Nociceptin inhibits airway microvascular leakage induced by HCl intra-oesophageal instillation

1. Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Our previous results suggest that microvascular leakage induced, in the guinea-pig airways, by intra-oesophageal hydrochloric acid (HCl) infusion was mainly dependent on the release of tachykinins. Nociceptin, an endogenous ligand of the opioid receptor NOP, has been shown to inhibit bronchoconstriction and cough in guinea-pig or cat by inhibiting tachykinin release. 2. The purpose of this study was to investigate the effects of nociceptin on the intra-oesophageal HCl-induced airway microvascular leakage evaluated by Evans blue dye extravasation measurement in anaesthetised guinea-pigs pretreated with propranolol, atropine and phosphoramidon. 3. Infusion of intra-oesophageal HCl led to a significant increase in plasma extravasation in the main bronchi and trachea. This increase was abolished when animals underwent a bilateral vagotomy. 4. Airway microvascular leakage was inhibited by nociceptin (3-30 microg x kg(-1) i.v.) in a dose-dependent manner (maximal inhibition at the dose of 30 microg x kg(-1): 19.76+/-1.13 vs 90.92+/-14.00 ng x mg(-1) tissue for nociceptin and HCl infusion, respectively, in the main bronchi, P<0.01). The NOP receptor agonist [Arg(14),Lys(15)]N/OFQ mimicked the inhibitory effect of nociceptin, but at a 10-fold lower dose (3 microg x kg(-1) i.v). The NOP receptor antagonist J-113397 had no effect on plasma protein extravasation by itself, but was able to block the inhibitory effect of nociceptin. 5. Morphine (1 mg x kg(-1)) had a similar inhibitory effect as that of nociceptin. Naloxone pretreatment abolished the effect of morphine, but was enable to block the inhibitory effect of nociceptin. 6. Under similar conditions, nociceptin, in the previous range of concentration, was unable to counteract the airway microvascular leakage induced by substance P (SP). 7. These results suggest that airway plasma extravasation induced by intra-oesophageal HCl instillation might be inhibited by specific stimulation of the NOP receptor with nociceptin. Nociceptin is likely to act at a pre-junctional level, by inhibiting tachykinin release, since it was unable to prevent SP-induced airway plasma extravasation.     

Nociceptin/orphanin FQ peptide receptors: pharmacology and clinical implications

The advance of functional genomics revealed the superfamily of G-protein coupled receptors (GPCRs). Hundreds of GPCRs have been cloned but many of them are orphan GPCRs with unidentified ligands. The first identified orphan GPCR is the opioid receptor like orphan receptor, ORL1. It was cloned in 1994 during the identification of opioid receptor subtypes and was de-orphanized in 1995 by the discovery of its endogenous ligand, nociceptin or orphanin FQ (N/OFQ). This receptor was renamed as N/OFQ peptide (NOP) receptor. Several selective ligands acting at NOP receptors or other anti-N/OFQ agents have been reported. These include N/OFQ-derived peptides acting as agonists (cyclo[Cys(10),Cys(14)]N/OFQ, [Arg(14), Lys(15)]N/OFQ, [pX]Phe(4)N/OFQ(1-13)-NH(2), UFP-102, [(pF)Phe(4),Aib(7), Aib(11),Arg(14),Lys(15)]N/OFQ-NH(2)) or antagonists (Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2), [Nphe(1)]N/OFQ(1-13)-NH(2), UFP-101, [Nphe(1), (pF)Phe(4),Aib(7),Aib(11),Arg(14),Lys(15)]N/OFQ-NH(2)), hexapeptides, other peptide derivatives (peptide III-BTD, ZP-120, OS-461, OS-462, OS-500), non-peptide agonists (NNC 63-0532, Ro 64-6198, (+)-5a compound, W-212393, 3-(4-piperidinyl)indoles, 3-(4-piperidinyl) pyrrolo[2,3-b]pyridines) and antagonists (TRK-820, J-113397, JTC-801, octahydrobenzimidazol-2-ones, 2-(1,2,4-oxadiazol-5-yl)-1 H-indole, N-benzyl-D-prolines, SB-612111), biostable RNA Spiegelmers specific against N/OFQ, and a functional antagonist, nocistatin. Buprenorphine and naloxone benzoylhydrazone are two opioid receptor ligands showing high affinity for NOP receptors. NOP receptor agonists might be beneficial in the treatment of pain, anxiety, stress-induced anorexia, cough, neurogenic bladder, edema, drug dependence, and, less promising, in cerebral ischemia and epilepsy, while antagonists might be of help in the management of pain, depression, dementia and Parkinsonism. N/OFQ is also involved in cardiovascular, gastrointestinal and immune regulation. Altered plasma levels of N/OFQ have been reported in patients with various pain states, depression and liver diseases. This review summarizes the pharmacological characteristics of, and studies with, the available NOP receptor ligands and their possible clinical implications.     

UFP-101, a peptide antagonist selective for the nociceptin/orphanin FQ receptor

Nociceptin/orphanin FQ modulates various biological functions at central and peripheral levels by selectively activating a G-protein coupled receptor named N/OFQ peptide (NOP) receptor. For extending our knowledge on the biological roles of the N/OFQ-NOP receptor system the identification of selective NOP ligands, especially antagonists, is mandatory. [Nphe1, Arg14, Lys15] N/OFQ-NH2 (UFP-101) is a novel NOP ligand that was designed by combining, in the same molecule, the [Nphe1] chemical modification which eliminates efficacy and the [Arg14, Lys15] substitution which increases ligand potency and duration of action in vivo. In the present article, we summarize the pharmacological features of UFP-101 as determined in a series of in vitro and in vivo assays. Moreover, some biological actions and possible therapeutic indications of NOP ligands are discussed on the basis of results obtained with UFP-101. Data obtained with this compound were compared with those generated using other NOP antagonists, especially J-113397 and [Nphe1]N/OFQ(1-13)-NH2, receptor or peptide knockout mice and other pharmacological tools useful for blocking N/OFQ - NOP receptor signaling. The analysis of the available data demonstrates that UFP-101 is a useful pharmacological tool for the investigation of the central and peripheral biological functions regulated by the N/OFQ-NOP receptor system and for defining the therapeutic potential of NOP receptor ligands.     

Quantitative study of [(pF)Phe4,Arg14,Lys15]nociceptin/orphanin FQ-NH2 (UFP-102) at NOP receptors in rat periaqueductal gray slices

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a novel member of the opioid receptor family with little affinity for traditional opioids. This receptor and its endogenous ligand, N/OFQ, are widely distributed in the brain and are implicated in many physiological functions including pain regulation. [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102) is a newly developed peptide agonist of NOP receptors. In this study, we quantitatively investigated the effect of UFP-102 at native NOP receptors of the ventrolateral periaqueductal gray (PAG), a crucial midbrain area involved in pain regulation and enriched with NOP receptors, using blind patch-clamp whole-cell recording technique in rat brain slices. UFP-102, like N/OFQ, induced an outward current in ventrolateral PAG neurons and increased the membrane current elicited by a hyperpolarization ramp from -60 to -140 mV. The current induced by UFP-102 was characterized with inward rectification and had a reversal potential near the equilibrium potential of K(+) ions, indicating that UFP-102 activates G-protein coupled inwardly rectifying K(+) channels. The effect of UFP-102 was concentration-dependent with the maximal effect similar to that of N/OFQ. The EC(50) value was 11+/-2 nM, which is 5 fold lower than that of N/OFQ. The effect of UFP-102 was not affected by naloxone while competitively antagonized by UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2)), a potent NOP receptor antagonist, with a pA(2) value of 6.7. These results suggest that UFP-102 is a full agonist at the postsynaptic NOP receptors of the midbrain of rats and is 5 fold more potent than N/OFQ.     

In vitro and in vivo pharmacological characterization of the nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-ol

It was recently reported that the hexapeptide Ac-RYYRIK-ol binds with high affinity nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors and competitively antagonizes N/OFQ actions in the mouse vas deferens assay. Here we further describe the in vitro and in vivo pharmacological features of this NOP receptor ligand. In mouse brain homogenate the degradation half life of Ac-RYYRIK-ol (2.48 min) was significantly higher than that of the parent compound Ac-RYYRIK-NH2 (1.20 min). In the electrically stimulated mouse vas deferens, Ac-RYYRIK-ol (10-1000 nM) competitively antagonized the inhibitory effect of N/OFQ (pA2=8.46), while in the isolated mouse colon the hexapeptide mimicked N/OFQ contractile effects thus behaving as a NOP receptor agonist (pEC50=9.09). This latter effect was no longer evident in colon tissues taken from mice knock out for the NOP receptor gene (NOP-/-). In vivo in mice, similarly to N/OFQ, Ac-RYYRIK-ol (dose range 0.001-1 nmol) produced: i) pronociceptive effects after intracerebroventricular (i.c.v.) administration and antinociceptive actions when given intrathecally (i.t.) in the tail withdrawal assay; ii) inhibition of locomotor activity and iii) stimulation of food intake after supraspinal administration. Finally, in the forced swimming test, Ac-RYYRIK-ol was inactive per se, but reversed the antidepressant-like effects elicited by the NOP receptor selective antagonist UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH2). Thus, in all these in vivo assays Ac-RYYRIK-ol mimicked the actions of N/OFQ showing however higher potency. In conclusion, Ac-RYYRIK-ol displayed a complex pharmacological profile which is likely due to the low efficacy agonist nature of this novel ligand of the NOP receptor. The high potency, selectivity of action, and in vivo effectiveness make Ac-RYYRIK-ol a useful pharmacological tool for future studies in the field of N/OFQ and its NOP receptor.     

Nphe1,Arg14,Lys15]N/OFQ-NH2 is a competitive antagonist of NOP receptors in the periaqueductal gray

Nociceptin/orphanin FQ (N/OFQ) and N/OFQ peptide (NOP) receptors are implicated in many physiological functions including pain regulation. This study quantitatively investigated the interaction of a novel NOP receptor antagonist, UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ-NH2), with N/OFQ in the ventrolateral periaqueductal gray, a crucial midbrain area for pain regulation. N/OFQ concentration-dependently activated G-protein coupled inwardly rectifying K+ (GIRK) channels in ventrolateral neurons of periaqueductal gray slices. UFP-101 antagonized N/OFQ-induced GIRK channel activation in a concentration-dependent manner and produced a parallel shift of the concentration-response curve of N/OFQ. The pA2 value estimated from Schild plot is 6.92+/-0.06. At concentrations up to 1 microM, UFP-101 had no effect on membrane current per se and did not affect the GIRK current activated by [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin, a mu-opioid receptor agonist. It is concluded that UFP-101 is a potent and competitive peptide antagonist of NOP receptors that mediate GIRK channel activation in ventrolateral periaqueductal gray neurons.     

Regulation of pancreatic secretion in vitro by nociceptin/orphanin FQ and opioid receptors: a comparative study.

The effects of nociceptin/orphanin FQ (N/OFQ) on gastrointestinal functions resemble those of classic analgesic opioid agonists. In this study, we compared changes in amylase release from guinea pig isolated pancreatic acini and lobules induced by the N/OFQ analogue [Arg(14),Lys(15)]N/OFQ and by the delta-receptor opioid agonist deltorphin. Carbachol strongly stimulated amylase release from isolated acini. Both peptides left baseline and carbachol-stimulated amylase secretion from pancreatic acini unchanged. Co-incubation of KCl-stimulated lobules with [Arg(14),Lys(15)]N/OFQ or deltorphin inhibited KCl-induced amylase release in a concentration-dependent manner. Although maximal inhibition of amylase release by [Arg(14),Lys(15)]N/OFQ and deltorphin had similar amplitude, [Arg(14),Lys(15)]N/OFQ was 100-fold more potent than deltorphin on a molar basis. The selective NOP-receptor antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101) antagonized [Arg(14),Lys(15)]N/OFQ-induced inhibition but left deltorphin-induced inhibition unchanged. The selective delta opiate receptor antagonist naltrindole had no effect on [Arg(14),Lys(15)]N/OFQ inhibition but partly prevented the inhibition by deltorphin. [Arg(14),Lys(15)]N/OFQ and deltorphin combined had no influence on each other. These findings show that [Arg(14),Lys(15)]N/OFQ inhibits pancreatic enzyme secretion by suppressing cholinergic transmission in intralobular nerve fibers, as previously reported for opioid agents. They suggest that [Arg(14),Lys(15)]N/OFQ inhibition of amylase release is mediated through the NOP receptor and not through the delta opioid receptor. The N/OFQ-NOP receptor system, like the delta opioid system, plays an inhibitory role in regulating exocrine pancreatic secretion.     

N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands

Previous structure-activity studies on nociceptin/orphanin FQ (N/OFQ) identified [Phe(1)Psi(CH(2)NH)Gly(2)]N/OFQ(1-13)-NH(2) and [Nphe(1)]N/OFQ(1-13)-NH(2) as a N/OFQ peptide receptor (NOP) partial agonist and pure antagonist, respectively. The addition of fluorine to the Phe(4) or the insertion of a further pair of basic amino acids Arg(14)-Lys(15) generate potent agonists. On the basis of these findings, we combined in the N/OFQ-NH(2) template the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) that increase the agonist potency with those conferring partial agonist (Phe(1)Psi(CH(2)NH)Gly(2)) or pure antagonist (Nphe(1)) properties. Twelve peptides were synthesized and pharmacologically evaluated in Chinese hamster ovary cells expressing the human recombinant NOP and in electrically stimulated mouse vas deferens and guinea pig ileum assays. All peptides behaved as NOP ligands; the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) increased ligand affinity/potency. Peptides with the normal Phe(1)-Gly(2) peptide bond behaved as full agonists, and those with the Phe(1)Psi(CH(2)NH)Gly(2) modification behaved as partial agonists, while those with the Nphe(1) modification behaved as partial agonists or pure antagonists depending on the presence or absence of the (pF)Phe(4) modification, respectively. The full agonist [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), the partial agonist [Phe(1)Psi(CH(2)NH)Gly(2),(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), and the pure antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) represent the most potent peptide ligands for NOP.     

UFP-101, a high affinity antagonist for the nociceptin/orphanin FQ receptor: radioligand and GTPγ<Superscript>35</Superscript>S binding studies

Studies of the pharmacology of nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) have been hampered by the lack of a range of high potency antagonists. In this study we have examined the effects of a novel N/OFQ analogue [Nphe(1),Arg(14),Lys(15)]N/OFQ NH(2) hereafter referred to as UFP-101. [(3)H]N/OFQ competition binding and GTPgamma(35)S binding assays were performed using CHO cells expressing the human NOP receptor (CHO(hNOP)). UFP-101 (pK(i) of 10.14+/-0.09) and a range of NOP selective agonists displaced [(3)H]N/OFQ binding with the following rank order of affinity: [Arg(14),Lys(15)]N/OFQ>[( pF)Phe(4)]N/OFQ(1-13)NH(2)>N/OFQ(1-13)NH(2)>UFP-101>N/OFQ>Ro64-6198>[Nphe(1)]N/OFQ(1-13)NH(2). N/OFQ, N/OFQ(1-13)NH(2), [( pF)Phe(4)]N/OFQ(1-13)NH(2), [Arg(14),Lys(15)]N/OFQ and Ro64-6198 also produced a concentration dependent (pEC(50) values of 8.75+/-0.11, 9.28+/-0.15, 9.69+/-0.04, 9.12+/-0.11 and 8.09+/-0.07 respectively) and saturable stimulation of GTPgamma(35)S binding and all were full agonists. UFP-101 did not stimulate GTPgamma(35)S binding per se, but produced a concentration dependent and parallel rightward shift in the concentration response curves to all agonists. UFP-101 yielded pA(2) values in the range 8.4-9.0. For comparison a pA(2) for [Nphe(1)]N/OFQ(1-13)NH(2) (the template for UFP-101) against N/OFQ of 7.33+/-0.08 was obtained. Slope factors for the Schild regression lines were approximately 1 indicating competitivity. When UFP-101 is compared with its template molecule [Nphe(1)]N/OFQ(1-13)NH(2), Arg(14),Lys(15) substitution produced approximately 1 log greater potency. We suggest that due to its high potency UFP-101 should prove a further useful tool in the evaluation of the N/OFQ-NOP receptor system.     

Nocistatin inhibits 5-hydroxytryptamine release in the mouse neocortex via presynaptic Gi/o protein linked pathways

BACKGROUND AND PURPOSE: Nocistatin (NST) is a neuropeptide generated from cleavage of the nociceptin/orphanin FQ (N/OFQ) precursor. Evidence has been presented that NST acts as a functional antagonist of N/OFQ, although NST receptor and transduction pathways have not yet been identified. We previously showed that N/OFQ inhibited [(3)H]5-hydroxytryptamine ([(3)H]5-HT) release from mouse cortical synaptosomes via activation of NOP receptors. We now investigate whether NST regulates [(3)H]5-HT release in the same preparation. EXPERIMENTAL APPROACH: Mouse and rat cerebrocortical synaptosomes in superfusion, preloaded with [(3)H]5-HT and stimulated with 1 min pulses of 10 mM KCl, were used. KEY RESULTS: Bovine NST (b-NST) inhibited the K(+)-induced [(3)H]5-HT release, displaying similar efficacy but lower potency than N/OFQ. b-NST action underwent concentration-dependent and time-dependent desensitization, and was not prevented either by the NOP receptor antagonist [Nphe(1) Arg(14),Lys(15)]N/OFQ(1-13)-NH(2) (UFP-101) or by the non-selective opioid receptor antagonist, naloxone. Contrary to N/OFQ, b-NST reduced [(3)H]5-HT release from synaptosomes obtained from NOP receptor knockout mice. However, both N/OFQ and NST were ineffective in synaptosomes pre-treated with the G(i/o) protein inhibitor, Pertussis toxin. NST-N/OFQ interactions were also investigated. Co-application of maximal concentrations of both peptides did not result in additive effects, whereas pre-application of maximal b-NST concentrations partially attenuated N/OFQ inhibition. CONCLUSIONS AND IMPLICATIONS: We conclude that b-NST inhibits [(3)H]5-HT release via activation of G(i/o) protein linked pathways, not involving classical opioid receptors and the NOP receptor. The present data strengthen the view that b-NST is, per se, a biologically active peptide endowed with agonist activity.     

Effects of nociceptin/orphanin FQ receptor ligands on blood pressure,heart rate,and plasma catecholamine concentrations in guinea pigs

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP) and has been shown previously to produce bradycardia and hypotension in rodents. In this study we have measured the effects of intravenous N/OFQ, and the NOP antagonists [Nphe(1)]N/OFQ(1-13)-NH(2) ([Nphe(1)]) and [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101) on cardiovascular parameters and plasma catecholamine concentrations. Female Hartley guinea pigs were anesthetized with pentobarbital and ventilated artificially. MAP and HR were measured via a femoral arterial catheter and ECG, respectively. Plasma catecholamine concentrations were measured by HPLC. Animals received saline, N/OFQ (0.25, 1.25, 6.25 and 25 nmol cumulatively at 10-min intervals), [Nphe(1)] (600 nmol) and UFP-101 (60 nmol) i.v. in various combinations. After establishing a stable baseline, MAP and HR measurements and blood sampling were performed at the beginning and 3 min following each drug administration. N/OFQ significantly decreased MAP, HR and the plasma noradrenaline concentrations in a dose dependent manner (maximally by 29.1+/-1.8%, 13.8+/-0.8% and 46.6+/-7.8%, respectively) To the contrary, N/OFQ tended to increase plasma adrenaline concentration but did not affect plasma dopamine concentrations. There was a significant correlation between percent change in MAP (0.69, P<0.01) or HR (0.84, P<0.01) and that in plasma noradrenaline. [Nphe(1)], but not UFP-101, alone significantly decreased MAP. [Nphe(1)] partially antagonized N/OFQ-induced hypotension, bradycardia and the decrease in plasma concentration of noradrenaline. UFP-101 fully prevented the effects of N/OFQ in this model. In conclusion, the present study shows that intravenous N/OFQ, via NOP receptors, elicits hypotension and bradycardia also in the anaesthetized guinea pig and that the decrease in MAP and HR are positively correlated with the decrease in the plasma noradrenaline level.     

Nphe1,Arg14,Lys15]nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor

1. Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions by activating a specific G-protein coupled receptor (NOP). Few molecules are available that selectively activate or block the NOP receptor. Here we describe the in vitro and in vivo pharmacological profile of a novel NOP receptor ligand, [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101). 2. UFP-101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high affinity (pK(i) 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP-101 competitively antagonizes the effects of N/OFQ on GTPgamma(35)S binding in CHO(hNOP) cell membranes (pA(2) 9.1) and on cyclic AMP accumulation in CHO(hNOP) cells (pA(2) 7.1), being per se inactive at concentrations up to 10 microM. 3. In isolated peripheral tissues of mice, rats and guinea-pigs, and in rat cerebral cortex synaptosomes preloaded with [(3)H]-5-HT, UFP-101 competitively antagonized the effects of N/OFQ with pA(2) values in the range of 7.3 - 7.7. In the same preparations, the peptide was inactive alone and did not modify the effects of classical opioid receptor agonists. 4. UFP-101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive effect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP-101 at 10 nmol produces per se a robust and long lasting antinociceptive effect. 5. UFP-101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ-NOP receptor system.     

Effect of Hoe 140, a new bradykinin receptor antagonist, on bradykinin- and platelet-activating factor-induced bronchoconstriction and airway microvascular leakage in guinea pig.

We have investigated the effect of a new bradykinin receptor antagonist, Hoe 140 (D-Arg- Hyp3,Thi5,D-Tic7,Oic8]-bradykinin), on bradykinin- and platelet-activating factor (PAF)-induced bronchoconstriction and airway microvascular leakage in anesthetized guinea pigs. Extravasation of Evans blue dye and lung resistance were measured simultaneously. Both i.v. (15 nmol/kg) and inhaled bradykinin (1 mM, 45 breaths) caused a significant increase in lung resistance and leakage of dye at all airway levels. Hoe 140 (100 nmol/kg i.v.) almost completely inhibited these airway responses induced by bradykinin except for dye extravasation in trachea induced by inhaled bradykinin. Inhaled PAF (3 mM, 30 breaths) significantly increased lung resistance and leakage of due at all airway levels, but Hoe 140 had no effect on these responses. Bradykinin-induced bronchoconstriction and airway microvascular leakage are predominantly mediated by activation of B2 receptor, since Hoe 140 is a B2 receptor antagonist. Bradykinin receptor-mediated mechanisms do not play an important role on inhaled PAF-induced bronchoconstriction and microvascular leakage.     

Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand,ZP120: in vitro and in vivo studies in mice

1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo.     

Binding of the novel radioligand [3H]UFP-101 to recombinant human and native rat nociceptin/orphanin FQ receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-(3)H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [(3)H]labeled NOP antagonist, [Nphe(1),4'-(3)H-Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) ([(3)H]UFP-101). We have characterized [(3)H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHO(hNOP)) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [(3)H]UFP-101 (antagonist) was compared with [(3)H]N/OFQ (agonist). The effects of GTPgammaS (10 microM) and Na(+) were investigated alone and in combination in competition experiments with both radioligands. In CHO(hNOP), B (max), and pK (D), values were 561 and 580 fmol mg protein(-1) and 9.97 and 10.19 for [(3)H]UFP-101 and [leucyl-(3)H]N/OFQ, respectively. In rat cerebrocortex B (max) and pK (D), values were 65 and 88 fmol mg protein(-1) and 10.12 and 10.34 for [(3)H]UFP-101 and [leucyl-(3)H]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation (r (2) 0.92 in Rat and 0.97 in CHO(hNOP)). Naloxone was inactive. The binding of both radioligands was Na(+)-dependent with [(3)H]UFP-101 being more sensitive (IC(50) approximately20 mM). Unlike the agonist [leucyl-(3)H]N/OFQ, the antagonist [(3)H]UFP-101 was unaffected by GTPgammaS. [(3)H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-(3)H]N/OFQ in competition experiments. In addition, the binding of [(3)H]UFP-101 is unaffected by GTPgammaS. This radioligand will be useful to further characterize NOP in a range of binding paradigms.     

MEN16132, a kinin B2 receptor antagonist, prevents the endogenous bradykinin effects in guinea-pig airways

Kinins have been suggested to be involved in human airway diseases such as asthma and rhinitis. MEN16132 is a non-peptide kinin B(2) receptor antagonist able to inhibit the responses produced by intravenous bradykinin into the airways, as bronchoconstriction and microvascular leakage; we tested the effect of MEN16132 on endogenously generated bradykinin through the dextran sulfate-induced contact activation of kinin-kallikrein cascade in guinea-pigs. After dextran sulfate administration (1.5 mg/kg i.v.), the pulmonary insufflation pressure was monitored and the microvascular leakage of upper and lower airways was assessed using Evans blue as tracer of plasma protein extravasation. Our results demonstrated that topical MEN16132 strongly inhibited the dextran sulfate-induced bronchoconstriction (0.3 mM solution aerosol for 5 min) and plasma protein extravasation in both lower airways (3-10 microM solution aerosol for 5 min) and nasal mucosa (0.3 nmol/nostril); Icatibant, the peptide antagonist of kinin B(2) receptor, exerted a 3-30-fold less potent inhibitory effect than MEN16132. We conclude that local application of MEN16132 into the airways abolishes the responses produced by the endogenous generation of bradykinin and it can be useful as new pharmacological tool to check the role of kinins in human diseases.     

Nociceptin/orphanin FQ inhibits capsaicin-induced guinea-pig airway contraction through an inward-rectifier potassium channel

Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid-like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin-induced bronchoconstriction in isolated guinea-pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea-pig nodose ganglia cells. Capsaicin increased intracellular Ca(2+) concentration in these cells through activation of vanilloid receptors. Capsaicin-induced Ca(2+) responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 microM). N/OFQ inhibitory effect on the Ca(2+) response in nodose ganglia cells was antagonized by tertiapin (0.5 microM), an inhibitor of inward-rectifier K(+) channels, but not by verapamil, a voltage gated Ca(2+) channel blocker, indicating that an inward-rectifier K(+) channel is involved in N/OFQ inhibitory effect. In isolated guinea-pig bronchus, N/OFQ (1 microM) inhibited capsaicin-induced airway contraction. Tertiapin (0.5 microM) abolished the N/OFQ inhibition of capsaicin-induced bronchial contraction. Capsaicin (10 microg) increased pulmonary inflation pressure in the isolated perfused guinea-pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 microM). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin-induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 microM) blocked the capsaicin-induced tachykinin release. These results indicate that N/OFQ-induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward-rectifier K(+) channel activation, accounts for the inhibition of capsaicin-evoked broncoconstriction.     

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles]: highly selective small-molecule nociceptin/orphanin FQ receptor agonists

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles that act as potent and selective orphanin FQ/nociceptin (N/OFQ) receptor (NOP) agonists were identified. The best compound, (+)-5a, potently inhibited 3H-N/OFQ binding to the NOP receptor (K(i) = 0.49 nM) but was >1000-fold less potent in binding to MOP, KOP, and DOP opiate receptors. Further, (+)-5a potently stimulated GTP gamma S binding to NOP membranes (EC50 = 65 nM) and inhibited forskolin-mediated cAMP accumulation in NOP-expressing cells (EC50 = 9.1 nM) with a potency comparable to that of the natural peptide agonist N/OFQ. These results indicate that (+)-5a is a highly selective and potent small-molecule full agonist of the NOP receptor.     

Orphanin FQ/nociceptin potentiates [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin-Induced mu-opioid receptor phosphorylation

In this study, we investigate the molecular mechanisms by which acute orphanin FQ/nociceptin (OFQ/N), acting through the nociceptin opioid peptide (NOP) receptor, desensitizes the mu-opioid receptor. We described previously the involvement of protein kinase C and G-protein-coupled receptor kinases (GRK) 2 and 3 in OFQ/N-induced mu receptor desensitization. Because phosphorylation of the mu receptor triggers the successive regulatory mechanisms responsible for desensitization, such as receptor uncoupling, internalization, and down-regulation, we investigated the ability of OFQ/N to modulate [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-induced mu receptor phosphorylation in BE(2)-C human neuroblastoma cells transfected with epitope-tagged mu receptors. OFQ/N treatment (100 nM, 60 min) potentiated DAMGO-induced mu receptor phosphorylation; inhibition of GRK2 or protein kinase C concomitant with OFQ/N treatment blocked the OFQ/N-mediated increase in DAMGO-induced phosphorylation. Inclusion of the NOP antagonist peptide III-BTD during OFQ/N pretreatment blocked the potentiation of DAMGO-induced phosphorylation by OFQ/N, which is consistent with the potentiation being mediated via actions of the NOP receptor. In addition, in cells expressing mu receptors in which the GRK-mediated phosphorylation site Ser(375) was mutated to alanine, OFQ/N treatment failed to potentiate DAMGO-induced mu receptor phosphorylation and failed to desensitize the mu receptor. However, DAMGO-induced mu receptor phosphorylation and OFQ/N-induced mu receptor desensitization occurred in cells expressing mu receptors lacking non-GRK phosphorylation sites. These data suggest that OFQ/N binds to NOP receptors and activates protein kinase C, which then increases the ability of GRK2 to phosphorylate the agonist-occupied mu receptor, heterologously regulating homologous mu receptor desensitization.     

Structure–activity studies on high affinity NOP‐active hexapeptides

Abstract: Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G‐protein coupled receptor ORL1 (NOP), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiologic functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited number of high‐affinity ligands to NOP, and particularly the lack of availability of useful specific antagonists. Herein, we describe the pharmacologic activity of a series of modified amino acid containing modifications of the hexapeptide Ac‐RYYRWR‐NH₂, with high affinity for NOP. These compounds were tested for binding affinity using [³H]N/OFQ binding to human NOP in CHO cells, and functional activity by measuring stimulation of [³⁵S]GTPγS‐binding in CHO cell membranes. These studies suggest that each Arg of the hexapeptide is required to maintain high‐binding affinity. The peptide maintains high affinity if the Tyr² or Tyr³ are modified, but at least one of these residues must maintain its hydroxyl group or there is a large decrease in intrinsic activity of the peptide.