QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.077339v1 312/3/1114    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carrà, G. Articles by Calo', G. Search for Related Content PubMed PubMed Citation Articles by Carrà, G. Articles by Calo', G.

NEUROPHARMACOLOGY

[(pF)Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ (UFP-102), a Highly Potent and Selective Agonist of the Nociceptin/Orphanin FQ Receptor

Department of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center (G.C., A.R., F.M., G.M., D.R., E.G., S.Z., D.R., M.M., G.C.) and Department of Pharmaceutical Sciences and Biotechnology Center (R.G., S.S.), University of Ferrara, Ferrara, Italy; Department of Cardiovascular Sciences, Pharmacology and Therapeutics Group (T.A.B., J.M., C.P.H., D.G.L.) and Department of Health Sciences, Division of Anaesthesia, Critical Care and Pain Management (D.J.R.), Leicester Royal Infirmary, Leicester, United Kingdom; and Department of Pharmacology (V.A.K., D.R.K.), Louisiana State University, Health Sciences Center, New Orleans, Louisiana

Received September 8, 2004; accepted October 25, 2004.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂ (UFP-102), has been generated by combining in the N/OFQ-NH₂ sequence two chemical modifications, [Arg¹⁴,Lys¹⁵] and [(pF)Phe⁴], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO_hNOP cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(±)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA₂ = 7.75–8.12) and UFP-101 ([Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂)(pA₂ = 6.91–7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP^–/–). In vivo, UFP-102 (0.01–0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1–10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP^–/– mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.

Understanding the roles that the N/OFQ-NOP receptor system plays in physiology and pathology is to a major extent dependent upon the identification of highly potent and selective ligands. Currently available ligands for the NOP receptor and their therapeutic potential have been recently reviewed by Zaveri (2003). These are 1) nonpeptide ligands generally discovered via high-throughput screening in industrial laboratories (e.g., the NOP-selective antagonists J-113397 (Ozaki et al., 2000) and SB-612111 (Zaratin et al., 2004); 2) small peptides identified by screening of synthetic peptide combinatorial libraries [e.g., the NOP-selective partial agonist Ac-RYYRWK-NH₂ (Dooley et al., 1997) or the nonselective NOP ligand peptide III-BTD (Becker et al., 1999)]; and 3) N/OFQ-related peptides identified by classical structure activity relationship studies (for review, see Guerrini et al., 2000), including the full agonists N/OFQ(1-13)-NH₂ and N/OFQ-NH₂ (Calo et al., 1996), the partial agonist [Phe¹(CH₂-NH)Gly²]N/OFQ(1-13)-NH₂ (Guerrini et al., 1998), and the pure antagonist [Nphe¹]N/OFQ(1-13)-NH₂ (Calo et al., 2000a).

In addition to these ligands, two highly potent NOP agonists have been identified by alternative strategies. In the first case, substitution of Phe⁴ with (pF)Phe⁴ was used to generate [(pF)Phe⁴]N/OFQ(1-13)-NH₂ (Bigoni et al., 2002; Rizzi et al., 2002b). (pF)Phe is a noncoded amino acid in which a hydrogen atom in the para-position of the phenyl ring is substituted with the fluorine atom. In the second case, an extra pair of the basic residues, Arg and Lys, was introduced into position 14 and 15 of N/OFQ to generate [Arg¹⁴,Lys¹⁵]N/OFQ (Okada et al., 2000). Considering these approaches, we have combined in the N/OFQ-NH₂ sequence the chemical modifications that reduce ([Phe¹(CH₂-NH)Gly²]) or eliminate ([Nphe¹]) agonist efficacy, with those that increase agonist potency ([(pF)Phe⁴] and [Arg¹⁴Lys¹⁵]). Among the novel NOP ligands that were synthesized, we identified the selective antagonist [Nphe¹,Arg¹⁴Lys¹⁵]N/OFQ-NH₂ (UFP-101) and the selective agonist [(pF)Phe⁴,Arg¹⁴,Lys¹⁵] N/OFQ-NH₂ (UFP-102). In previous studies, we have evaluated the pharmacological properties of UFP-101 in various in vitro and in vivo assays (Calo et al., 2002; Gavioli et al., 2003; Marti et al., 2003; McDonald et al., 2003b; Mela et al., 2004).

In the present study, we investigated the pharmacological profile of UFP-102 in vitro using cells expressing the human recombinant NOP receptor, various isolated tissues, and cerebral cortex synaptosomes that release 5-HT, and in vivo in the tail withdrawal and locomotor activity assays in mice and in cardiovascular and renal function studies performed in rats.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
In Vitro Studies
Receptor Binding and Functional [Guanosine 5'-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPS) Binding and cAMP] Assays on CHO Cells Expressing the Human NOP Receptor. CHO_hNOP cells were maintained in Dulbecco's minimal essential medium:Ham's F-12 (1:1) supplemented with 5% FCS, penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (2.5 µg/ml). Media for stock (nonexperimental) cultures were further supplemented with hygromycin B (200 µg/ml) and G418 (Geneticin) (200 µg/ml). CHO cells expressing the human recombinant KOP (), DOP (), and MOP (µ) opioid receptors were cultured in Ham's F-12 with 10% fetal calf serum penicillin (100 IU/ml), streptomycin (100 µg/ml), and fungizone (2.5 µg/ml). Stock cultures additionally contained G418 (200 µg/ml) for maintenance of the receptor plasmid. Cell cultures were kept at 37°C in 5% CO₂/humidified air and used for experimentation when confluent (3–4 days). For [³⁵S]GTP S and [leucyl-³H]N/OFQ competition binding studies, membrane fragments obtained through homogenization of cell suspensions followed by centrifugation (20,000g for 10 min at 4°C) in buffer consisting either of Tris (50 mM) with or without MgSO₄ (5 mM) (competition binding for NOP or classical opioid, respectively) or Tris (50 mM) EGTA (0.2 mM) ([³⁵S]GTPS binding) were used. Membrane fragments were further homogenized and centrifuged for a total of three cycles. Whole cells in Krebs-HEPES buffer were used for studies of the inhibition of forskolin-stimulated cAMP formation (Okawa et al., 1999).

Competition Binding Assay. CHO_hNOP membrane protein (10 µg) was incubated in 0.5 ml of homogenization buffer containing 0.5% bovine serum albumin (BSA), 10 µM of a cocktail of peptidase inhibitors (captopril, amastatin, bestatin, and phosphoramidon), 0.2 nM [³H]N/OFQ, and increasing concentrations (10^–12–10^–5 M) of the nonradiolabeled peptides under study. Nonspecific binding was defined in the presence of 1 µM N/OFQ. For opioid receptor selectivity studies of UFP-102, the ability of increasing concentrations to displace the binding of [³H]diprenorphine was measured at classical human recombinant opioid receptors. Between 30 and 45 µg of CHO_hKOP/DOP/MOP homogenated was incubated in homogenization buffer containing 10 µM peptidase inhibitors (as described above) and 0.4 nM [³H]diprenorphine. Nonspecific binding was defined in the presence of 10 µM naloxone. All competition binding studies were incubated for 1 h at room temperature and bound and free radioactivity was separated by vacuum filtration using a Brandel cell harvester using WhatmanGF/B filters soaked in polyethylenimine (0.5%) to reduce nonspecific binding.

[³⁵S]GTPS Binding Assay. Experimentation was performed essentially as described by Berger et al. (2000). Freshly prepared CHO_hNOP membranes (20 µg) were incubated in 0.5-ml volumes of buffer consisting Tris (50 mM), EGTA (0.2 mM), GDP (100 µM), bacitracin (0.15 mM), BSA (1 mg/ml), peptidase inhibitors (amastatin, bestatin, captopril, and phosphoramidon; 10 µM), [³⁵S]GTPS (150 pM), and peptides in the concentration range of 10^–12 to 10^–5 M. Nonspecific binding was determined in the presence of 10 µM unlabeled [³⁵S]GTPS. Assays were incubated for 1 h at 30°C with gentle shaking, and bound and free radiolabel were separated by vacuum filtration onto Whatman GF/B filters. Polyethylenimine was not used. In all cases, radioactivity was determined after filter extraction (8 h; Optiphase Safe; PerkinElmer Wallac, Gaithersburg, MD) using liquid scintillation spectroscopy.

Cyclic AMP Assay. Inhibition of forskolin-stimulated cAMP was measured in whole CHO_hNOP cells. Volumes (0.3 ml) of cell suspension in Krebs-HEPES buffer were incubated in the presence of 3-isobutyl-1-methylxanthine (1 mM), forskolin (1 µM), and varying concentrations of the peptides under study for 15 min at 37°C. Reactions were terminated by the addition of HCl (10 M) and then neutralized with 10 M NaOH and 1 mM Tris, pH 7.4. The concentration of cAMP was measured using the protein binding method described by Brown et al. (1971).

Bioassay on Isolated Tissues. Tissues were taken from male Swiss mice (30–35 g), albino guinea pigs (300–350 g), and Sprague-Dawley rats (300–350 g). The mouse vas deferens and colon, the guinea pig ileum, and the rat vas deferens were prepared as described previously (Bigoni et al., 1999; Rizzi et al., 1999). The mouse and rat vas deferens and the guinea pig ileum were continuously stimulated through two platinum ring electrodes with supramaximal voltage rectangular pulses of 1-ms duration and 0.05-Hz frequency. The electrically evoked contractions (twitches) were measured isotonically with a strain gauge transducer (Basile 7006) and recorded with the PC-based acquisition system Autotrace 2.2 (RCS, Florence, Italy). After an equilibration period of about 60 min, the contractions induced by electrical field stimulation were stable; at this time, cumulative concentration-response curves to N/OFQ or UFP-102 were performed (0.5 log-unit step) in the absence or in presence of antagonists (UFP-101, J-113397, or naloxone; 15-min preincubation time). In mouse colon experiments, contractions elicited by agonists were measured isometrically. The concentration-response curves to UFP-102 were performed consecutively, adding to the bath different concentrations of the peptide every 20 min followed by washing. Separate series of experiments were performed in tissues (colon and vas deferens) taken from CD1/C57BL6/J-129 mice with NOP receptor gene knockout (NOP^–/–) or wild type (NOP^+/+). These mice were genotyped by polymerase chain reaction. Details of the generation and breeding of mutant mice have been published previously (Nishi et al., 1997; Gavioli et al., 2003).

Neurochemical Experiments on Mouse Cerebral Cortex Synaptosomes. Male Swiss, CD1/C57BL6/J-129 NOP^+/+, and NOP^–/– mice (20–25 g) were used for these studies. On the morning of the experiment, mice were decapitated under light ether anesthesia, and the fronto-parietal cortex was isolated. Synaptosomes were prepared as described previously (Mela et al., 2004). Briefly, the cortex was homogenized in ice-cold 0.32 M sucrose buffer at pH 7.4 and then centrifuged for 10 min at 1000g_max (4°C). The supernatant was then centrifuged for 20 min at 12,000g_max (4°C) with the synaptosomal pellet being resuspended in oxygenated (95% O₂,5%CO₂) Krebs' solution (118.5 mM NaCl, 4.7 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, and 10 mM glucose) containing ascorbic acid (0.05 mM) and disodium EDTA (0.03 mM). Synaptosomes were preloaded with [³H]5-HT by incubation (25 min) in medium containing 50 nM [³H]5-HT (specific activity of 27.8 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA). One-milliliter aliquots of the suspension (protein concentration of about 0.35 mg of protein/ml) were slowly injected into nylon syringe filters (outer diameter 13 mm, 0.45-µm pore size, internal volume approximately 100 µl; Phenomenex, Torrance, CA) connected to a peristaltic pump. Filters were maintained at 36.5°C in a thermostatic bath and superfused at a flow rate of 0.4 ml/min with a preoxygenated Krebs' solution. Sample collection (every 3 min) was initiated after a 20-min period of filter washout. K⁺ stimulation (1-min pulse) was applied at the 38th min. Under these experimental conditions, previous studies demonstrated that perfusion with tetrodotoxin or omission of Ca²⁺ from the superfusion medium reduced to about 50% or virtually abolished, respectively, the 10 mM K⁺-evoked [³H]5-HT overflow (Mela et al., 2004). N/OFQ and UFP-102 were added to the superfusion medium 9 min before the K⁺ pulse and maintained until the end of the experiment. At the end of the experiment, radioactivity retained in the superfusate samples and filters (dissolved with 1 ml of 1 M NaOH followed by 1 ml of 1 M HCl) was determined by liquid scintillation spectrophotometry using a Beckman LS 1800 beta-spectrometer and Ultima Gold XR scintillation fluid (Packard Instruments B.V., Groningen, The Netherlands).

In Vivo Studies
Animals. Male Swiss albino and CD1/C57BL6/J-129 NOP^+/+ and NOP^–/– mice weighing 20 to 25 g were used. Mice were handled according to guidelines published in the European Communities Council directives (86/609/EEC). They were housed in 425 x 266 x 155-mm cages (Tecniplast, Milan, Italy), eight animals/cage, under standard conditions (22°C, 55% humidity, 12-h light/dark cycle, light on at 7:00 AM) with food (MIL, standard diet; Morini, Reggio Emilia, Italy) and water ad libitum for at least 2 days before experiments began. Each mouse was used once. Mice were i.c.v. injected (injection volume 2 µl) under light ether anesthesia (i.e., just sufficient for losing the righting reflex) using the "free hand" technique described by Laursen and Belknap (1986). In brief, a 27-gauge needle attached via a polyethylene tube to a 10-µl Hamilton syringe was used for the injection at approximate 45° angle, at 2 mm lateral to the bregma midline. Each mouse only received one i.c.v. injection.

Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 275 to 300 g were used for the cardiovascular and renal function studies. Rats were housed in groups of five or fewer under a 12-h light/dark cycle (light on at 7:00 AM) until the day of the experiments. All rats were fed with normal sodium diets (sodium content, 163 mEq/kg) and were allowed tap water ad libitum. All the procedures were conducted in accordance with the Louisiana State University Health Sciences Center and the National Institutes of Health Guidelines for the Care and Use of Animals.

Tail Withdrawal Assay. All experiments were started at 10:00 AM and performed according to the procedure described previously (Calo et al., 1998). Briefly, the mice were placed in a holder and the distal one-half of the tail was immersed in water at 48°C. Withdrawal latency time was measured by an experienced observer blind to drug treatment. A cut-off time of 20 s was chosen to avoid tissue damage. For each series of experiments, at least 12 mice were randomly assigned to each treatment. Tail withdrawal latency was determined immediately before and 5, 15, 30, 60, and 90 min after i.c.v. injection of 2 µl of saline (control) or N/OFQ, or UFP-102.

Locomotor Activity Assay. Experiments were carried out between 2:00 and 6:00 PM, following the procedure described by Rizzi et al. (2001a). Briefly, the mice were routinely tested 3 min after i.c.v. injection. Locomotor activity was assessed using Basile activity cages, which consist of a four-channel resistance detector circuit which converts the bridges "broken" by the animal paws into pulses that are summed by an electronic counter every 5 min. Total number of impulses were recorded every 5 min for 30 min. Mice were not accustomed to the cages before drug treatment and the experiment was performed in a quiet and dimly illuminated room. For each series of experiments, at least 12 mice were randomly assigned to each treatment.

Cardiovascular and Renal Function Studies. Five to 6 days before experimentation, rats were anesthetized with ketamine (40 mg/kg i.m.; Vedco Inc., St. Joseph, MO) in combination with xylazine (5 mg/kg i.m.; Butler, Columbus, OH) and chronically implanted with a stainless steel guide cannula in the lateral cerebroventricle for i.c.v. injection of drug/vehicle. On the day of the study, rats were anesthetized with sodium methohexital (75 mg/kg i.p. and supplemented with 10 mg/kg i.v. as needed; King Pharmaceuticals, Bristol, TN) and instrumented with left femoral artery (blood pressure measurement), vein (isotonic saline infusion), and urinary bladder (urine collection) catheters and a recording electrode to measure renal sympathetic nerve activity, using standard techniques described previously (Kapusta et al., 1997; Kapusta and Kenigs, 1999). Rats were then placed in a rat holder (a chamber with Plexiglas ends connected by stainless steel rods) that permits forward and backward movement of the rat, allows for collection of urine, and protects the renal nerve recording preparation. An i.v. infusion of isotonic saline (55 ml/min) was then started and continued throughout the experiment. The experimental protocol commenced after rats regained full consciousness and cardiovascular and renal excretory function stabilized. After collection of baseline control measurements for each parameter, UFP-102 (0.055 nmol; n = 6) or isotonic saline vehicle (5 µl; n = 8) was injected i.c.v. Immediately after central administration, experimental urine samples (10-min consecutive periods) were collected for 90 min. Data depicted in Fig. 7 for N/OFQ are from previously published studies (Kapusta and Kenigs, 1999) and are superimposed for comparison to findings with UFP-102.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Cardiovascular, renal excretory, and renal nerve responses produced by i.c.v. N/OFQ (5.5 nmol), UFP-102 (0.055 nmol), and vehicle (5 µl) in conscious Sprague-Dawley rats. Values are means ± S.E.M. of at least eight rats. Urine samples (consecutive 10-min periods) were collected during control (C, 20 min) and immediately after drug/vehicle injection for 70 (N/OFQ) or 90 min (UFP-102 and vehicle). Data for N/OFQ, from a previously published study (Kapusta et al., 1999), are superimposed for comparison to findings with UFP-102. HR, heart rate; MAP, mean arterial pressure; RSNA, renal sympathetic nerve activity; UNaV, urinary sodium excretion; V, urine flow rate; *, p < 0.05, statistically different from corresponding control value, according to ANOVA followed by the Dunnett's test.

Drugs. The peptides used in this study were prepared and purified as described previously (Guerrini et al., 1997). J-113397 was prepared as a racemic mixture, according to De Risi et al. (2001). Captopril, amastatin, bestatin, phosphoramidon, naloxone, cAMP, 3-isobutyl-1-methylxanthine, BSA, [³⁵S]GTPS, GDP, bacitracin, and forskolin were from Sigma Chemical (Poole, Dorset, UK). All tissue culture media and supplements were from Invitrogen (Paisley, UK). [2,8-³H]cAMP (28.4 Ci mmol^–1), [³⁵S]GTPS (1250 Ci mmol^–1), and [15,16-³H]diprenorphine (1.85–2.59 Ci mmol^–1) were from PerkinElmer Life and Analytical Sciences and [leucyl-³H]N/OFQ was from Amersham Biosciences (Piscataway, NJ). For in vitro experiments, the compounds were solubilized in H₂O and stock solutions (2 mM) were stored at –70°C until use; for in vivo studies, the substances were solubilized in physiological medium just before performing the experiment.

Data Analysis and Terminology. All data are expressed as mean ± S.E.M., and the number of separate experiments is reported for each series of data. Data have been analyzed statistically with the Student's t test or one-way ANOVA followed by the Dunnett's test, as specified in table and figure legends; p values less than 0.05 were considered significant. Concentration of ligands producing 50% inhibition of specific binding (IC₅₀) was corrected for the competing mass of radioligand using the Cheng and Prusoff equation to yield K_i values. Curve fitting was performed using PRISM 3.0 (GraphPad Software Inc., San Diego, CA). Agonist potencies were expressed as pEC₅₀, which is the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist. The E_max is the maximal effect that an agonist can elicit in a given tissue/preparation. In mouse vas deferens experiments, pA₂ of UFP-101 versus N/OFQ and UFP-102 was evaluated by Schild analysis, whereas in the other preparations, pK_B values were calculated using the Gaddum Schild equation pK_B = –log((CR – 1)/[antagonist]), where CR is concentration ratio, assuming a slope equal to unity. Spontaneous 5-HT release from synaptosomes was expressed as fractional release (i.e., tritium efflux expressed as percentage of the tritium content in the filter at the onset of the corresponding collection period), whereas K⁺-evoked tritium overflow was calculated by subtracting the estimated spontaneous efflux (obtained by interpolation between the samples preceding and following the stimulation) from the total efflux observed in the stimulated sample.

In vivo data from studies in mice were analyzed as follows: raw data from tail withdrawal experiments were converted to the area under the curve (90 min); the AUC data were used for statistical analysis; locomotor activity data were analyzed statistically using the data expressed as cumulative impulses over the 30-min observation period. Cardiovascular and renal function data from rat studies were analyzed using repeated measures ANOVA.

	Results

Top Abstract Materials and Methods Results Discussion References

 
In Vitro Studies
Competition Binding, [³⁵S]GTPS Binding Stimulation, and Inhibition of cAMP Accumulation at Human NOP Receptors. The ability of UFP-102 to bind to opioid receptors was evaluated using membranes of CHO cells expressing human recombinant NOP and classical opioid receptors (MOP, DOP, and KOP). In CHO_hNOP membranes, UFP-102 produced a concentration-dependent inhibition of [³H]N/OFQ binding with a pK_i value of 10.67, displaying an affinity for the NOP receptor 17-fold higher than that of N/OFQ (pK_i = 9.44). UFP-102 also binds to classical opioid receptors albeit with reduced affinity by 223-fold at KOP (pK_i = 8.32), by 3300-fold at MOP (pK_i = 7.15), and by >17,000-fold at DOP (pK_i = 6.42) receptors.

In line with previous findings, in CHO_hNOP membranes N/OFQ stimulated [³⁵S]GTPS binding and in whole CHO_hNOP cells inhibited forskolin-stimulated cAMP accumulation. In both these assays, UFP-102 mimicked the effects of the natural ligand behaving as a full agonist, although UFP-102 was more potent than N/OFQ by 26-fold in [³⁵S]GTPS and by 2-fold in the cAMP assay (Table 1).

Bioassays in Isolated Tissues. In the electrically stimulated mouse and rat vas deferens and guinea pig ileum, UFP-102 concentration dependently inhibited twitches, eliciting maximal effects similar to N/OFQ but being 20- to 48-fold more potent (Table 1).

The kinetics of the inhibitory effects of N/OFQ and UFP-102 on the mouse vas deferens electrically induced twitch response can be seen in Fig. 1. The inhibitory effect (40% inhibition) induced by 10 nM N/OFQ occurred immediately after adding the peptide to the bath and was rapidly (1 min) reversible after washing of the tissue. In contrast, an equiefficacious concentration of UFP-102 (i.e., 0.3 nM) induced a slower inhibitory effect, which reached a plateau only after 10 min; moreover, the recovery of the tissue to the control twitch took greater than 10 min.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Typical tracings showing the effect of equieffective concentrations of N/OFQ and UFP-102 in the electrically stimulated mouse vas deferens. Abscissa, time in minutes; ordinate, control twitch.

The selective NOP receptor antagonist UFP-101 was used to probe the nature of the agonist response to both N/OFQ and UFP-102 in the mouse vas deferens. UFP-101 did not modify per se the electrically induced twitches, but displaced to the right the concentration-response curve to N/OFQ and UFP-102 in a concentration-dependent manner (Fig. 2, top). Curves obtained in the presence of UFP-101 were parallel to the control and reached similar maximal effects even in the presence of the highest concentration of antagonist (i.e., 10 µM). The corresponding Schild plots were linear with slopes not significantly different from unity and yielded pA₂ values of 7.18 and 7.22 against N/OFQ and UFP-102, respectively (Fig. 2, bottom).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Electrically stimulated mouse vas deferens. Top, concentration-response curve to N/OFQ (left) and UFP-102 (right) obtained in the absence (control) and presence of increasing concentrations of UFP-101 (0.1, 1, and 10 µM). Corresponding Schild plots are shown (bottom). Points indicate the means and vertical lines indicate the S.E.M. of at least four experiments.

Single concentrations of the NOP-selective antagonists (1 µM UFP-101 and 0.1 µM J-113397) and the nonselective opioid receptor antagonist naloxone (1 µM) were challenged against the inhibitory effects induced by UFP-102 in the rat and mouse vas deferens and in the guinea pig ileum. In all the tissues, the antagonists did not modify per se the control twitches and naloxone did not affect the concentration-response curve to UFP-102. In contrast, both UFP-101 and J-113397 displaced to the right the concentration-response curve to UFP-102 without modifying its maximal effects. pK_B values calculated from these experiments are summarized in Table 2 and compared with those obtained testing the same antagonists against N/OFQ.

The effects of N/OFQ, UFP-102, and the DOP-selective agonist deltorphin-I were investigated in the electrically stimulated mouse vas deferens taken from wild-type (NOP^+/+) and NOP receptor knockout (NOP^–/–) mice. In NOP^+/+ tissues, UFP-102 mimicked the inhibitory effects of N/OFQ (E_max 91 ± 1%; pEC₅₀ 7.62), showing similar maximal effects (86 ± 2%) but higher potencies (pEC₅₀ 9.40). In tissues taken from NOP^–/– mice, N/OFQ and UFP-102 were inactive up to 1 µM. In the same series of experiments, the DOP receptor selective agonist deltorphin-I displayed similar high potency and efficacy in tissues from NOP^+/+ and NOP^–/– mice (data not shown).

In the isolated mouse colon, UFP-102 mimicked the contractile effect of N/OFQ showing similar maximal effects but 2-fold higher potency (Table 1). The effects of UFP-102 were also investigated in colon tissues taken from NOP^+/+ and NOP^–/– mice. UFP-102 (10 nM) produced a contraction of colon tissues of NOP^+/+ mice amounting to 55 ± 6% of contraction induced by 10 µM carbachol, whereas it was found inactive up to 1 µM in tissues taken from NOP^–/– mice. Carbachol produced similar contractile effects in tissues from NOP^+/+ and NOP^–/– mice (data not shown).

[³H]5-HT Overflow in Mouse Cerebral Cortex Synaptosomes. As previously reported by Mela et al. (2004), 1-min pulse of KCl (10 mM) evoked a [³H]5-HT overflow from mouse cerebral cortex synaptosomes that was inhibited in a concentration-dependent manner by N/OFQ (0.1–1000 nM). Analysis of the concentration-response curve yielded a pEC₅₀ value of 8.64. Under the same experimental conditions, UFP-102 mimicked the effects of N/OFQ, showing similar maximal effects but being 7-fold more potent than the natural ligand (Table 1). To determine whether the effects of UFP-102 were dependent on activation of NOP receptors, maximally effective peptide concentrations were tested in synaptosomes obtained from NOP^–/– mice. Basal and K⁺-evoked [³H]5-HT overflow from NOP^+/+ and NOP^–/– mice was not different from that measured in Swiss mice according to previously reported data (Mela et al., 2004). In NOP^+/+ mice, 1 µM UFP-102 inhibited K⁺-evoked tritium overflow approximately to the same extent as in Swiss mice (67 ± 4% of control values), whereas in NOP^–/– mice the peptide was ineffective (101 ± 7% of control values).

In Vivo Studies
Tail Withdrawal Assay. Intracerebroventricular injection of 0.01 nmol of UFP-102 in mice did not induce any effect on gross behavior. In contrast, mice treated with 0.1 and 0.3 nmol showed a decrease in locomotor activity, ataxia, and loss of the righting reflex, in a similar manner to that which occurs after i.c.v. injection of high doses of N/OFQ (i.e., 10 nmol) (Reinscheid et al., 1995; Calo et al., 1998; Rizzi et al., 2001a). However, although the N/OFQ effects seemed to occur immediately after i.c.v. injection, those produced by UFP-102 were only evident after 30 min.

Results summarized in Fig. 3 show that the tail withdrawal latencies of saline injected mice were stable at 4 to 5 s over the time course of the experiment. UFP-102 at 0.01 nmol was inactive, whereas it induced statistically significant pronociceptive effects at 0.1 and 0.3 nmol, which peaked at 30 to 60 min postinjection. The pronociceptive effects induced by UFP-102 were long-lasting and were still evident 180 min after the i.c.v. injection (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 3. Mouse tail withdrawal assay. Dose-response curve to UFP-102 (0.01–0.3 nmol) injected i.c.v. Points indicate the means and vertical lines indicate the S.E.M. of five experiments. *, p < 0.05 versus saline according to ANOVA followed by the Dunnett's test performed on area under the curve data (see Materials and Methods).

We compared the effects of high doses of N/OFQ (10 nmol) and UFP-102 (0.3 nmol) in NOP^+/+ and NOP^–/– mice. As shown in Fig. 4, top, tail withdrawal latencies of saline injected NOP^+/+ mice were stable at 5 to 6 s over the time course of the experiment and were similar to those found in Swiss mice injected with saline. NOP^–/– mice displayed basal tail withdrawal latencies similar to wild-type mice; however, in these animals injection of saline produced a short-lasting increase in tail withdrawal latencies. Both N/OFQ and UFP-102 induced a statistically significant pronociceptive effect in NOP^+/+ mice (Fig. 4, top), whereas they were completely inactive in mice lacking the NOP receptor gene (Fig. 4, bottom).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Mouse tail withdrawal assay. Effects of i.c.v. injection of N/OFQ (10 nmol), and UFP-102 (0.3 nmol) in NOP+/+ (top) and NOP–/– mice (bottom). Points indicate the means and vertical lines the S.E.M. of four experiments. *, p < 0.05 versus saline according to ANOVA followed by the Dunnett's test performed on area under the curve data (see Materials and Methods).

Locomotor Activity Assay. As shown in Fig. 5, mice treated with saline displayed a progressive reduction in spontaneous locomotor activity from 277 ± 40 impulses/5 min to 101 ± 22 impulses/5 min during the 30-min experiment. Also in this assay, UFP-102 mimicked the effect of N/OFQ (Rizzi et al., 2001a), eliciting a dose-dependent (0.01–0.3 nmol) reduction of locomotor activity. UFP-102 was inactive at 0.01 nmol, whereas at 0.1 and 0.3 nmol produced a statistically significant reduction in locomotor spontaneous activity compared with saline-injected mice. N/OFQ (1 nmol) produced a rapid and short-lasting inhibition of spontaneous locomotor activity (Rizzi et al., 2001a), whereas the action of the equiefficacious dose of UFP-102 (0.1 nmol) was slow in onset yet produced a long-lasting inhibitory response (Fig. 5). Indeed, when spontaneous locomotor activity was measured for 30 min 3 h after injection, N/OFQ was completely inactive (saline 915 ± 88; 1 nmol of N/OFQ 727 ± 94), whereas 0.1 nmol UFP-102 virtually abolished motor activity (192 ± 56).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Mouse locomotor activity assay. Top, dose-response curve to UFP-102 (0.01–0.3 nmol) injected i.c.v. Points indicate the means and vertical lines the S.E.M. of five experiments. Bottom, cumulative impulses over the 30-min observation were used for statistical analysis. *, p < 0.05 versus saline, according to ANOVA followed by the Dunnett's test.

The effects of 10 nmol N/OFQ and 0.3 nmol UFP-102 on locomotor spontaneous activity were investigated in NOP^+/+ and NOP^–/– mice. As shown in Fig. 6, top, N/OFQ and UFP-102 essentially suppressed locomotor activity in NOP^+/+ mice, whereas they were inactive in mice lacking the NOP receptor gene (Fig. 6, bottom).

View larger version (22K): [in this window] [in a new window]   Fig. 6. Mouse locomotor activity assay. Effects of N/OFQ (10 nmol) and UFP-102 (0.3 nmol) on spontaneous locomotor activity in NOP+/+ (top) and in NOP–/– (bottom) mice. Points indicate the means and vertical lines indicate the S.E.M. of four experiments. Cumulative impulses over the 30-min observation were used for statistical analysis. *, p < 0.05 versus saline, according to ANOVA followed by the Dunnett's test.

Cardiovascular and Renal Functions in Conscious Sprague-Dawley Rats. The cardiovascular, renal excretory, and renal nerve responses to i.c.v. injection of N/OFQ and UFP-102 are shown in Fig. 7. As depicted, i.c.v. UFP-102 produced a significant decrease in heart rate, mean arterial pressure, urinary sodium excretion, and renal sympathetic nerve activity, and an increase in urine flow rate. In general, the directional changes in cardiovascular and renal function produced by i.c.v. UFP-102 were the same as those elicited by i.c.v. N/OFQ [N/OFQ data from Kapusta and Kenigs (1999) are shown in Fig. 7 for comparative purposes]. There are, however, several differences in the responses produced by these two compounds. First, based on the dose tested in these studies, UFP-102 (0.055 nmol) seems to be approximately 100-fold more potent than N/OFQ (5.5 nmol). In addition, although both N/OFQ and UFP-102 produced a decrease in mean arterial pressure over the first 10 min after injection, the hypotensive response produced by central UFP-102, but not N/OFQ, was sustained for the duration of the 90-min study. Similarly, although each ligand produced a comparable onset and magnitude of increase in urine flow rate, the diuresis produced by i.c.v. UFP-102 was sustained for a substantially longer period (approximately 40 min) than that elicited by central N/OFQ. Finally, unlike N/OFQ that produced a gradual decrease in renal sympathetic nerve activity that was significant by 30 min, i.c.v. UFP-102 did not evoke a renal sympathoinhibitory effect until 60 min after drug administration.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In vitro and in vivo data obtained from the present study demonstrate that UFP-102 is a highly potent and selective full agonist for the NOP receptor. Indeed, this is the most potent NOP ligand identified to date.

UFP-102 binds with high affinity to the recombinant human NOP receptor and produces maximal effects similar to those of N/OFQ in both the [³⁵S]GTPS and cAMP assays performed in CHO_hNOP cells. The full agonist behavior of UFP-102 was confirmed in animal tissue preparations expressing NOP receptors from both peripheral (isolated tissues) and central (synaptosomes) sites of origin. Typically, UFP-102 displayed increases in potency of >10-fold relative to the natural peptide; the only exceptions were the cAMP assay and the mouse colon bioassay where UFP-102 was only 2-fold more potent than N/OFQ and in synaptosomes where the equieffective concentration ratio was 7. It should be noted that the latter preparations are characterized by very high stimulus-response coupling efficiencies (large receptor reserve) as demonstrated by the fact that the NOP partial agonist [Phe¹(CH₂-NH)Gly²]N/OFQ(1-13)-NH₂ (Guerrini et al., 1998) behaves as a full agonist in these preparations (Okawa et al., 1999; Rizzi et al., 1999; Mela et al., 2004) and as a partial agonist or as an antagonist in the other preparations (Bigoni et al., 1999; McDonald et al., 2003a). Thus, in systems with large receptor reserve, the difference in potency between UFP-102 and N/OFQ will tend to be underestimated.

In tissues, UFP-102 kinetics of action was slower and its effects more resistant to washout than those elicited by the natural ligand N/OFQ. Similar kinetics of action has been reported for other NOP ligands, such as the peptide ZP120 (Rizzi et al., 2002a) and the nonpeptide Ro 64-6198 (Rizzi et al., 2001b). The reason(s) for the differences in kinetic behavior of these NOP agonists compared with N/OFQ are at present unknown.

Both pharmacological and knockout findings in vitro converge indicating that UFP-102 is a highly selective ligand whose effects are exclusively due to NOP receptor activation. First, UFP-102 binds with relatively low affinity to classical opioid receptors, being at least 200-fold selective for NOP; second, in functional assays, the actions of UFP-102 were resistant to naloxone but inhibited by the selective NOP antagonists UFP-101 (Calo et al., 2002) and J-113397 (Bigoni et al., 2000; Ozaki et al., 2000) with pA₂/pK_B values similar to those obtained against N/OFQ; and third, the effects of UFP-102 in addition to those of N/OFQ were no longer evident in tissues (colon, vas deferens, and cerebral cortex) taken from mice lacking the NOP receptor gene.

The high potency and selectivity of action of UFP-102 were confirmed in in vivo studies. UFP-102 mimicked the pronociceptive and locomotor inhibitory effects in mice and the cardiovascular and renal effects in rats produced by supraspinal administration of N/OFQ (Kapusta et al., 1997; Calo et al., 1998; Rizzi et al., 2001a). In all these assays, UFP-102 displayed higher potency than the natural peptide (between 10- and 100-fold) and produced slow onset but longer lasting effects. These kinetic features of UFP-102, also revealed by the in vitro experiments, may be attributed to a slower but stronger binding to the NOP receptor compared with N/OFQ; at least in part, they may due also to a decrease susceptibility of UFP-102 to degradation by peptidases, a feature that may be conferred to the molecule by the presence, toward the C terminus, of the cationic residues Arg¹⁴,Lys¹⁵ (Rizzi et al., 2002c).

The high selectivity of action of UFP-102 was also confirmed in vivo in mice. In fact, knockout studies, which represent the acid test for in vivo selectivity of action, clearly demonstrated that the biological effects of UFP-102 are solely due to NOP receptor activation. Indeed, no differences were recorded in NOP^–/– mice injected with saline or UFP-102 in either the tail withdrawal or the locomotor activity assay.

In conclusion, an extensive range of experimental data obtained in a variety of in vitro preparations and in vivo studies in mice and rats studies demonstrate that UFP-102 is a highly potent and selective full agonist at NOP receptors. In comparison with the native ligand N/OFQ, UFP-102 showed a gain of potency, a slow onset, and a relatively long duration of action, with these responses being observed in both in vitro and especially in in vivo assays. This unique pharmacological profile makes UFP-102 a very valuable pharmacological tool to elucidate the different biological functions regulated by the N/OFQ-NOP receptor system, particularly in vivo where prolonged duration of action and high potency are features that will reduce by severalfold the biologically active dose of the drug.

	Acknowledgements

 
We apologize to all authors whose work could not be cited due to space limitations.

	Footnotes

 
This work was financially supported by the University of Ferrara (60% grant to G.C.), the Italian Ministry of the University (Grant FIRB 2001 to D.R.), by the International Association for the Study of Pain (collaborative travel grant Leicester/Ferrara), and by National Institutes of Health (collaborative Grant NHLBI HL-71212 to D.R.K. and D.R., and NIDDK DK-43337 and DK-02605 to D.R.K.).

doi:10.1124/jpet.104.077339.

ABBREVIATIONS: N/OFQ, nociceptin/orphanin FQ; NOP, nociceptin/orphanin FQ peptide receptor; J-113397, (±)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; UFP-101, [Nphe¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂; UFP-102, [(pF)Phe⁴,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂; 5-HT, 5-hydroxytryptamine; CHO, Chinese hamster ovary; KOP, -opioid peptide receptor; DOP, -opioid peptide receptor; MOP, µ-opioid peptide receptor; [³⁵S]GTPS, guanosine 5'-O-(3-[³⁵S]thio)triphosphate; BSA, bovine serum albumin; ANOVA, analysis of variance; SB-612111, (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol; Ro 64-6198, (1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one; ZP120, Ac-RYYRWK₍₇₎-NH₂.

¹ G.C. and A.R. contributed equally to this work.

Address correspondence to: Dr. Girolamo Calo', Department of Experimental and Clinical Medicine, Section of Pharmacology, via Fossato di Mortara 19, 44100 Ferrara, Italy. E-mail: g.calo{at}unife.it

	References

Top Abstract Materials and Methods Results Discussion References

 

Becker JA, Wallace A, Garzon A, Ingallinella P, Bianchi E, Cortese R, Simonin F, Kieffer BL, and Pessi A (1999) Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library. J Biol Chem 274: 27513–27522.[Abstract/Free Full Text]

Berger H, Calo G, Albrecht E, Guerrini R, and Bienert M (2000) [Nphe(1)]NC(1-13)NH(2) selectively antagonizes nociceptin/orphanin FQ-stimulated G-protein activation in rat brain. J Pharmacol Exp Ther 294: 428–433.[Abstract/Free Full Text]

Bigoni R, Calo G, Rizzi A, Guerrini R, De Risi C, Hashimoto Y, Hashiba E, Lambert DG, and Regoli D (2000) In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist. Naunyn-Schmiedeberg's Arch Pharmacol 361: 565–568.[CrossRef][Medline]

Bigoni R, Giuliani S, Calo G, Rizzi A, Guerrini R, Salvadori S, Regoli D, and Maggi CA (1999) Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies. Naunyn-Schmiedeberg's Arch Pharmacol 359: 160–167.[CrossRef][Medline]

Bigoni R, Rizzi D, Rizzi A, Camarda V, Guerrini R, Lambert DG, Hashiba E, Berger H, Salvadori S, Regoli D, et al. (2002) Pharmacological characterisation of [(pX)Phe(4)]nociceptin(1-13)amide analogues1. In vitro studies. Naunyn-Schmiedeberg's Arch Pharmacol 365: 442–449.[CrossRef][Medline]

Calo G, Guerrini R, Bigoni R, Rizzi A, Marzola G, Okawa H, Bianchi C, Lambert DG, Salvadori S, and Regoli D (2000a) Characterization of [Nphe(1)]nociceptin(1-13)NH(2), a new selective nociceptin receptor antagonist. Br J Pharmacol 129: 1183–1193.[CrossRef][Medline]

Calo G, Guerrini R, Rizzi A, Salvadori S, and Regoli D (2000b) Pharmacology of nociceptin and its receptor—a novel therapeutic target. Br J Pharmacol 129: 1261–1283.[CrossRef][Medline]

Calo G, Rizzi A, Bogoni G, Neugebauer V, Salvadori S, Guerrini R, Bianchi C, and Regoli D (1996) The mouse vas deferens: a pharmacological preparation sensitive to nociceptin. Eur J Pharmacol 311: R3–R5.[CrossRef][Medline]

Calo G, Rizzi A, Marzola G, Guerrini R, Salvadori S, Beani L, Regoli D, and Bianchi C (1998) Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay. Br J Pharmacol 125: 373–378.[CrossRef][Medline]

Calo G, Rizzi A, Rizzi D, Bigoni R, Guerrini R, Marzola G, Marti M, McDonald J, Morari M, Lambert DG, et al. (2002) [Nphe1,Arg14,Lys15]nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br J Pharmacol 136: 303–311.[CrossRef][Medline]

Cox BM, Chavkin C, Christie MJ, Civelli O, Evans C, Hamon MD, Hoellt V, Kieffer B, Kitchen I, McKnight AT, et al. (2000) Opioid receptors, in The IUPHAR Compendium of Receptor Characterization and Classification (Girdlestone D ed) pp 321–333. IUPHAR Media Ltd., London.

Brown BL, Albano JD, Ekins RP, and Sgherzi AM (1971) A simple and sensitivesaturation assay method for the measurement of adenosine 3':5'-cyclic monophosphate. Biochem J 121: 561–562.[Medline]

De Risi C, Piero Pollini G, Trapella C, Peretto I, Ronzoni S, and Giardina GA (2001) A new synthetic approach to 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dih ydro-benzimidazol-2-one (J-113397), the first nonpeptide ORL-1 receptor antagonist. Bioorg Med Chem 9: 1871–1877.[CrossRef][Medline]

Dooley CT, Spaeth CG, Berzetei-Gurske IP, Craymer K, Adapa ID, Brandt SR, Houghten RA, and Toll L (1997) Binding and In vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1. J Pharmacol Exp Ther 283: 735–741.[Abstract/Free Full Text]

Gavioli EC, Marzola G, Guerrini R, Bertorelli R, Zucchini S, De Lima TC, Rae GA, Salvadori S, Regoli D, and Calo G (2003) Blockade of nociceptin/orphanin FQ-NOP receptor signalling produces antidepressant-like effects: pharmacological and genetic evidences from the mouse forced swimming test. Eur J Neurosci 17: 1987–1990.[CrossRef][Medline]

Guerrini R, Calo G, Rizzi A, Bianchi C, Lazarus LH, Salvadori S, Temussi PA, and Regoli D (1997) Address and message sequences for the nociceptin receptor: a structure-activity study of nociceptin-(1-13)-peptide amide. J Med Chem 40: 1789–1793.[CrossRef][Medline]

Guerrini R, Calo G, Rizzi A, Bigoni R, Bianchi C, Salvadori S, and Regoli D (1998) A new selective antagonist of the nociceptin receptor. Br J Pharmacol 123: 163–165.[CrossRef][Medline]

Guerrini R, Calo G, Rizzi A, Bigoni R, Rizzi D, Regoli D, and Salvadori S (2000) Structure-activity relationships of nociceptin and related peptides: comparison with dynorphin A. Peptides 21: 923–933.[CrossRef][Medline]

Kapusta DR and Kenigs VA (1999) Cardiovascular and renal responses produced by central orphanin FQ/nociceptin occur independent of renal nerves. Am J Physiol 277: R987–R995.

Kapusta DR, Sezen SF, Chang JK, Lippton H, and Kenigs VA (1997) Diuretic and antinatriuretic responses produced by the endogenous opioid-like peptide, nociceptin (orphanin FQ). Life Sci 60: PL15–PL21.[CrossRef][Medline]

Laursen SE and Belknap JK (1986) Intracerebroventricular injections in mice. Some methodological refinements. J Pharmacol Methods 16: 355–357.[CrossRef][Medline]

Marti M, Stocchi S, Paganini F, Mela F, De Risi C, Calo G, Guerrini R, Barnes TA, Lambert DG, Beani L, et al. (2003) Pharmacological profiles of presynaptic nociceptin/orphanin FQ receptors modulating 5-hydroxytryptamine and noradrenaline release in the rat neocortex. Br J Pharmacol 138: 91–98.[CrossRef][Medline]

McDonald J, Barnes TA, Okawa H, Williams J, Calo G, Rowbotham DJ, and Lambert DG (2003a) Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone-inducible mammalian expression system. Br J Pharmacol 140: 61–70.[CrossRef][Medline]

McDonald J, Calo G, Guerrini R, and Lambert DG (2003b) UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPgamma(35)S binding studies. Naunyn-Schmiedeberg's Arch Pharmacol 367: 183–187.[CrossRef][Medline]

Mela F, Marti M, Ulazzi L, Vaccari E, Zucchini S, Trapella C, Salvadori S, Beani L, Bianchi C, and Morari M (2004) Pharmacological profile of nociceptin/orphanin FQ receptors regulating 5-hydroxytryptamine release in the mouse neocortex. Eur J Neurosci 19: 1317–1324.[CrossRef][Medline]

Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monserrat B, et al. (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature (Lond) 377: 532–535.[CrossRef][Medline]

Mogil JS and Pasternak GW (2001) The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family.Pharmacol Rev 53: 381–415.[Abstract/Free Full Text]

Nishi M, Houtani T, Noda Y, Mamiya T, Sato K, Doi T, Kuno J, Takeshima H, Nukada T, Nabeshima T, et al. (1997) Unrestrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptin/orphaninFQ receptor. EMBO (Eur Mol Biol Organ) J 16: 1858–1864.[CrossRef][Medline]

Okada K, Sujaku T, Chuman Y, Nakashima R, Nose T, Costa T, Yamada Y, Yokoyama M, Nagahisa A, and Shimohigashi Y (2000) Highly potent nociceptin analog containing the Arg-Lys triple repeat. Biochem Biophys Res Commun 278: 493–498.[CrossRef][Medline]

Okawa H, Nicol B, Bigoni R, Hirst RA, Calo G, Guerrini R, Rowbotham DJ, Smart D, McKnight AT, and Lambert DG (1999) Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin (1-13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors. Br J Pharmacol 127: 123–130.[CrossRef][Medline]

Ozaki S, Kawamoto H, Itoh Y, Miyaji M, Azuma T, Ichikawa D, Nambu H, Iguchi T, Iwasawa Y, and Ohta H (2000) In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist. Eur J Pharmacol 402: 45–53.[CrossRef][Medline]

Reinscheid RK, Nothacker HP, Bourson A, Ardati A, Henningsen RA, Bunzow JR, Grandy DK, Langen H, Monsma FJ, and Civelli O (1995) Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science (Wash DC) 270: 792–794.[Abstract/Free Full Text]

Rizzi A, Bigoni R, Calo G, Guerrini R, Salvadori S, and Regoli D (1999) [Nphe(1)]nociceptin-(1-13)-NH(2) antagonizes nociceptin effects in the mouse colon. Eur J Pharmacol 385: R3–R5.[CrossRef][Medline]

Rizzi A, Bigoni R, Marzola G, Guerrini R, Salvadori S, Regoli D, and Calo G (2001a) Characterization of the locomotor activity-inhibiting effect of nociceptin/orphanin FQ in mice. Naunyn-Schmiedeberg's Arch Pharmacol 363: 161–165.[CrossRef][Medline]

Rizzi A, Rizzi D, Marzola G, Regoli D, Larsen BD, Petersen JS, and Calo G (2002a) Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice. Br J Pharmacol 137: 369–374.[CrossRef][Medline]

Rizzi A, Salis B, Ciccocioppo R, Marzola G, Bigoni R, Guerrini R, Massi M, Madeddu P, Salvadori S, Regoli D, et al. (2002b) Pharmacological characterisation of [(pX)Phe(4)]nociceptin(1-13)NH(2) analogues2. In vivo studies. Naunyn-Schmiedeberg's Arch Pharmacol 365: 450–456.[CrossRef][Medline]

Rizzi D, Bigoni R, Rizzi A, Jenck F, Wichmann J, Guerrini R, Regoli D, and Calo G (2001b) Effects of Ro 64-6198 in nociceptin/orphanin FQ-sensitive isolated tissues. Naunyn-Schmiedeberg's Arch Pharmacol 363: 551–555.[CrossRef][Medline]

Rizzi D, Rizzi A, Bigoni R, Camarda V, Marzola G, Guerrini R, De Risi C, Regoli D, and Calo G (2002c) [Arg14,Lys15]nociceptin, a highly potent agonist of the nociceptin/orphanin FQ receptor: in vitro and in vivo studies. J Pharmacol Exp Ther 300: 57–63.[Abstract/Free Full Text]

Zaratin PF, Petrone G, Sbacchi M, Garnier M, Fossati C, Petrillo P, Ronzoni S, Giardina GA, and Scheideler MA (2004) Modification of nociception and morphine tolerance by the selective opiate receptor-like orphan receptor antagonist (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). J Pharmacol Exp Ther 308: 454–461.[Abstract/Free Full Text]

Zaveri N (2003) Peptide and nonpeptide ligands for the nociceptin/orphanin FQ receptor ORL1: research tools and potential therapeutic agents. Life Sci 73: 663–678.[CrossRef][Medline]

This article has been cited by other articles:

				T. V. Khroyan, N. T. Zaveri, W. E. Polgar, J. Orduna, C. Olsen, F. Jiang, and L. Toll SR 16435 [1-(1-(Bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a Novel Mixed Nociceptin/Orphanin FQ/{micro}-Opioid Receptor Partial Agonist: Analgesic and Rewarding Properties in Mice J. Pharmacol. Exp. Ther., February 1, 2007; 320(2): 934 - 943. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.104.077339v1 312/3/1114    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carrà, G. Articles by Calo', G. Search for Related Content PubMed PubMed Citation Articles by Carrà, G. Articles by Calo', G.