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As part of our continuing studies on the structure–activity relationships of cyclic pentapeptides based on the structure of endomorphin-2, we report here the synthesis and biological activities of a new series of analogs incorporating 2′, 3′ or 4′-methylphenylalanine (MePhe) residues into positions 3 or 4 of the parent cyclopeptide, Dmt-c[ d -Lys-Phe-Phe-Asp]NH 2 (Dmt = 2′,6′-dimethyltyrosine). Analogs with MePhe in position 4 showed a row of magnitude increased μ-opioid receptor (MOP receptor) affinity as compared with a parent compound. The in vitro potencies of the new analogs were determined in calcium mobilization assay performed in Chinese Hamster Ovary (CHO) cells expressing human recombinant opioid receptors and chimeric G proteins. All analogs were strong μ/κ (MOP/KOP) receptor agonists and weak δ (DOP) receptor agonists. In the in vivo hot-plate test in mice, the MePhe 4 -modified peptides showed remarkable antinociceptive activity after intracerebroventricular (i.c.v.) administration which was most likely due to the concomitant activation of more than one opioid receptor type.

Neuropeptide S (NPS) is a 20-aminoacid peptide that selectively activates a G-protein coupled receptor named NPSR. Preclinical studies have shown that NPSR activation promotes anxiolysis, hyperlocomotion, arousal and weakfullness. Previous findings suggest that dopamine neurotransmission plays a role in the actions of NPS. Based on the close relationship between dopamine and Parkinson disease (PD) and on the evidence that NPSR are expressed on brain dopaminergic nuclei, the present study investigated the effects of NPS in motor deficits induced by intracerebroventricular (icv) administration of the dopaminergic neurotoxin 6-OHDA in the mouse rotarod test. 6-OHDA injection evoked motor deficits and significantly reduced tyrosine hidroxylase (TH)-positive cells in the substantia nigra (SN) and ventral tegmental area. However, a positive correlation was found only between the motor performance of 6-OHDA-injected mice and the number of TH-positive cells in SN. The systemic administration of l-DOPA+benserazide (25+6.25 mg/kg) counteracted 6-OHDA-induced motor deficits in mice. Similar to L-DOPA, the icv injection of NPS (0.1 and 1 nmol) reversed motor deficits evoked by 6-OHDA. In conclusion, NPS attenuated 6-OHDA-induced motor impairments in mice assessed in the rota-rod test. We discussed the beneficial actions of NPS based on a putative facilitation of dopaminergic neurotransmission in the brain. Finally, these findings candidate NPSR agonists as a potential innovative treatment for PD.

A series of N-substituted analogs based upon the spiropiperidine core of the lead compound Spiroxatrine was synthesized. In particular, the new compounds were obtained by replacing the benzodioxane moiety of the Spiroxatrine with several 2-substituted 1,3-dioxolanes. Thus the designed derivatives were synthesized and evaluated as possible NOP receptor ligands. As a conclusion of these studies, the new triazaspirodecanone derivatives showed unique and significant SAR as NOP receptor agonists. In particular, the present study demonstrated that 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one portion together with appropriate 1,3-dioxolane substituents could lead to a new promising class of NOP receptor ligands.

Neuropeptide S (NPS) regulates different biological functions by selectively activating a G protein coupled receptor named NPSR. Previous studies identified [d-Pen5]NPS as a NPSR antagonist. This study investigated the structure activity relationship of [d-Pen5]NPS by alkylation of the thiol group of d-Pen with different aliphatic and aromatic moieties. Nine compounds were synthesised and assayed in calcium mobilization studies, performed in HEK293 cells expressing the recombinant mouse NPSR. All compounds behaved as pure NPSR antagonists of moderate to high potency, in a similar manner to the reference peptide. The most potent compounds were obtained using alkylating groups as cyclic aromatic moieties. [d-Pen(p- t BuBzl)5]NPS, the best compound of this series, was further investigated in vivo, where it fully prevented the stimulatory effects of supraspinal NPS on mouse locomotor activity. [d-Pen(p- t BuBzl)5]NPS is proposed as a novel pharmacological tool which could be useful to investigate in vitro and in vivo biological functions regulated by the NPS/NPSR receptor system.

Nociceptin/orphanin FQ (N/OFQ) acts as an anxiolytic-like agent in the rat and behaves as a functional antagonist of corticotropin-releasing factor (CRF) due to its ability to oppose CRF biological actions. In response to stress, CRF triggers changes in neurotransmitter systems including serotonin (5-HT). The role of 5-HT1A receptor in anxiety has been supported by preclinical and clinical studies. The present study investigated the possible functional antagonism between N/OFQ (1nmol/rat) and CRF (0.2nmol/rat) in anxiety-related conditions in rats, using elevated plus maze and defensive burying tests, in order to confirm previous literature results. Moreover, possible changes in the serotonergic system were studied in areas rich of serotonergic neurons: frontal cortex and pons. In both tests N/OFQ showed anxiolytic-like effects while CRF displayed anxiogenic-like effects. N/OFQ before CRF treatment counteracted the anxiogenic-like effects evoked by CRF. In frontal cortex, N/OFQ significantly decreased 5-HT levels but did not modify the hydroxyindoleacetic acid (5-HIAA) ones; CRF modified neither 5-HT nor 5-HIAA content but counteracted changes induced by N/OFQ alone. In pons, N/OFQ induced no change in serotonergic activity while CRF significantly decreased 5-HT levels and increased 5-HIAA content. The two peptides' combination reinstated serotonergic parameters to controls. In frontal cortex, N/OFQ increased the 5HT1A receptor density but reduced its affinity, while CRF alone did not induce any change. In pons, CRF decreased 5HT1ABmax and KD whereas N/OFQ was ineffective. All biochemical modifications were reverted by N/OFQ plus CRF treatment. The present study confirms that N/OFQ counteracts CRF anxiogenic-like effects in the behavioral tests evaluated. These effects may involve central serotonergic mechanisms since N/OFQ plus CRF induces a reversion of serotonergic changes provoked by single peptide. Our data support the hypothesis that N/OFQ may behave as functional CRF antagonist, this action being of interest for the treatment of anxiety disorders.

A series of triazospirodecanone derivatives were synthesized as potential NOP ligands. 8-(Chroman-4-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (4) and its 5-fluoro analogue (18) proved to be active as agonists with EC50 values in the submicromolar range. Single enantiomers of compound 4 were separated and tested as NOP agonists; the eutomer R-(+)-4 showed a pEC50 of 7.34. Finally docking studies were performed on the NOP receptor to identify the most significant stereospecific interactions.

Nociceptin/orphanin FQ (NC) and its receptor (OP 4 ) have been implicated in the regulation of various functions including nociception. [Nphe 1 ]NC(1–13)NH 2 (Nphe) is a selective OP 4 antagonist which prevents the pronociceptive effects of supraspinal NC and causes per se a naloxone-insensitive antinociceptive effect. In the present study, we tested Nphe in wild type (WT) and OP 4 receptor knock out mice and found that a clear antinociceptive effect of the antagonist was evident only in WT mice. Moreover, we evaluated, over 5 days of treatment, the antinociceptive effects of Nphe in comparison with those of DAMGO and found that tolerance develops to the effects of the opioid receptor agonist but not to Nphe. These data demonstrate that the antinociceptive action of Nphe is due to the block of OP 4 receptors and that no tolerance develops to this kind of antinociception.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.

Nociceptin/orphanin FQ (NC) and its receptor (OP4) have been implicated in pain transmission. The aim of the present study was to investigate the role of the NC/OP4 system in stress-induced analgesia (SIA). The tail-withdrawal assay was performed in mice stressed by forced swimming in water at 15 degrees C (high severity swims) or 32 degrees C (low severity swims). High severity swims produced a naloxone-insensitive antinociceptive effect which was blocked by supraspinal NC (1 nmol). The selective OP4 receptor antagonist, [Nphe1]NC(-13)NH2 (30 nmol), was inactive by itself, but prevented the effect of NC. Low severity swims produced a milder analgesic effect that was partially antagonized by naloxone, completely blocked by NC and potentiated by [Nphe1]NC(-13)NH2. These findings confirm the anti-analgesic role of supraspinal NC and suggest that endogenous NC signaling counteracts the opioid component of SIA.

In this study we administered nociceptin/orphanin FQ (NC) ionotophoretically onto neurons located in functionally distinct thalamic structures of urethane-anesthetized rats. Extracellular single unit recordings were made in the medial and lateral ventroposterior nucleus, posterior thalamic nucleus, zona incerta, lateral posterior nucleus, laterodorsal nucleus, ventrolateral nucleus and reticular nucleus. NC decreased the firing rate in 60% of thalamic neurons. This decrease in firing rate was accompanied by a significant reduction in the number of high threshold bursts. In about 20% of the neurons NC increased the firing rate. In most cells NC-induced increases in discharge rate could be blocked by the GABAA receptor antagonists bicuculline and SR 95531. The NC receptor ligands [Phe1Ψ(CH2-NH)Gly2] nociceptin(1-13)NH2, Ac-RYYRIK-NH2 and [Nphe1]NC(1-13)NH2 were also evaluated. All these peptides inhibited NC-induced changes in firing rate. In addition, in some neurons where NC inhibited firing, [Nphe1]NC(1-13)NH2 and Ac-RYYRIK-NH2 elicited per se an increase in firing rate, suggesting the existence of tonic innervation of thalamic neurons by NC-containing fibres. In NC-inhibited neurons nocistatin induced a significant increase in firing rate. The present study demonstrated that NC regulates various thalamic nuclei related not only to somatosensory, but also to the visual and motor functions. British Journal of Pharmacology (2001) 134, 333–342; doi:10.1038/sj.bjp.0704264

Aims To examine the effects of nociceptin (NC) and endomorphin 1 (EM1) on electrical field stimulation (EFS)-induced contractions of the human vas deferens (hVD). Methods Concentration-response curves to NC and EM1 were constructed in the absence and in presence of peptidase inhibitors (PI). In some experiments a NC receptor antagonist. [Phe 1 -(CH 2 -NH)Gly 2 ]NC(1-13)NH 2 [F/G], 10 μM) or naloxone (1 μM) were included. Results All data are mean(95%CI). In the presence of PI, NC inhibited twitches (E max = 67(44,90)%; pEC 50 = 7.28(6.95,7.61)). NC inhibition was sensitive to [F/G]. EM1 also inhibited twitches both in the absence (E max = 82(73,91)% pEC 50 = 7.07 (6.92,7.22)) and presence (E max = 83(76.90)%; pEC 50 = 7.00(6.91,7.09)) of PI. EM1 inhibition was sensitive to naloxone. Conclusions These data suggest that bVD express NC and opioid receptors that inhibit neurogenic contractions.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.

Studies of nociceptin/orphanin FQ (NC) have been hampered by the paucity of available ligands with activity at the nociceptin receptor (NCR). In this study we have compared the agonist profile of NC and a novel NCR agonist, Ro65-6570, in a series of radioligand binding studies and effects on forskolin-stimulated cAMP formation in Chinese hamster ovary (CHO) cells expressing the recombinant human NCR (CHOhNCR). In addition, we report the effects of three antagonists, [Nphe1]NC(1–13)NH2, J-113397 and III-BTD, on these responses. In radioligand binding studies Ro65-6570, [Nphe1]NC(1–13)NH2, J-113397 and III-BTD displaced [3H]NC with similar pK i values (8.4–8.8). This compares with a pK D of 10.2 for NC in a direct saturation experiment. [Nphe1]NC(1–13)NH2 and J-113397 showed at least 100-fold selectivity over classical opioid receptors. Both NC and Ro65-6570 produced a concentration-dependent inhibition of cAMP formation with pEC50 values of 9.56±0.06 and 8.68±0.04, respectively. Maximum inhibition achieved was 100%. [Nphe1]NC(1–13)NH2, J-113397 and III-BTD produced a parallel rightward shift in the concentration-response curves to both NC and Ro65-6570 with pK B values of ~6.5, ~7.5 and ~7.7, respectively. Importantly, all three antagonists were devoid of residual agonist activity. Collectively, these data indicate the value of Ro65-6570, [Nphe1]NC(1–13)NH2, J-113397 and III-BTD in studies of the physiological role played by NC. However, due to the relatively poor selectivity of Ro65-6570 and III-BTD caution should be exercised when using tissues that co-express µ-opioid receptors.

The present study was undertaken to investigate the effects of the novel nociceptin receptor antagonist, [Nphe1]-Nociceptin (1-13)-NH2 (bilateral intrahippocampal injection, 50 nmole rat−1) on purported nociceptin-induced (bilateral intrahippocampal injection, 5 nmole rat−1) deficits in spatial learning in the rat Morris water maze task. In addition, experiments were performed in an ‘open field’ to investigate possible peptide-induced changes in exploratory behaviour. Nociceptin significantly impaired the ability of the animal to locate the hidden platform throughout training (P

Peptide III-BTD has been recently identified as a non selective nociceptin/orphanin FQ receptor ligand by screening of a synthetic peptide combinatorial library. In the present study we evaluated the pharmacological profile of peptide III-BTD in isolated tissues (mouse and rat vas deferens, guinea pig ileum) sensitive to both nociceptin and opioid peptides. In the mouse vas deferens and guinea pig ileum, III-BTD concentration dependently inhibited the electrically induced twitch (pEC50 5.91 and 6.18, respectively; Emax 94 ± 1% and 94 ± 2%) and this effect was blocked by naloxone (1 μM). In the rat vas deferens, III-BTD was inactive in most of the tissues, while in few others it elicited a slight inhibition only at the highest concentration tested (10 μM). In the presence of 1 μM naloxone, 1 μM III-BTD shifted to the right the concentration response curve of nociceptin in a parallel manner, showing pKB values in the range 6.6–6.9. These data confirm on native nociceptin receptors the pharmacological profile of peptide III-BTD which behaved as a mixed nociceptin receptor antagonist / opioid receptor agonist in the [35S]GTPγS binding assay performed on cells expressing the recombinant human receptors.

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH(2) was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH(2) inhibited spontaneous locomotor activity in mice with ID(50) of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH(2) could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH(2), [Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-NH(2) ([F/G]NC(1-13)NH(2)) inhibited the noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH(2) in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH(2) and of [F/G]NC(1-13)NH(2), it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.

When suspended in vitro in isolated organ baths, segments of the rabbit ileum show a fairly strong and stable spontaneous activity, which derives from the continuous release of acetylcholine and the activation of muscarinic receptors, since the activity is completely eliminated by atropine. Dynorphin A (pEC(50): 8.6+/-0.07), neuropeptide Y and its congener human pancreatic polypeptide (pEC(50): 9.40+/-0.10), and nociceptin (pEC(50): 8.08+/-0.12) dose-dependently inhibit the spontaneous activity through the activation of receptors, which are specifically antagonised respectively by naloxone (pA(2): 7.17+/-0.12), 2-(naphtalen-1-ylamino)-3-phenylpropionitrile (JCF 104; pA(2): 5. 80+/-0.10), and [Nphe(1)]nociceptin-(1-13)NH(2) (pA(2): 6.17+/-0.19). This last compound, a selective nociceptin-receptor (OP(4)) antagonist, inhibits the effect of nociceptin in a competitive manner, as demonstrated by Schild analysis. [Nphe(1)]nociceptin-(1-13)NH(2) also antagonizes the effects of other OP(4) receptor ligands such as the full agonist, nociceptin-(1-13)-NH(2), and the partial agonists, [Phe(1)psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) (intrinsic activity (alpha(E))=0.5) and Ac-RYYWK-NH(2) (alpha(E)=0.5), with pA(2) values ranged from 5.8 to 6.2. These results indicate that the functional site mediating the inhibitory effect of nociceptin in the rabbit ileum, is pharmacologically identical to the OP(4) sites of other species (mouse, rat, guinea pig, man), since the potencies (pA(2) values) of the pure and competitive antagonist [Nphe(1)]nociceptin-(1-13)NH(2) is very similar to the values obtained in the other species. Moreover, the rabbit ileum is one of the few isolated organs that allow classifying compounds, which interact with OP(4) receptors as full agonists, partial agonists, or pure antagonists.

The modulation of [3H]-5-hydroxytryptamine ([3H]-5-HT) efflux from superfused rat cortical synaptosomes by delta, kappa, mu and ORL1 opioid receptor agonists and antagonists was studied. Spontaneous [3H]-5-HT efflux was reduced (20% inhibition) by either 0.5 μM tetrodotoxin or Ca2+-omission. Ten mM K+-evoked [3H]-5-HT overflow was largely Ca2+-dependent (90%) and tetrodotoxin-sensitive (50%). The delta receptor agonist, deltorphin-I, failed to modulate the K+-evoked neurotransmitter efflux up to 0.3 μM. The kappa and the mu receptor agonists, U-50,488 and endomorphin-1, inhibited K+-evoked [3H]-5-HT overflow (EC50=112 and 7 nM, respectively; Emax=28 and 29% inhibition, respectively) in a norBinaltorphimine- (0.3 μM) and naloxone- (1 μM) sensitive manner, respectively. None of these agonists significantly affected spontaneous [3H]-5-HT efflux. The ORL1 receptor agonist nociceptin inhibited both spontaneous (EC50=67 nM) and K+-evoked (EC50=13 nM; Emax=52% inhibition) [3H]-5-HT efflux. The effect of NC was insensitive to naloxone (up to 10 μM), but was antagonized by [Nphe1]nociceptin(1-13)NH2 (a novel selective ORL1 receptor antagonist; pA2=6.7) and by naloxone benzoylhydrazone (pA2=6.3). The ORL1 ligand [Phe1ψ(CH2-NH)Gly2]nociceptin(1-13)NH2 also inhibited K+ stimulated [3H]-5-HT overflow (EC50=64 nM; Emax=31% inhibition), but its effect was partially antagonized by 10 μM naloxone. It is concluded that the ORL1 receptor is the most important presynaptic modulator of neocortical 5-HT release within the opioid receptor family. This suggests that the ORL1/nociceptin system may have a powerful role in the control of cerebral 5-HT-mediated biological functions. British Journal of Pharmacology (2000) 130, 425–433; doi:10.1038/sj.bjp.0703321

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA 2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pK i of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA 2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA 2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 µM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA 2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system.

Nociceptin (NC), alias Orphanin FQ, has been recently identified as the endogenous ligand of the opioid receptor-like 1 receptor (OP4). This new NC/OP4 receptor system belongs to the opioid family and has been characterized pharmacologically with functional and binding assays on native (mouse, rat, guinea-pig) and recombinant (human) receptors, by using specific and selective agonists (NC, NC(1–13)NH2) and a pure and competitive antagonist, [Nphe1]NC(1–13)NH2. The similar order of potency of agonists and affinity values of the antagonist indicate that the same receptor is present in the four species. OP4 is expressed in neurons, where it reduces activation of adenylyl cyclase and Ca2+ channels while activating K+ channels in a manner similar to opioids. In this way, OP4 mediates inhibitory effects in the autonomic nervous system, but its activities in the central nervous system can be either similar or opposite to those of opioids. In vivo experiments have demonstrated that NC modulates a variety of biological functions ranging from nociception to food intake, from memory processes to cardiovascular and renal functions, from spontaneous locomotor activity to gastrointestinal motility, from anxiety to the control of neurotransmitter release at peripheral and central sites. These actions have been demonstrated using NC and various pharmacological tools, as antisense oligonucleotides targeting OP4 or the peptide precursor genes, antibodies against NC, an OP4 receptor selective antagonist and with data obtained from animals in which the receptor or the peptide precursor genes were knocked out. These new advances have contributed to better understanding of the pathophysiological role of the NC/OP4 system, and ultimately will help to identify the therapeutic potential of new OP4 receptor ligands.

Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G-protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe1]nociceptin(1-13)NH2 acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. [Nphe1]nociceptin(1-13)NH2 binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pKi 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA2 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea-pig with pA2 values ranging from 6.0 to 6.4. [Nphe1]nociceptin(1-13)NH2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe1]nociceptin(1-13)NH2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine-induced analgesia. Collectively our data indicate that [Nphe1]nociceptin(1-13)NH2, acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics. British Journal of Pharmacology (2000) 129, 1183–1193; doi:10.1038/sj.bjp.0703169

1 Stimulation of the opioid receptor-like1 (ORL-1) receptor by nociceptin (NC) produces hyperalgesia and reverses the antinociceptive effects induced by opioids. Most studies concerning the central effects of NC were conducted using acute pain models. The role NC may play in chronic inflammation remains unelucidated. 2 The present study was undertaken to assess the action of NC in the Freund's adjuvant-induced monoarthritic rat model. The effects of drugs known to act as analgesics in this model were evaluated. The effects of NC, NCNH2, and the ORL-1 ligand, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2), were also studied alone or in association with morphine. 3 NC (1 - 30 nmol, i. c.v.) was inactive, whilst NCNH2 (10 nmol, i.c.v.) exerted hyperalgesic effects (-4.5+/-0.9 vs -0.7+/-0.8 s of vehicle-treated animals). [F/G]NC(1-13)NH2 (0.01 - 10 nmol, i.c.v.) induced hyperalgesia in the arthritic paw (-3.3+/-0.6 vs -0.3+/-0.5 s of vehicle-treated animals; 10 nmol). 4 Both NC (0.01 - 10 nmol, i.c.v. ) and [F/G]NC(1-13)NH2 (0.01 - 1 nmol, i.c.v), 30 min after morphine (3 mg kg-1, s.c.) induced an immediate and short-lived reversal of morphine effects (2.6+/-0.3 vs 10.4+/-1.0 and 1.2+/-1.5 vs 9.3+/-1.1 s of morphine alone, respectively), therefore displaying anti-opioid activity. 5 In the Freund's adjuvant-induced rat model of arthritis, both NC and [F/G]NC(1-13)NH2 act as anti-opioid peptides. Furthermore, NCNH2 and [F/G]NC(1-13)NH2 induce hyperalgesia when given alone. Further investigations and the identification of a centrally acting ORL-1 antagonist are necessary to better understand the role of NC in pain mechanisms.

WE studied the effects of intrathecal (i.t.) nocistatin, a peptide identified from the precursor of orphanin FQ/ nociceptin (OFQ) on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats and its interaction with i.t. OFQ. Nocistatin induced a moderate, non-dose-dependent facilitation of the flexor reflex without producing reflex depression whereas i.t. OFQ induced a biphasic dose-dependent facilitatory and inhibitory effect. The facilitatory effect of low dose (0.55 pmol) OFQ was significantly increased by nocistatin. On the other hand, the duration, but not magnitude, of reflex depression induced by a high (550 pmol) dose of OFQ was significantly shortened by 5.5 nmol nocistatin. Thus, nocistatin interacts with OFQ in a complex fashion, increasing excitation and reducing inhibition. No evidence was obtained for an antinociceptive effect of nocistatin in rat spinal cord.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1ΨC(CH2-NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms. British Journal of Pharmacology (1999) 127, 123–130; doi:10.1038/sj.bjp.0702539

we studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe 1 ψ(CH 2 -NH)Gly 2 ]NC(1–13)NH 2 ([ F G ]NC(1–13)NH 2 ). NC inhibited in a concentration dependent manner (pEC 50 7.14; E max − 87 ± 3% of control values) EPS induced contractions. NC effect was mimicked by the NC analogues, NCNH 2 and NC(1–13)NH 2 , but not by NC(1–9)NH 2 NC (1 μM) did not affect the contractile effects of exogenously applied neurokinin A (1 μM). [ F G ]NC(1–13)NH 2 (10 μM) completely prevented the inhibition induced by NC (1 μM), whereas naloxone (1 μM) was found inactive. Both naloxone and ([ F G ]NC(1–13)NH 2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.

1 The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol−1 2 Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nm and Bmax of 94 fmol mg−1 of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs=0.104 min−1; K1=0.034 min−1; T1/2=20 min; K+1=0.07 min nm−1. 3 Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(δ), OP2(κ) and OP3(μ). 4 Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1ψ(CH2-NH)Gly2]NC(1–13)-NH2 ([F/G]NC(1–13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH=NC-NH2=NC(1–13)-NH2 >> NC(1–12)-NH2 > NC(1–13)-OH >> NC(1–11)-NH2, and for NC and NC(1–13)-NH2 analogues: [Tyr1]NC-NH2 [Leu1]NC(1–13)-NH2 [Tyr1]NC(1–13)-NH2 [F/G]NC(1–13)-NH2 >> [Phe3]NC(1–13)-NH2 > [DF/G]NC(1–13)-NH2. 5 Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 μm indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor. British Journal of Pharmacology (1998) 125, 1485–1490; doi:10.1038/sj.bjp.0702226

Purpose: Smooth muscle reactivity is one of the factors involved in the pathogenesis of varicose veins. We investigated the myotropic effects of the 3 main vasoconstrictor agents—norepinephrine (NE), angiotensin II (Ang II), and endothelin-1 (ET-1)—in isolated human saphenous veins. Methods: Human saphenous veins were collected from 23 patients with primary chronic venous insufficiency who underwent elective varicose vein resections and who were stratified into the following 3 groups: group 1, 7 patients in clinical class 2; group 2, 9 patients in clinical classes 3 and 4; and group 3, 7 patients in clinical classes 5 and 6. Moreover, 6 patients who underwent arterial bypass grafting procedures represented the control group. The tissues were suspended in organ baths that contained Krebs solution, and their mechanical responses were measured isometrically. The cumulative concentration–response curves to Ang II, NE, and ET-1 were performed at 90-minute intervals in each tissue. Results: In the control tissues, NE, Ang II, and ET-1 induced concentration-dependent contractions with apparent affinities (pEC50, the negative logarithm to base 10 of the molar concentration of the agonist, which produces the 50% of the maximal effect) and maximal effects (maximum effect, g of contraction) that were equal to 7.06 ± 0.23, 8.53 ± 0.34, 7.63 ± 0.10, and 2.21 ± 0.33, 1.65 ± 0.31, 2.60 ± 0.77, respectively. Two main findings were evident in comparison of varicose veins with control tissues. First, the maximum effect that was evoked by all of the stimulants was reduced progressively with the increasing severity of the disease, which raised the third group to statistical significance for both NE and Ang II (P < .05). Second, a marked reduction of Ang II apparent affinity was already evident in tissues that were taken from patients in an early stage of the disease (P < .05). Conclusion: The demonstration of a significant reduction in Ang II and NE contractile activities and the important reduction of that of ET-1 in the diseased veins as compared with the control tissues extends the previous observations regarding the impairment of smooth muscle contractility in primary chronic venous insufficiency. Moreover, the dramatic reduction of Ang II affinity, which appears in an early stage of the disease, supports the hypothesis that such abnormality within the venous wall could play a role in the pathogenesis of primary varicose vein disease. (J Vasc Surg 1998;28:855-61.)

1 The newly discovered neuropeptide nociceptin (NC) has recently been reported to be the endogenous ligand of the opioid-like orphan receptor. Despite its structural similarity to opioids, when injected intracerebroventricularly (i.c.v.) in the mouse, NC exerts a direct hyperalgesic effect and reverses opioid-induced analgesia. In the present investigation, these two effects of NC were evaluated under the same experimental conditions; in addition, a pharmacological characterization of the receptor mediating these central effects of NC was attempted. 2 NC caused a dose dependent (0.1–10 nmol/mouse), naloxone-insensitive reduction of tail withdrawal latency with a maximal effect of about 50% of the reaction time observed in saline injected mice. In the same range of doses, NC inhibited morphine (1 nmol/mouse) induced analgesia. 3 The effects of the natural peptide were mimicked by NCNH2 and NC(1–13)NH2 (all tested at 1 nmol/mouse) while 1 nmol NC(1–9)NH2 was found to be inactive either in reducing tail withdrawal latency or in preventing morphine analgesia. 4 [Phe1ψ(CH2-NH)Gly2]NC(1–13)NH2 ([F/G]NC(1–13)NH2), which has been shown to antagonize NC effects in the mouse vas deferens, acted as an agonist, mimicking NC effects in both the experimental paradigms. In addition, when NC and [F/G]NC(1–13)NH2 were given together, their effects were additive. 5 These results demonstrate that both the direct hyperalgesic action and the anti-morphine effect of NC can be studied under the same experimental conditions in the mouse tail withdrawal assay. Moreover, the pharmacological characterization of the NC functional site responsible for these actions compared with the peripherally active site, indicates the existence of important differences between peripheral and central NC receptors. British Journal of Pharmacology (1998) 125, 373–378; doi:10.1038/sj.bjp.0702087

In conclusion, although a case of combination intoxication is less informative than that involving a single drug alone, the specific type of ECG alteration observed (never previously reported for drugs without harmful cardiotoxic effects), and its complete regression and disappearance commensurate with normalisation of serum drug concentrations, are quite unique and worthy of note.

1 The human umbilical vein responds to bradykinin (BK) with contractions that are mediated by B2 receptors. In the present study, the corresponding vascular smooth muscle B2 binding sites have been investigated. 2 [3H]-BK, a full agonist labelled ligand, was used to demonstrate a single binding site giving a Kd value of 0.51±0.02 nM and a Bmax of 24±1 fmol mg−1 protein. Scatchard plots were linear (r=0.98) in the 0.05–5 nM range of concentrations. Non-specific binding was found to be 30% of total binding. 3 Competition binding curves gave the following order of potency for various B2 receptor agonists: BK-[Hyp3]-BKLys-BK>>[Aib7]-BK>>>[desArg9]-BK, which is typical of B2 receptors. There was no binding to B1 receptors since the selective B1 receptor ligand, Lys-[desArg9]BK was inactive up to 10 μM (n=4). 4 Characterization of the binding site with antagonists, performed with three chemically distinct series of peptide and non-peptide compounds, revealed a high affinity of Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-BK) (Ki 0.17 nM; n=4) which was more potent that FR 173657 ([(E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide]) (Ki 1.94 nM; n=4), D-Arg-[Hyp3,D-Phe7,Leu8]-BK (Ki 256 nM; n=4) and Win 64338 (phosphonium, [[4[[2[[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopropyl]amino]phenyl]methyl]tributyl, chloride, monohydrochloride) (Ki 1,450 nM; n=4). 5 The present study describes and characterises B2 receptor binding sites in the vascular smooth muscle of the human umbilical vein. The binding assay appears to be suitable for studying new agonists or antagonists designed to activate or block the B2 receptor class that mediate the majority of the physiopathological effects of kinins in man.

The coronary artery and renal vein of the adult pig are sensitive and reliable monoreceptor systems for studying kinin receptors. The pig coronary artery with intact endothelium is highly sensitive to bradykinin (BK, pEC50 8.6), while being insensitive to the B1 receptor agonist, LysdesArg9BK. The tissue responds to BK with concentration-dependent relaxation, which is prevented by B2 receptor antagonists, particularly dArg[Hyp3, Thi5, dTic7, Oic8]BK (HOE 140, pKB 9.3), (E)-3-(6-acetoamido-3-pyridyl)-N-(N-{2, 4-dichloro-3-[(2-methyl-8-quinolinyl)oxy-methyl]phenyl}-N-methylaminocarbonylmethyl)acrylamide (FR 173657), a new non peptide compound (pK B 9.3), while B1 receptor antagonists (e.g. Lys[Leu8]desArg9BK) are inactive. The order of potency of kinin-related peptides in this vessel is: LysBK ≥ BK > [Hyp3]BK>[Aib7]BK, a sequence typical of a B2 receptor system. Antagonists such as HOE 140 and FR 173657, at high concentrations reduce the maximum effect of BK and thus behave as noncompetitive antagonists. The kinin B1 receptor was studied in the pig renal vein without endothelium and incubated for several hours in order to allow for the de novo formation of this functional site. After 7–8 h in vitro incubation, the vessel shows high sensitivity to LysdesArg9BK (pEC50 8.3) and is insensitive to BK. The pig renal vein responds to B1 receptor agonists with concentration-dependent contraction which maintains a stable plateau and is prevented by selective B1 receptor antagonists such as Lys[Leu8]desArg9BK (pKB 6.7). The most active antagonist has been found to be desArg9HOE 140 (pA2 7.6) which acts as competitive antagonist in this preparation. Some B2 antagonists (e.g. HOE 140) show weak (pK B 6.1) anti-B1 receptor activity while the non-peptide compound FR 173657 is inactive on the B1 receptor and therefore acts as a potent and selective kinin B2 receptor antagonist in the pig. The data obtained in this study allow us to compare the porcine B2 and B1 receptors with those of other species including man, and underline some interesting features that are unique to the porcine functional sites.

High throughput calcium mobilization assays are extensively used for pharmacological characterization of GPCR ligands. These approaches, initially developed for G(q)-coupled receptors, can be extended to G(i) coupled GPCRs using chimeric G proteins. Here we used the Gα(qi5) protein to force the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, as well as the classical opioid receptors to signal through the PLC-IP(3)-Ca(2+) pathway in CHO cells. Calcium levels were monitored using the fluorometric imaging plate reader FlexStation II and the Ca(2+) dye Fluo 4 AM. For investigating the pharmacology of the NOP receptor a panel of full and partial agonists and antagonists were assessed, while a small panel of agonists and antagonists was used for evaluating the pharmacological profile of opioid receptors. Some limitations of this assay and differences in the results obtained in comparison with those with G(i) based biochemical assays are described. Overall, the present results confirm that the chimeric G protein strategy is useful for studying the pharmacological activity of G(i) coupled receptor ligands and that the aberrant signaling does not produce any measurable change in the pharmacological profile of the receptor under study. Thus, this G protein strategy is extremely useful for setting up primary screening assays for NOP and classical opioid receptors and likely for other members of the GPCR family.

It is our contention that the concept of a probiotic as a living bacterium providing unspecified health benefits is inhibiting the development and establishment of an evidence base for the growing field of pharmacobiotics. We believe this is due in part to the current regulatory framework, lack of a clear definition of a probiotic, the ease with which currently defined probiotics can be positioned in the market place, and the enormous profits earned for minimum investment in research. To avoid this, we believe the following two actions are mandatory: international guidelines by a forum of stakeholders made available to scientists and clinicians, patient organizations, and governments; public research funds made available to the scientific community for performing independent rigorous studies both at the preclinical and clinical levels.

Rationale Previous studies have shown that activation of brain neuropeptide S receptor (NPSR) facilitates reinstatement of cocaine seeking elicited by environmental cues predictive of drug availability. This finding suggests the possibility that blockade of NPSR receptors may be of therapeutic benefit in cocaine addiction. To evaluate this hypothesis, we investigated the effect of two newly synthetized NPSR antagonists, namely the quinolinone-amide derivative NPSR-QA1 and the NPS peptidic analogue [D-Cys (tBu) 5 ]NPS on cocaine self-administration and on discriminative cue-induced relapse to cocaine seeking in the rat. Methods Separate groups of rats self-administered food and cocaine 0.25 mg/kg/inf in FR1 and FR5 (fixed ratio reinforcement schedules) for 30-min and 2-h sessions per day. After food and cocaine intake reached baseline levels, the effect of NPSR-QA1 was tested on cocaine and food selfadministration. The NPSR-QA1 was injected intraperitoneally and its effect on discriminative cue-induced reinstatement was evaluated, while [D-Cys(tBut) 5 ]NPS was injected intracranially, intra-lateral hypothalamus, intra-perifornical area of the hypothalamus, and intra-central amygdala. The effect of the NPSR-QA1 on extinction of cocaine seeking was also assessed. Results Intraperitoneal administration of NPSR-QA1 (15– 30 mg/kg) did not affect cocaine self-administration. Conversely, NPSR-QA1 (15–30 mg/kg) decreased discriminative cue-induced cocaine relapse. At the lowest dose, this effect was specific, while at the highest dose, NPSR-QA1 also reduced food self-administration. The efficacy of NPSR antagonism on cocaine seeking was confirmed with [D-Cys (tBu) 5 ]NPS (10–30 nmol/rat) as it markedly inhibited relapse behavior following site-specific injection into the lateral hypothalamus and the perifornical area of the hypothalamus but not into the central amygdala. Conclusions The identification of the NPS/NPSR system as an important new element involved in the physiopathology of cocaine addiction and the discovery of the anti-addictive properties of NPSR antagonists opens the possibility of exploring a new mechanism for cocaine addiction treatment.

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 (HEK293mNPSR) and human (HEK293hNPSRIle107 and HEK293hNPSRAsn107) 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 (pKB 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 mgkg(-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.

Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) were identified in the mid 90s as a novel peptidergic system structurally related to opioids. A growing body of preclinical evidence suggests that blockade of NOP receptors evokes antidepressant-like actions. These have been explored using a range of compounds (peptide and non peptide antagonists), across different species (rat and mouse) and assays (behavioral despair and chronic mild stress) suggesting a robust and consistent antidepressant-like effect. Moreover, rats and mice knockout for the NOP receptor gene display an antidepressant-like phenotype in behavioral despair assays. Electrophysiological, immunohistochemical and neurochemical studies point to an important role played by monoaminergic systems, particularly 5-HTergic, in mediating the antidepressant-like properties of NOP antagonists. However other putative mechanisms of action, including modulation of the CRF system, circadian rhythm and a possible neuroendocrine-immune control might be involved. A close relationship between the N/OFQ-NOP receptor system and stress responses is well described in the literature. Stressful situations also alter endocrine, behavioral and neurochemical parameters in rats and chronic administration of a NOP antagonist restored these alterations. Interestingly, clinical findings showed that plasma N/OFQ levels were significantly altered in major and post-partum depression, and bipolar disease patients. Collectively, data in the literature support the notion that blockade of NOP receptor signaling could be a novel and interesting strategy for the development of innovative antidepressants.

Species-related specific differences in the pharmacological profile of vasopressin V1a receptors have been reported. Thus, the aim of the present study was to identify a vascular preparation of human origin expressing V1a receptors. Vasopressin was found to contract human gastric artery strips without endothelium with high affinity (pEC50 8.9). The maximal effect induced by vasopressin was inversely related to the diameter of the vessel. Oxytocin was found to contract the human gastric artery strips with low potency (pEC50 7.2). Contraction induced by vasopressin was competitively antagonized by the non peptide vasopressin receptor antagonists SR 49059 (pA2 9.2), OPC 21268 (pA2 6.2) and OPC 31260 (pA2 7.1). The order of potency of agonists (vasopressin > > oxytocin) and of antagonists (SR 49059 > > OPC 31260 > OPC 21268) indicate the contraction induced by vasopressin in the isolated human gastric artery is mediated by the V1a receptor type. The present data are similar to those obtained in different preparations expressing the native human V1a receptor as well as to those obtained in cell transfected with this receptor. The human gastric artery is a monoreceptor system of great utility for studying the effects of new drugs interacting with the human V1a receptor.

Our manuscript by Piekielna et al. entitled “Synthesis of mixed opioid affinity cyclic endomorphin-2 analogues with fluorinated phenylalanines” (DOI: 10.1021/acsmedchemlett.5b00056) was published in ACS Medicinal Chemistry Letters in April 2015.

High throughput calcium mobilization assays are extensively used for pharmacological characterization of GPCR ligands. These approaches, initially developed for G(q)-coupled receptors, can be extended to G(i) coupled GPCRs using chimeric G proteins. Here we used the Gα(qi5) protein to force the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, as well as the classical opioid receptors to signal through the PLC-IP(3)-Ca(2+) pathway in CHO cells. Calcium levels were monitored using the fluorometric imaging plate reader FlexStation II and the Ca(2+) dye Fluo 4 AM. For investigating the pharmacology of the NOP receptor a panel of full and partial agonists and antagonists were assessed, while a small panel of agonists and antagonists was used for evaluating the pharmacological profile of opioid receptors. Some limitations of this assay and differences in the results obtained in comparison with those with G(i) based biochemical assays are described. Overall, the present results confirm that the chimeric G protein strategy is useful for studying the pharmacological activity of G(i) coupled receptor ligands and that the aberrant signaling does not produce any measurable change in the pharmacological profile of the receptor under study. Thus, this G protein strategy is extremely useful for setting up primary screening assays for NOP and classical opioid receptors and likely for other members of the GPCR family.

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.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.

The novel NK(1) receptor ligand Netupitant has been characterized in vitro and in vivo. In calcium mobilization studies CHO cells expressing the human NK receptors responded to a panel of agonists with the expected order of potency. In CHO NK(1) cells Netupitant concentration-dependently antagonized the stimulatory effects of substance P (SP) showing insurmountable antagonism (pK(B) 8.87). In cells expressing NK(2) or NK(3) receptors Netupitant was inactive. In the guinea pig ileum Netupitant concentration-dependently depressed the maximal response to SP (pK(B) 7.85) and, in functional washout experiments, displayed persistent (up to 5h) antagonist effects. In mice the intrathecal injection of SP elicited the typical scratching, biting and licking response that was dose-dependently inhibited by Netupitant given intraperitoneally in the 1-10mg/kg dose range. In gerbils, foot tapping behavior evoked by the intracerebroventricular injection of a NK(1) agonist was dose-dependently counteracted by Netupitant given intraperitoneally (ID(50) 1.5mg/kg) or orally (ID(50) 0.5mg/kg). In time course experiments in gerbils Netupitant displayed long lasting effects. In all the assays Aprepitant elicited similar effects as Netupitant. These results suggest that Netupitant behaves as a brain penetrant, orally active, potent and selective NK(1) antagonist. Thus this molecule can be useful for investigating the NK(1) receptor role in the control of central and peripheral functions. Netupitant has clinical potential in conditions such as chemotherapy induced nausea and vomiting, in which the blockade of NK(1) receptors has been demonstrated valuable for patients.

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.

The increased attention to biologic rhythms and clinical manifestations of several diseases has recently led to the identification of "chronorisks" for certain pathologic events. To evaluate whether a temporal pattern exists for acute gastrointestinal bleeding, 369 consecutive patients with acute gastrointestinal bleeding observed at the emergency department (ED) of S. Anna Hospital of Ferrara, Italy, during a 3-year period (1988-1990) were studied. On presentation to the ED, 287 patients had upper gastrointestinal tract bleeding (UGB), and 82 patients had lower gastrointestinal tract bleeding (LGB). In 270 cases of UGB and in 75 cases of LGB, the hour of symptom onset was known and the data were analyzed for the number of observed versus expected events during the four 6-hour intervals of a day. The majority of events occurred in the morning (32.2% of UGB and 42.7% of LGB). Cosinor analysis yielded a circadian rhythmicity with a peak in the late morning to early afternoon for both UGB and LGB independent of sex and site of bleeding.

BACKGROUND: Neuropeptide S (NPS) and its receptor (NPSR) is a novel neuropeptide system that regulates arousal and anxiety. A link between natural sleep and general anesthesia has been suggested. Therefore, we hypothesized that the NPS neuronal system may also modulate general anesthesia. METHODS: The effects of intracerebroventricular NPS and [D-Cys(tBu)5]NPS, a peptide NPSR antagonist, on ketamine and thiopental anesthesia time were measured in rats. Anesthesia time was defined as the interval between the loss of righting reflex and its recovery. RESULTS: Intracerebroventricular NPS 1 to 30 nmol significantly reduced ketamine anesthesia time, showing a bell-shaped dose-response curve. [D-Cys(tBu)5]NPS 20 nmol antagonized NPS 1 nmol effects and was per se able to increase ketamine anesthesia time. Similar results were obtained investigating thiopental anesthesia time that was significantly reduced by NPS and prolonged by [D-Cys(tBu)5]NPS. CONCLUSION: NPS via selective NPSR activation stimulates the wakefulness-promoting pathway, thus reducing anesthesia duration. The endogenous NPS/NPSR system seems to tonically control these pathways.

Aims Mucosal mast cells (MMC) are mediators of the stress responses in the gastrointestinal tract. We examined the effect of acute cold-restraint stress and of the neuropeptide nociceptin/orphanin FQ (N/OFQ), implicated in the modulation of stress responses, on MMC density in the rat colon. Main methods Stress was induced by restraining the animals into individual cages at 3 °C for 3 h. N/OFQ and the selective N/OFQ peptide (NOP) receptor antagonist, UFP-101, were infused subcutaneously via Alzet osmotic minipumps. Segments of the distal colon were collected. MMCs were identified immunohistochemically with a monoclonal antibody to rat mast cell protease (RMCP) II and with a rabbit polyclonal antibody to CD117/c-kit receptor. Key findings Acute stress caused a decrease in the density of MMCs in the rat colonic mucosa. Short-term peripheral infusion of N/OFQ (0.1 to 10 μg/kg/h for 4 h) caused a dose-related reduction of MMC density. Peak reduction occurred after the 4-h infusion of N/OFQ, 1 μg/kg/h. Reduction was maintained after the 52-h infusion period and declined following 7 and 14 days of infusion. The infusion of N/OFQ (1 μg/kg/h for 4 h) in rats exposed to acute stress caused a decrease in MMC density comparable to that obtained with the single treatments. UFP-101, at the doses of 1 and 10 μg/kg/h, which itself had no significant effect on MMC density, when concurrently infused in stress-exposed rats, abolished the stress-induced decrease of MMC density. Significance Present results indicate that the peripheral N/OFQ-NOP system is involved in stress-induced reduction of MMC density.

Activation of adenosine receptors modifies the action of classic neurotransmitters (i.e. dopamine, glutamate and acetylcholine) and other neuromodulators, like vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide S (NPS). Similarly to adenosine, NPS is involved in the regulation of stimulus and response to fear and arousal. Thus, the present study investigates the effects of NPS on locomotor activity in mice treated with or without α,β-methylene adenosine 5′-diphosphate (AOPCP), the inhibitor of ecto-5′-nucleotidase. Additionally, we evaluate the activity of ecto-5′-nucleotidase in brain slices of mice treated with or without NPS. Male adult CF-1 mice received i.c.v. NPS as 0.1 nmol injection with or without pre-treatment with 1 nmol α,β-methylene adenosine 5′-diphosphate (AOPCP), the selective inhibitor of ecto-5′-nucleotidase, to evaluate locomotor activity. In another set of experiments, mice received i.c.v. infusion of 0.1 nmol NPS to assay enzymatic activity in brain slices. The results demonstrated that the pre-treatment with AOPCP, which was inactive per se, prevented NPS-induced hyperlocomotion in mice. The dose of 0.1 nmol NPS was efficient to induce hyperlocomotion in animals during the observation period in the activity cage. Regarding enzymatic activity, i.c.v. NPS injection did not induce any significant alterations in ATP and AMP hydrolysis in striatum and hippocampus brain slices of mice. The present study shows that the hyperlocomotor effect of NPS depends on the ecto-5′-nucleotidase activity.