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.     

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.     

Effects of [(pF)Phe(4)]nociceptin/orphanin FQ-(1-13)NH(2) on GTPgamma(35)S binding and cAMP formation in Chinese hamster ovary cells expressing the human nociceptin/orphanin FQ receptor

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP). In this study using Chinese hamster ovary (CHO) cells expressing the human NOP (CHO(hNOP)) and GTPgamma(35)S binding and cAMP inhibition assays, we have characterised a novel N/OFQ ligand, [(pF)Phe(4)]N/OFQ-(1-13)NH(2), ([(pF)Phe(4)]). [(pF)Phe(4)] was produced by insertion of a fluorine atom into the para position of the phenyl ring of Phe(4) of the truncated N/OFQ peptide N/OFQ-(1-13)NH(2). In CHO(hNOP) membranes [(pF)Phe(4)] and N/OFQ-(1-13)NH(2) stimulated GTPgamma35S binding with pEC(50) (mean+/-S.E.M.) values of 9.55+/-0.01 and 8.94+/-0.5 (P<0.05), respectively. In whole CHO(hNOP) cells [(pF)Phe(4)] and N/OFQ-(1-13)NH(2) inhibited forskolin stimulated cAMP formation with pEC(50) values of 10.19+/-0.06 and 9.60+/-0.04, respectively (P<0.05). [(pF)Phe(4)] was more potent ( approximately 4 fold) than N/OFQ-(1-13)NH(2). In both assays, the effects of [(pF)Phe(4)] and N/OFQ-(1-13)NH(2) were pertussis toxin sensitive and reversed by the NOP antagonists J-113397 (pA(2)/pK(B) values 7.89-8.53) and III-BTD (pA(2)/pK(B) values 7.27-7.96). [(pF)Phe(4)] is therefore a potent full agonist at NOP receptors that will be useful as pharmacological tool for defining the role of N/OFQ-NOP system in health and disease.     

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.     

Chronic treatment with the selective NOP receptor antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) reverses the behavioural and biochemical effects of unpredictable chronic mild stress in rats

Introduction

The present study was designed to assess the antidepressant effects of UFP-101, a selective nociceptin/orphanin FQ peptide (NOP) receptor antagonist, in a validated animal model of depression: the chronic mild stress (CMS).

Materials and methods and Results

UFP-101 (5, 10 and 20 nmol/rat; i.c.v., once a day for 21 days) dose- and time-dependently reinstated sucrose consumption in stressed animals without affecting the same parameter in non-stressed ones. In the forced swimming test, UFP-101 reduced immobility of stressed rats from day 8 of treatment. After a 3-week treatment, rats were killed for biochemical evaluations. UFP-101 abolished increase in serum corticosterone induced by CMS and reverted changes in central 5-HT/5-HIAA ratio. The behavioural and biochemical effects of UFP-101 mimicked those of imipramine, the reference antidepressant drug, administered at the dose of 15 mg/kg (i.p.). Co-administration of nociceptin/orphanin FQ (5 nmol/rat, from day 12 to 21) prevented the effects of UFP-101. Brain-derived neurotrophic factor mRNA and protein in hippocampus were not reduced by CMS nor did UFP-101 modify these parameters.

Discussion and Conclusion

This study demonstrated that chronic treatment with UFP-101 produces antidepressant-like effects in rats subjected to CMS supporting the proposal that NOP receptors represent a candidate target for the development of innovative antidepressant drugs.     

Nphe(1)]N/OFQ-(1-13)-NH(2) is a competitive and selective antagonist at nociceptin/orphanin FQ receptors mediating K(+) channel activation in rat periaqueductal gray slices.

A novel member of the opioid related receptor family, the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was identified and demonstrated to be involved in many physiological functions including pain regulation. [Nphe(1)]N/OFQ-(1-13)-NH(2) (Nphe) is a novel peptide antagonist of NOP receptors, developed using peripheral preparations. We have quantitatively investigated the interaction of Nphe with N/OFQ, the endogenous ligand of NOP receptors, in the midbrain ventrolateral periaqueductal gray (PAG), a crucial brain region for N/OFQ-induced reversal of opioid analgesia, using the patch-clamp recording technique in brain slices. N/OFQ concentration-dependently activated an inwardly rectifying K(+) current in response to hyperpolarization ramps from -60 to -140 mV. Nphe concentration-dependently attenuated the K(+) current activated by N/OFQ without changing its reversal potential. The presence of Nphe right-shifted the concentration-response curve to N/OFQ in a parallel manner. The Schild plot analysis yielded a slope of 1.16 and a pA(2) value of 6.64 that is similar to those obtained in peripheral preparations. At concentrations up to 3 microM, Nphe affected neither the membrane current per se, nor the inwardly rectifying K(+) current activated by [D-Ala(2), N-Me-Phe(4),Gly-ol(5)]-enkephalin or baclofen, a mu-opioid and GABA(B) receptor agonist, respectively. It is concluded that Nphe acts as a pure, selective and competitive antagonist at native NOP receptors of ventrolateral PAG neurons.     

Functional heterogeneity of nociceptin/orphanin FQ receptors revealed by (+)-5a Compound and Ro 64-6198 in rat periaqueductal grey slices

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is a non-opioid branch of the opioid receptor family implicated in several neurological and psychological disorders, such as pain, anxiety, depression, involuntary movement, addiction, seizure and dementia. Heterogeneity of NOP receptors has been proposed based on the findings of splicing variants and from binding and functional studies. We have previously reported that Ro 64-6198, a NOP receptor agonist, activated a subset, but not all, of N/OFQ-sensitive NOP receptors in midbrain ventrolateral periaqueductal grey (vlPAG). In this study, we found that a new NOP receptor ligand, (+)-5a Compound ((3aS, 6aR)-1-(cis-4-isopropylcyclohexyl)-5'-methyl-2'-phenylhexahydrospiro[piperidine-4,1'-pyrrolo[3, 4-c]pyrrole]), also activated a subset of NOP receptors in vlPAG neurons. (+)-5a Compound (0.1-30 μm) concentration-dependently activated G-protein-coupled inwardly-rectifying potassium (GIRK) channels mediated through the NOP receptors in about 35% of the recorded vlPAG neurons. (+)-5a Compound (EC50: 605 nm) was less potent (1/12) and efficacious (47%) than N/OFQ. In (+)-5a Compound-insensitive neurons, Ro 64-6198 was also ineffective, and vice versa, but N/OFQ activated GIRK channels through NOP receptors. In (+)-5a Compound-sensitive neurons, (+)-5a Compound precluded the effect of Ro 64-6198. Immunofluorecent and morphometric studies showed that most of the (+)-5a Compound-sensitive neurons were multipolar with intensive dendritic arborization and immunoreactive to glutamic acid decarboxylase-67. It is suggested that (+)-5a Compound activates a subset of NOP receptors, similar to the Ro 64-6198-sensitive subset, in the vlPAG neurons which are mostly GABAergic. These results further support the presence of functional heterogeneity of NOP receptors in the midbrain PAG.     

Quantitative study of the antagonistic effect of (-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111) on nociceptin/orphanin FQ-mediated potassium channel activation in rat periaqueductal gray slices

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, a non-opioid branch of the opioid receptor family, shows structural similarities to traditional opioid receptors but binds opioid with very poor affinity. This receptor has been implicated in many physiological functions including pain regulation. This study quantitatively investigated the effect of (-)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1 -yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111), a novel non-peptide ligand of NOP receptor, on the native NOP receptors in the midbrain ventrolateral periaqueductal gray (vlPAG), a crucial region for pain regulation. SB-612111 concentration-dependently antagonized N/OFQ-induced G-protein coupled inwardly rectifying K(+) (GIRK) current in vlPAG neurons. The IC(50) value of SB-612111 estimated from dose-response curves is 87.7±1.2nM. SB-612111 had no intrinsic agonistic activity and did not affect the GIRK current induced by [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin, a mu-opioid receptor agonist, when tested at concentrations of up to 1μM. It is concluded that SB-612111 is a pure, potent and selective antagonist of NOP receptors that mediate GIRK channel activation in the vlPAG neurons.     

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.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1(psi)(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 (CHO(NCR)) 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 CHO(NCR) 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 CHO(NCR) cells was NCNH2> or =NC=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 alpha = 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.     

Phe1psi(CH2-NH)Gly2]nociceptin-(1 - 13)-NH2 activation of an inward rectifier as a partial agonist of ORL1 receptors in rat periaqueductal gray.

1. [Phe1psi(CH2-NH)Gly2]nociceptin-(1 - 13)-NH2 (Phepsi), a tridecapeptide analogue of orphanin FQ/nociceptin (OFQ/N), was introduced as a competitive antagonist of opioid receptor-like orphan receptor (ORL1) in guinea-pig ileum and mouse vas deferens preparations in vitro but was recently found to act as an agonist in vivo. 2. In the periaqueductal gray, a site enriched with both OFQ/N and ORL1 and involved in OFQ/N-induced hyperalgesia and anti-analgesia, the effects of Phepsi and OFQ/N on the membrane current were studied using whole cell patch clamp recording technique in rat brain slices. 3. OFQ/N (0.01 - 1 microM) activated an inwardly rectifying type of K+ channels in ventrolateral neurons of PAG. Phepsi (0.03 - 1 microM), like OFQ/N, also activated this inward rectifier but had only 30% efficacy of OFQ/N. 4 At maximal effective concentration (1 microM), Phepsi reversed the increment of K+ conductance induced by OFQ/N (300 nM) by 46%. On the other hand, Phepsi also prevented the effect of OFQ/N if pretreated before OFQ/N. 5 It is suggested that Phepsi acts as a partial agonist of ORL1 that mediates the activation of inwardly rectifying K+ channels in ventrolateral neurons of rat periaqueductal gray.     

Equilibrium constants for (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) acting at recombinant NR1/NR2A and NR1/NR2B N-methyl-D-aspartate receptors: Implications for studies of synaptic transmission

We have quantified the effects of the N-methyl-d-aspartate (NMDA) receptor antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) at rat recombinant N-methyl-D-aspartate receptor (NR)1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either preapplied or coapplied with glutamate. The IC50 values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses, being 31 +/- 2 nM (with glutamate at its EC50 concentration) and 214 +/- 10 nM (at 10 times the EC50 concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K(B)) of 15 +/- 2 nM. Furthermore, Schild analysis of an NMDA receptor carrying a threonine-to-alanine point mutation in the NR2A ligand binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K(B) increased by around 15-fold. At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC50 value of 215 +/- 13 nM was obtained in the presence of the EC50 concentration of glutamate (1.5 microM), whereas a value of 2.2 +/- 0.14 microM was obtained with higher (15 microM) glutamate concentrations. Schild analysis gave a K(B) for NVP-AAM077 at NR2B-containing receptors of 78 +/- 3 nM. Finally, using a kinetic scheme to model "synaptic-like" activation of NMDA receptors, we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors.