) receptor (NPSR) ligands QA1 and PI1 was investigated. In vitro QA1 and PI1 were tested in calcium mobilization studies performed in HEK293 cells expressing the recombinant mouse (HEK293_mNPSR) and human (HEK293_hNPSRIle107 and HEK293_hNPSRAsn107) NPSR receptors. In vivo the compounds were studied in mouse righting reflex (RR) and locomotor activity (LA) tests. NPS caused a concentration dependent mobilization of intracellular calcium in the three cell lines with high potency (pEC₅₀ 8.73–9.14). In inhibition response curve and Schild protocol experiments the effects of NPS were antagonized by QA1 and PI1. QA1 displayed high potency (pK_B 9.60–9.82) behaving as a insurmountable antagonist. However in coinjection experiments QA1 produced a rightward swift of the concentration response curve to NPS without modifying its maximal effects; this suggests that QA1 is actually a slow dissociating competitive antagonist. PI1 displayed a competitive type of antagonism and lower values of potencies (pA₂ 7.74–8.45). In vivo in mice NPS (0.1 nmol, i.c.v.) elicited arousal promoting action in the RR assay and stimulant effects in the LA test. QA1 (30 mg kg⁻¹) was able to partially counteract the arousal promoting NPS effects, while PI1 was inactive in the RR test. In the LA test QA1 and PI1 only poorly blocked the NPS stimulant action. The present data demonstrated that QA1 and PI1 act as potent NPSR antagonists in vitro, however their usefulness for in vivo investigations in mice seems limited probably by pharmacokinetic reasons.     

Neuropeptide S stimulates human monocyte chemotaxis via NPS receptor activation

Neuropeptide S (NPS) produces several biological actions by activating a formerly orphan GPCR, now named NPS receptor (NPSR). It has been previously demonstrated that NPS stimulates murine leukocyte chemotaxis in vitro. In the present study we investigated the ability of NPS, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human monocyte chemotaxis. At a concentration of 10⁻⁸ M fMLP significantly stimulated chemotaxis. NPS produced a concentration dependent chemotactic action over the concentration range 10⁻¹² to 10⁻⁵ M. The NPSR antagonists [d-Cys(^tBu)⁵]NPS, [^tBu-d-Gly⁵]NPS and SHA 68 were used to pharmacologically characterize NPS action. Monocyte chemoattractant effect of NPS, but not fMLP, was completely blocked by either peptide antagonists or SHA with the nonpeptide molecule being more potent. None of the NPSR antagonists modified per se random cell migration. Thus, the present study demonstrated that NPS is able to stimulate human monocyte chemotaxis and that this effect is entirely due to selective NPSR activation.     

Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone and progesterone was analyzed in common carp Cyprinus carpio. Carp kidney leukocytes were cultured in RPMI 1640 medium containing 0.1, 1, 10, 100 or 1000 nM concentration of each hormone. The production of superoxide anion, nitric oxide (NO) and phagocytosis were measured in vitro. Similar concentrations of cortisol were used as control. Phagocytic activities of carp macrophages was suppressed by treatment with beta-estradiol, progesterone and 11-ketotestosterone. The production of NO in carp macrophages was suppressed by progesterone and 11-ketotestosterone. However, carp macrophages incubated with beta-estradiol, progesterone and 11-ketotestosterone did not show any difference in the production of superoxide anion in comparison with control macrophages in the absence of hormones. Carp macrophages treated with cortisol suppressed phagocytosis and the production of nitric oxide and superoxide anion.     

Vertebrate cytokines interleukin 12 and gamma interferon, but not interleukin 10, enhance phagocytosis in the annelid Eisenia hortensis

Phagocytosis assays employing class I [interleukin 12 (IL-12)], and class II [gamma interferon (gIFN) and IL-10] human recombinant cytokines were carried out to determine the biological effects of these molecules on innate immune responses in the earthworm Eisenia hortensis. Coelomocytes from E. hortensis were pre-incubated with the cytokines for 16-20 h in vitro followed by introduction of Escherichia coli expressing green fluorescent protein (E. coli/GFP). The pro-inflammatory cytokines IL-12 and gIFN stimulated statistically significant (p ? 0.05) enhanced phagocytosis of E. coli/GFP by hyaline amoebocytes as determined by flow cytometry; 10 out of 21 earthworms (48%) responded to IL-12, while eight out of 21 (38%) responded to gIFN. In contrast, the anti-inflammatory cytokine IL-10 neither stimulated nor inhibited phagocytosis in nine earthworms tested. These results demonstrate that vertebrate pro-inflammatory cytokines influence invertebrate cellular responses of immune cells causing enhanced phagocytic activity in earthworm coelomocytes.     

Structure-function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I

Neuropeptide S (NPS) and its receptor (NPSR) are thought to have a role in asthma pathogenesis; a number of single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalance of asthma. One such single nucleotide polymorphism leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. To gain insight into structure-function relationships within NPS and NPSR, we first carried out a limited structural characterization of NPS and subjected the peptide to extensive mutagenesis studies. Our results show that the NH(2)-terminal third of NPS, in particular residues Phe-2, Arg-3, Asn-4, and Val-6, are necessary and sufficient for activation of NPSR. Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked N107I variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS. Whereas this interaction may be at the root of the increase in potency associated with the N107I variant, we show here that the mutation also causes an increase in cell-surface expression of the mutant receptor, leading to a concomitant increase in the maximal efficacy (E(max)) of NPS. Our results identify the key residues of NPS involved in NPSR activation and suggest a molecular basis for the functional effects of the N107I mutation and for its putative pathophysiological link with asthma.     

Anxiolytic-like effect of neuropeptide S in the rat defensive burying

Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1–10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5 mg/kg, i.p.) and caffeine (20 mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.     

Morphine dependence is associated with changes in neuropeptide S receptor expression and function in rat brain

Neuropeptide S (NPS) is a newly identified ligand for the previously discovered G-protein coupled receptor 154 now named NPSR. Recently, it has been found that NPSR gene expression is altered during ethanol withdrawal. In this study we tried to elucidate if NPSR gene expression is modified in response to morphine withdrawal and its protracted abstinence. To induce opioid dependence Wistar rats were treated for 7 days with morphine. Twelve hours and 7 days after the last morphine administration brains were removed and the expression of NPSR mRNA was analyzed by in situ hybridization (ISH). Successful induction of opioid dependence was confirmed by the naloxone-precipitated withdrawal test 2 h after the last morphine administration. Moreover, 7 days after the last morphine dose animals were checked for signs of anxiety and for intracerebroventricular (ICV) NPS (0.3 and 1.0 nmol) induced anxiolytic effects by elevated plus maze (EPM). Results showed that in morphine treated rats strong somatic signs of naloxone-precipitated withdrawal occurred. ISH data revealed changes in NPSR gene expression in the ventral tegmental area as well as in the basolateral amygdaloid and bed nucleus of stria terminalis at 12 h and 7 days into abstinence, respectively. At 7 days into abstinence post dependent animals showed higher levels of anxiety than controls which were significantly attenuated by NPS. These results demonstrated that morphine dependence induction led to (i) changes in NPSR mRNA expression; (ii) increased anxiety; and (iii) more potent anxiolytic-like effect of NPS.     

The asthma candidate gene <Emphasis Type="Italic">NPSR1</Emphasis> mediates isoform specific downstream signalling

Background

Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B.

Methods

HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²⁺ assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays.

Results

NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells.

Conclusions

We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

     

Molecular evolution of the neuropeptide S receptor

The neuropeptide S receptor (NPSR) is a recently deorphanized member of the G protein-coupled receptor (GPCR) superfamily and is activated by the neuropeptide S (NPS). NPSR and NPS are widely expressed in central nervous system and are known to have crucial roles in asthma pathogenesis, locomotor activity, wakefulness, anxiety and food intake. The NPS-NPSR system was previously thought to have first evolved in the tetrapods. Here we examine the origin and the molecular evolution of the NPSR using in-silico comparative analyses and document the molecular basis of divergence of the NPSR from its closest vertebrate paralogs. In this study, NPSR-like sequences have been identified in a hemichordate and a cephalochordate, suggesting an earlier emergence of a NPSR-like sequence in the metazoan lineage. Phylogenetic analyses revealed that the NPSR is most closely related to the invertebrate cardioacceleratory peptide receptor (CCAPR) and the group of vasopressin-like receptors. Gene structure features were congruent with the phylogenetic clustering and supported the orthology of NPSR to the invertebrate NPSR-like and CCAPR. A site-specific analysis between the vertebrate NPSR and the well studied paralogous vasopressin-like receptor subtypes revealed several putative amino acid sites that may account for the observed functional divergence between them. The data can facilitate experimental studies aiming at deciphering the common features as well as those related to ligand binding and signal transduction processes specific to the NPSR.     

A Single-Nucleotide Polymorphism of Human Neuropeptide S Gene Originated from Europe Shows Decreased Bioactivity

Using accumulating SNP (Single-Nucleotide Polymorphism) data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS), a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L⁶-NPS) causing non-synonymous substitution on the 6^th position (V to L) of the NPS mature peptide region. L⁶-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ∼25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L⁶-NPS exhibits a significant lower bioactivity than the wild type NPS, with ∼20-fold higher EC₅₀ values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6^th position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.     

Bacterial sphingophospholipids containing non-hydroxy fatty acid activate murine macrophages via Toll-like receptor 4 and stimulate bacterial clearance

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance.     

神经肽S功能与构效关系研究进展

神经肽S (neuropeptide S,NPS) 是通过反向药理学策略鉴定的由20 个氨基酸组成的新神经肽,它激活G蛋白偶联受体(NPSR) 而发挥作用。NPS及其受体主要分布于中枢上与觉醒和应激相关的脑区。目前的研究表明NPS 系统具有广泛的生理作用,如增加觉醒、抑制睡眠、抗焦虑作用、抑制摄食和调节免疫功能等,成为相关疾病的一个重要的新药物靶点。同时,研究神经肽S 的构效关系并寻找新的高效激动剂和选择性拮抗剂,对揭示其作用机理有重要意义。     

Breed-linked polymorphisms of porcine toll-like receptor 2 (TLR2) and TLR4 and the primary investigation on their relationship with prevention against Mycoplasma pneumoniae and bacterial LPS challenge

Toll-like receptors (TLRs) play a crucial role in innate immunity, serving as pattern-recognition receptors and the first barrier in host defense against microbial infections. Genetic variations of TLR2 and TLR4 are closely associated with a variety of infectious diseases, particularly lung diseases. In this study, we detected six and four single nucleotide polymorphisms (SNPs) in the coding sequences of porcine TLR2 and TLR4 genes, respectively. Only SNP 1027C>A of TLR4 was shown to be markedly biased in Western and Oriental pig populations. Hence, the susceptibility of pigs with different genotype at position 1027C>A to Mycoplasma hyopneumoniae (Mhp) infection was investigated, and changes to the expression of TLR2, TLR4, TNF-α and IL-1β were monitored. The results showed that there was no significant difference in susceptibility to Mhp infection between AA and CC individuals despite expression levels for all detected genes of the challenge groups being significantly higher than the corresponding control groups. Furthermore, porcine alveolar macrophages of different genotype were collected and stimulated by lipopolysaccharide. We found that the expression of TLR2, TLR4, TNF-α and IL-1β genes were enhanced to different levels by lipopolysaccharide stimulation. TLR2 and TLR4 gene expressions and their rates of increase of 1027CC pigs were significantly higher than for 1027AC pigs (P?<?0.01), while TNF-α and IL-1β expressions were significantly lower than for 1027AC pigs (P?<?0.01). We predict that allele C at position 1027 of the TLR4 gene contributes to the pig's immune response to gram-negative bacterial infections.