Pharmacological Characterization of the Human NPS Receptor. (A) Dose response curve of [Ca2+]i mobilization induced by human, rat, and mouse NPS in an HEK cell line stably expressing human NPS receptor. (B) Saturation binding of [125I] Tyr¹⁰-NPS (4 pM to 1.7 nM) to CHO cells stably expressing human NPS receptor. (C) Displacement of 0.15 nM [125I] Y10-NPS by increasing concentrations of unlabeled human NPS. Data from triplicate experiments are shown as means ± SEM.

Tissue Distribution of NPS Precursor and NPS Receptor mRNA in Rat Tissues. Quantitative RT-PCR was used to measure transcript levels of NPS precursor (left) and NPS receptor mRNA (right) in 45 rat tissues. Transcript levels were normalized to ?actin. pbl, peripheral blood leucocytes.

Expression of NPS Precursor mRNA in the Pontine Area of the Rat Brain. (A) Schematic drawing of the section shown in (B). The level is at bregma -9.80 mm (Paxinos and Watson, 1997, reprinted with permission from Elsevier). (B) Representative autoradiogram of NPS mRNA expression in LC area. (C–E) Dark-field images of double in situ hybridization of NPS precursor mRNA (white) and TH mRNA (dark blue) in LC area. (D) Higher magnification of the area indicated by an arrow in (C). (E) Higher magnification of a more caudal section. (F–H) Dark-field images of double in situ hybridization of NPS precursor mRNA (white) and CRF mRNA (dark blue) at mid-level of LC area (F) and rostral LC (G). (H) Higher magnification of the area indicated by an arrow in (G). TH, tyrosine hydroxylase; NPS, neuropeptide S; CRF, corticotropin-releasing factor. Landmarks: Cb, cerebellum; 4V, fourth ventricle. Scale bar, 500 µm in (C), 250 µm in all other pictures.

Distribution of NPS Precursor mRNA Expression in Rat Brain. (A, D, and G) Drawings of the sections illustrated in (B) and (C) (Bregma -9.68 mm), (E) and (F) (Bregma -2.80 mm), and (H) and (I) (Bregma -3.14 mm), respectively (Paxinos and Watson, 1997). (B, C, E, F, H, and I) Dark-field images of NPS precursor mRNA expression in coronal sections of rat brain. (E and H) Expression of NPS precursor mRNA in boxed regions in (D) and (G), respectively. (C, F, and I) Higher magnification of the area indicated by an arrow in (B), (E), and (H), respectively. Arrows in (F) and (I) indicate single cells showing hybridization signals for NPS precursor mRNA. LPB, lateral parabrachial nucleus; Pr5, principle sensory 5 nucleus; DMH, dorsomedial hypothalamic nucleus; Amg, amygdala. Landmarks: Cb, cerebellum; 3V, third ventricle; opt, optic tract. Scale bar, 500 µm.

Distribution of NPS Receptor mRNA Expression in Rat Brain. (A, D, G, and J) Schematic drawings of the sections shown in (B) and (C) (Bregma, 3.20 mm), (E) and (F) (Bregma -1.80 mm), (H) and (I) (Bregma -2.80 mm), and (K) and (L) (Bregma -4.52 mm), respectively (Paxinos and Watson, 1997). (B, E, H, and K) Autoradiograms of NPSR mRNA expression in coronal rat brain sections. Arrows in (B), (E), (H), and (K) indicate endopiriform nucleus (En). Arrowheads in (E), (H), and (K) refer to secondary motor cortex (M2), retrosplenial agranular cortex (RSA)/M2, and RSA, respectively. (C, F, and I) Dark-field images of boxed regions in (B), (E), and (H), respectively. (L) Dark field image of midline thalamic regions of section (K). (M and N) Dark-field image of cortical regions in section (E). Arrows in (N) indicate scattered cells expressing NPSR mRNA in somatosensory cortex. (O) Dark-field image of cortical and subicular regions in section (K). AON, anterior olfactory nucleus; DEn, dorsal endopiriform nucleus; CM, central medial thalamic nucleus; IAM, interanteromedial thalamic nucleus; Rh, rhomboid thalamic nucleus; Re, reuniens thalamic nucleus; Amg, amygdala; Hyp, hypothalamus; S, subiculum; Prc, precommissural nucleus; PVP, paraventricular thalamus nucleus, posterior; PH, posterior hypothalamus. Landmarks: aca, anterior commissure, anterior part; pt, paratenial thalamic nuclei; opt, optic tract; D3V, dorsal third ventricle; 3V, third ventricle; Hip, hippocampus. Scale bar, 500 µm.

Central Administration of NPS Produces Behavioral Arousal and Wakefulness. (A) Hyperlocomotion effects of NPS in naive and habituated mice. Naive mice were new to the test chamber, while habituated animals were acclimatized for 1 hr prior to the injection. In naive mice, 0.1 and 1 nmole NPS induce significant hyperlocomotion (F3,324 = 92.83, p < 0.0001, two-way ANOVA for repeated measures). The same doses of NPS also produced significant effects in habituated animals (F3,336 = 135.59, p < 0.0001). (B) Arousal promoting effects of NPS in rats. NPS increases the amount of wakefulness and decreases SWS1, SWS2, and REM sleep in rats (n = 8 for each dose). **p < 0.01, 0.1 nmole and 1.0 nmole compared with saline; *p < 0.01, 1.0 nmole compared with saline (ANOVA followed by Scheffe's post hoc test).

Anxiolytic-like Effects of NPS in Mice.

NPS produces dose-dependent anxiolytic-like effects in C57Bl/6 mice exposed to the open field (A), light-dark box (B), elevated plus maze (C), and marble burying paradigm (D). Doses and groups: all doses are in nmole per animal; open field (n = 8 for each dose); light-dark box (PBS, n = 10; 0.01 nmole, n = 5; 0.03 nmole, n = 5; 0.1 nmole, n = 5; 0.3 nmole, n = 11; 1 nmole, n = 5; 3 nmole, n = 8); elevated plus maze (n = 5 for all doses); marble burying (PBS and 0.01 nmole, n = 10; 0.1 and 1 nmole, n = 9). **p < 0.01, *p < 0.05 compared to PBS control, ANOVA followed by Dunnett's test for multiple comparisons. All data are presented as means ± SEM.

Radioligand Binding Assay

Tyr¹⁰-NPS was labeled with 125I. CHO cells stably expressing human NPSR were seeded into 24-well plates and cultured for 48 hr. For saturation binding experiment, [125I] Tyr¹⁰-NPS at concentrations from 4 pM to 1.7 nM were used. For displacement binding, increasing concentrations of unlabeled human NPS (1 pM to 3 µM) were used to compete with 0.15 nM [125I] Tyr¹⁰-NPS. Nonspecific binding was determined in the presence of 1 µM unlabeled human NPS. The binding assay was carried out as described (Sakurai et al., 1998). In brief, cells were washed with PBS first and then incubated with radioligand with or without unlabeled NPS peptide in DMEM medium containing 0.1% bovine serum albumin at 20°C for 1.5 hr. Cells were washed five times with cold PBS and lysed with 1 N NaOH. Bound radioactivity was counted in a MicroBeta liquid scintillation counter (EG&G Wallac, Gaithersburg, MD) and corrected for counting efficiency. Data from triplicate incubations were analyzed using PRISM.

Structural characterization of human NPS by NMR. B) Putative structural conformation of NPS in the context of receptor binding, showing an a-helix in the region determined to contain a nascent helix in the unbound, solubilized peptide.

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Activation of NPSR-A by truncated mutants of human NPS.

Concentration-response curves in the Ca++-mobilization assay were generated as described in “Experimental Procedures” and the EC50 values thus obtained are shown in Table II. A) Activation by C-terminal-truncated NPS peptides. Each point is the mean ± S.D. of triplicate determinations. Representative experiments of selected mutant peptides are shown. WT (filled squares);1-13 (empty squares); 1-7 (fille d circles); 1-6 (empty circles); 1-5 (filled triangles). B) Activation by N-terminal-truncated NPS peptides. Each point is the mean ± S.D. of triplicate determinations. Representative experiments are shown. WT (filled squares); 2-20 (empty squares); 3-20 (filled circles); 4-20 (empty circle s). C) Summary of EC50 values obtained for truncated NPS mutant peptides. Each value is the mean ± S.E.M. of at least three separate determinations for each peptide and corresponds to values shown in Table II. Peptides for which EC50 values could not be obtained due to low activity and absence of a maximal plateau (1-5, 3-20 and 4-20) are shown as having an EC50 of 2000 nM. Values above bars represent % of maximal activity (based on WT peptide) obtained for these mutant peptides at a concentration of 2000 nM.

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Activation of NPSR-A by alanine point mutants of human NPS.

Concentration-response curves in the Ca++-mobilization assay were generated as described in “Experimental Procedures” and the EC50 values thus obtained are shown in Table II. A) Representative curves of selected mutant peptide. Each point is the mean ± S.D. of triplicate determinations. WT (filled squares); N4A (empty squares); V6A (filled circles); G7A (empty circles). C) Summary of EC50 values obtained for alanine point mutant peptides. Each value is the mean ± S.E.M. of at least three separate determinations for each peptide and corresponds to values shown in Table II. Peptides for which EC50 values could not be obtained due to low activity and absence of a maximal plateau (F2A, R3A, N4A and G7A) are shown as having an EC50 of 2000 nM. Values above bars represent % of maximal activity (based on WT peptide) obtained for these mutant peptides at a concentration of 2000 nM.

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Comparison of EC50 values of mutant NPS peptides on NPSR-A and NPSR-A-N107I variants.

Concentration-response curves in the Ca++-mobilization assay were generated as described in “Experimental Procedures” and the EC50 values thus obtained are shown in Table II. EC50 values of the various mutants were normalized to those obtained for WT peptide on each variant and are expressed as fold-increase over WT peptide. Each value is the mean ± S.E.M. of at least three separate determinations for each peptide. Peptides for which the fold-increase in EC50 is higher than 200 are shown as having a 200-fold increase. NPSR-A (filled squares); N107I-NPSR-A (empty squares). A) Summary of foldincreases in EC50 values for C-terminal-truncated peptides. B) Summary of fold-increases in EC50 values for alanine point mutant peptides.

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Pharmacological analysis of FLAG-NPSR and FLAG-NPSR-N107I variants in transientlytransfected

HEK 293 T cells. A) Concentration-response curves in the Ca++-mobilization assay were generated as described in “Experimental Procedures” and the EC50 values thus obtained are shown in Table I. Each point is the mean ± S.D. of quadruplicate determinations. A representative experiment is shown. FLAG-NPSR-A (filled squares); FLAG-NPSR-A-N107I (empty squares); FLAG-NPSR-B (filled circles); FLAG-NPSR-B-N107I (empty circles). *p<0.01 using Stduent’s t test. B) Concentrationdependent binding of 125I-NPS to whole cells expressing NPSR-A or NPSR-A-N107I variants. Specific binding corresponds to the difference in binding in the absence and presence of excess unlabeled NPS (See “Experimental Procedures”). Data is expressed as percent of calculated Bmax. A representative experiment is shown. FLAG-NPSR-A (filled squares); FLAG-NPSR-A-N107I (empty squares).

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

7 Cell surface expression of NPSR-A and NPSR-A-N107I variants in transiently-transfected

EK 293 T cells. A) Immunofluorescence microscopy of permeabilized and non-permeabilized cells transiently expressing FLAG-NPSR-A and FLAG-NPSR-A-N107I variants was carried out as described in “Experimental Procedures”. “NP” = non permeabilized cells; “P” = permeabilized cells B) ELISA determinations on non-permeabilized (upper left panel) and permeabilized (lower left panel) cells transiently expressing FLAG-NPSR-A (filled bars) and FLAG-NPSR-A-N107I (empty bars) variants were carried out as described in “Experimental Procedures”. Right panel: normalized cell surface expression of NPSR-A variants, expressed as percent of total receptor expression (100 x OD492 for nonpermeabilized cells / OD492 for permeabilized cells). *p<0.01 compared to NPSR-A using Student’s t-test.

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Effect of receptor expression levels on EC50 and Emax. A) NPS concentration-response curves

(Ca++-mobilization assay) are shown in cells transiently transfected with 0 (empty circles), 0.003 (empty squares), 0.01 (filled squares), 0.03 (filled circles) and 0.1 (filled triangles) mg of pcDEF3(FLAG-NPSRA- N107I) vector DNA, supplemented with pcDEF3 vector to maintain a constant total DNA concentration, as described in “Results”. Each point is the mean ± S.D. of quadruplicate determinations. A representative experiment is shown. B) Emax (filled bars) and EC50 (empty bars) values (± S.E.M.) obtained in transfections using various amounts of pcDEF3(FLAG-NPSR-A-N107I) DNA, corresponding to the representative experiment shown in A).

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Neuropeptide S: A Neuropeptide Promoting Arousal and Anxiolytic-like Effects

Arousal and anxiety are behavioral responses that involve complex neurocircuitries and multiple neurochemical components. Here, we report that a neuropeptide, neuropeptide S (NPS), potently modulates wakefulness and could also regulate anxiety. NPS acts by activating its cognate receptor (NPSR) and inducing mobilization of intracellular Ca2+. The NPSR mRNA is widely distributed in the brain, including the amygdala and the midline thalamic nuclei. Central administration of NPS increases locomotor activity in mice and decreases paradoxical (REM) sleep and slow wave sleep in rats. NPS was further shown to produce anxiolytic-like effects in mice exposed to four different stressful paradigms. Interestingly, NPS is expressed in a previously undefined cluster of cells located between the locus coeruleus (LC) and Barrington's nucleus. These results indicate that NPS could be a new modulator of arousal and anxiety. They also show that the LC region encompasses distinct nuclei expressing different arousal-promoting neurotransmitters.

Xu Y.L., et al. Neuron, Vol 43, 487-497, 19 August 2004 (All peptides used in this publication are manufactured by Phoenix Pharmaceuticals)

Neuropeptide S: A Novel Activating Anxiolytic?

Many different neuropharmacological agents modulate arousal and anxiety, yet to date, few endogenous substances have produced arousal with an anxiolytic effect. In this issue of Neuron, Xu et al. describe the localization and characterization of a novel neuropeptide, neuropeptide S (and its cognate receptor), that is unique in its arousing and anxiolytic-like properties.

George F. Koob, and Thomas N. Greenwell. Neuron, Vol 43, 487-497, 19 August 2004

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 (SNPs) within NPSR have been shown to be associated with an increased prevalance of asthma. One such SNP leads to the missense mutation N107I, which results in an increase in the potency of NPS for NPSR. In order 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 N-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. While 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 (Emax) 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.

Bernier V, et al. J Biol Chem. 2006 Jun 20; [Epub ahead of print]

Mapping in Human Brain Tissue by NPS (1-10) (Human) - Antibody (H-005-81)

Mapping in Mouse & Rat Brain Tissue by NPS, prepro (23-67) (Mouse) - Antibody (H-005-99)

Pharmacological characteristics of NPS Receptor and Neuropeptides

Primary Structures of Neuropeptide S from Human, Chimpanzee, Rat, Mouse, Dog, and Chicken. Amino acids divergent from the human sequence are shown in bold type. Sequences were deduced from GenBank entries BD168686 (human), BD168712 (rat), BD168690 (mouse), BU293859 (chicken), and genome sequencing traces 231487919 (chimpanzee) and 250468833 (dog).

Xu Y.L., et al. Neuron, Vol 43, 487-497, 19 August 2004