Nociceptin Research Articles

Central roles of nociceptin/orphanin FQ and nocistatin: allodynia as a model of neural plasticity

Publisher Summary This chapter discusses the roles of nociceptin/orphanin FQ and nocistatin. Opioid peptides are a family of neuropeptides with modulatory functions in nociception and inflammation. The heptadecapeptide called nociceptin or orphanin FQ (Noc/OFQ) has recently been identified as an endogenous ligand of the opioid receptor homologue, designated as ORL 1 in human and its rat counterparts LC132 and ROR-C. The Noc/OFQ receptor is expressed widely in the nervous system and it is becoming clear that Noc/OFQ is likely to participate in a broad range of central functions such as nociception, neuroendocrine secretion, and cognitive processes. The structure of Noc/OFQ and neuropeptide nocistatin (NST) is given. Central functions of Noc/OFQ and NST is briefly described. At the cellular level, Noc/OFQ exerts several cellular actions common with classical opioids, such as inhibiting CAMP production, increasing inwardly rectifying K + currents and depressing Ca 2+ currents. These actions were expected to reduce neuron excitability and to modulate neurotransmitter release through the Noc/OFQ receptors on presynaptic terminals of nerve endings. The multiple functions of Noc/OFQ and NST produced from the same precursor provide new insights into brain physiology, such as neural plasticity and may lead to therapeutic applications to the management of pain and neural disorders.

Target site of a newly synthesized nociceptin (orphanin FQ) antagonist J-113397 in its analgesic effect in the rat carrageenin test

Target site of a newly synthesized nociceptin (orphanin FQ) antagonist J-113397 in its analgesic effect in the rat carrageenin test

Evidence that N-terminal fragments of nociceptin modulate nociceptin-induced scratching, biting and licking in mice.

The intrathecal (i.t.) injection of 3.0 fmol nociceptin (orphanin FQ) elicited scratching, biting and licking responses in mice. N-terminal fragments of nociceptin, nociceptin (1-7), nociceptin (1-9) and nociceptin (1-13), induced no characteristic behavioral response. When these N-terminal fragments of nociceptin were injected simultaneously with nociceptin, the behavioral response induced by nociceptin was reduced dose-dependently. Nociceptin (1-13) was much more potent than nociceptin (1-7) and nociceptin (1-9) and antagonized nociceptin-induced response at equimolar doses. No significant effects of the N-terminal fragments were observed against the scratching, biting and licking response elicited by i.t. administration of substance P or N-methyl-D-aspartate. These results suggest that N-terminal fragments formed endogenously in the spinal cord may have an antagonistic effect on nociceptin-induced behavioral responses.

Analgesic effects of J-113397, a novel nociceptin (orphanin FQ) antagonist, in persistent-pain models but not in reflex-type pain models

Analgesic effects of J-113397, a novel nociceptin (orphanin FQ) antagonist, in persistent-pain models but not in reflex-type pain models

Vasodilator responses to the endomorphin peptides, but not nociceptin/OFQ, are mediated by nitric oxide release.

The endomorphin peptides, endogenous ligands for the mu-opioid receptor, and nociceptin (orphanin FQ; OFQ), an endogenous ligand for the ORL1 receptor, have substantial vasodilator activity in the rat. The roles of nitric oxide, vasodilator prostaglandins, and the opening of K+ATP channels in mediating vasodilator responses to these novel agonists were investigated in the hindquarters vascular bed of the rat. Under constant-flow conditions, injections of the mu-selective agonists, endomorphin 1 and 2, PL017 ([N-MePhe3, D-Pro4]-morphiceptin), and DAMGO, and the ORL1 receptor agonist, nociceptin/OFQ, produced dose-dependent decreases in hindquarters perfusion pressure. Vasodilator responses to endomorphin 1, PL017, and DAMGO were attenuated by the nitric oxide synthase inhibitor L-NAME at a time when vasodilator responses to nociceptin/OFQ were not altered. Responses to endomorphin 1 and 2, PL017, DAMGO, and nociceptin/OFQ were not altered by the cyclooxygenase inhibitor sodium meclofenamate or the K+ATP channel blocker U-37883A. The results of these studies indicate that responses to endomorphin 1 and 2, PL017, and DAMGO are mediated in large part by the release of nitric oxide, while responses to nociceptin/OFQ are mediated by an L-NAME-insensitive mechanism. Moreover, these results demonstrate that responses to these peptides are not mediated by the release of vasodilator prostaglandins or K+ATP channel opening in the hindquarters vascular bed.

ChemInform Abstract: 8‐Acenaphthen‐1‐yl‐1‐phenyl‐1,3,8‐triaza‐spiro[4.5]decan‐4‐one Derivatives as Orphanin FQ Receptor Agonists.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Nociceptin and the ORL-1 ligand [Phe1psi (CH2-NH)Gly2]nociceptin(1-13)NH2 exert anti-opioid effects in the Freund's adjuvant-induced arthritic rat model of chronic pain.

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

Structure–activity studies on nociceptin/orphanin FQ: from full agonist, to partial agonist, to pure antagonist

A heptadecapeptide (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln) was identified from rat brain and from porcine brain as a ligand for OP 4, a new G-protein coupled receptor that is similar in sequence to opioid receptors. The OP 4 receptor is widely expressed in the nervous system where it mediates a broad range of physiological functions. The new peptide, nociceptin (NC), has a primary sequence recalling that of opioid peptides. Despite the homologies (a) of the OP 4 receptor with known opioid receptors, especially the OP 2 (κ) receptor, and (b) of NC with opioid peptides, particularly dynorphin A, the two biological systems have different anatomical locations and chemical requirements for activation. NC does not bind to opioid receptors, and mammalian opioid peptides do not interact with the OP 4 receptor. The presence of Phe in position 1 and Arg in position 8, appear to be instrumental to exclude NC from interacting with the opioid receptors. Contrary to opioid peptides which strikly require Tyr in position 1, the active core that activates the OP 4 appears to be towards the centre of the peptide molecule and includes Phe 4. Based on the message/address model, several changes have been made in the N-terminal tetrapeptide Phe-Gly-Gly-Phe (message) and a few also in the C-terminal of the template NC(1–13)NH 2, a fragment that acts as a full agonist both in vitro and in vivo. Subtle changes of the N-terminal sequence, especially at Phe 1, led to the discovery of peptide antagonists ([Phe 1Ψ(CH 2NH)Gly 2]NC(1–13)NH 2 and [Nphe 1]NC(1–13)NH 2). The first compound has been widely used to characterize NC actions in the periphery and in the central nervous system. It has been shown to act mainly as an antagonist outside the brain and as an agonist in the central nervous system. [Nphe 1]NC(1–13)NH 2 on the contrary, acts as antagonist both in the periphery and in the brain. These first peptide prototypes may soon be followed by non-peptide compounds, some of which, are already described in patent literature.

Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.

1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system.

Effects of intrathecal nocistatin on the flexor reflex and its interaction with orphanin FQ nociceptin.

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

Opioid receptor‐like (ORL1) receptor distribution in the rat central nervous system: Comparison of ORL1 receptor mRNA expression with 125I‐[14Tyr]‐orphanin FQ binding

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the μ, κ, and δ opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with 125I-[14Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes. J. Comp. Neurol. 412:563–605, 1999. © 1999 Wiley-Liss, Inc.

Modification of acetylcholine release by nociceptin in conscious rat striatum

Nociceptin (NOC), an endogenous ligand for the orphan opioid receptor ORL 1 (ORL 1), has recently been recognized as a neuropeptide. We used brain microdialysis and on-line high performance liquid chromatography (HPLC) to examine the effect of NOC on the basal outflow of acetylcholine (ACh) in the freely moving rat striatum in vivo. ACh release was reduced by nociceptin at a concentration of 10 −5 M to 79% of control release. This effect of NOC was attenuated by [Phe 1Ψ(CH 2–NH)Gly 2]nociceptin-(1-13)–NH 2 (PheΨ), suggesting that NOC activates the ORL 1 receptor and (PheΨ) acts as an antagonist on ORL 1 in rat striatum in vivo. These findings indicate that NOC may act as a neuropeptide which inhibits ACh release in the striatum via ORL 1.

Nociceptin augments K(+) currents in hippocampal CA1 neurons by both ORL-1 and opiate receptor mechanisms.

We previously reported (see also the accompanying paper) that dynorphin A significantly enhanced the voltage-dependent K(+) M-current (I(M)) in CA3 and CA1 hippocampal pyramidal neurons (HPNs). Because the opioid-receptor-like-1 (ORL-1) receptor shares a high sequence homology with opioid receptors and is expressed in rat hippocampus, we examined the effects of orphanin FQ or nociceptin, the endogenous ligand for the ORL-1 receptor, using the rat hippocampal slice preparation and intracellular voltage-clamp recording. Current-voltage (I-V) relationships from CA1 HPNs revealed that nociceptin superfusion induced an outward current reversing near the equilibrium potential for K(+) ions. Ba(2+) (2 mM) blocked this effect. The nociceptin-induced current was largest at depolarized membrane potentials, where I(M) is largely activated. Nociceptin concentrations of 0.5-1 microM (but not 0.1 microM) significantly increased I(M) relaxation amplitudes with recovery on washout. Interestingly, both the general opiate antagonist naloxone and the kappa receptor antagonist nor-binaltorphimine (nBNI) inhibited the nociceptin-induced I(M) increases and outward currents in the depolarized range but not the inward current induced at hyperpolarized potentials. The putative ORL-1 receptor antagonist, [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) (hereafter ORLAn), blocked most of the nociceptin current near rest but not the I(M) increase. However, ORLAn alone had direct effects similar to those of nociceptin, indicating that ORLAn might be a partial agonist. Our results suggest that nociceptin postsynaptically modulates the excitability of HPNs through ORL-1 and kappa-like opiate receptors linked to different K(+) channels.

Synthesis, structure-activity relationship of nociceptin and its fragments

Nociceptin (NC) and its 4 fragments have been synthesized by solid phase peptide synthesis. Their hypotensive activity and mechanism, MVD assay and structure-activity relationship have been investigated. Results show that NC(1–13)NH2 is the smallest fragment that shares the same activity with NC. The truncation of C-terminal not only leads the decrease of receptor affinity but also the changes of receptor selectivity. The entire sequence may not be required for the full activity since NC(1–13)NH2 is as active as NC. Arg-Lys at the 12–13 position of C-terminal plays an important role in the activity of NC. The hypotensive activity of NC does not antagonize the hypertensive activity of renin-angiotensin system.

Orphanin FQ suppresses NMDA receptor-dependent long-term depression and depotentiation in hippocampal dentate gyrus.

We reported previously that orphanin FQ (OFQ) inhibited NMDA receptor-mediated synaptic currents and consequently suppressed induction of long-term potentiation (LTP) in the hippocampal dentate gyrus. This study examines the effect of OFQ on several other forms of long-term synaptic plasticity in the lateral perforant path of mouse hippocampal dentate gyrus. (1) Long-term depression (LTD): a low frequency stimulation (1 Hz, 15 min) applied to the lateral perforant path induced a long-lasting reduction in the dentate field potentials in slices from 22- to 30-day-old mice. This LTD was sensitive to the NMDA receptor blocker D-AP5, and could be significantly attenuated by bath application of OFQ (1 microM, 25 min). (2) Primed LTD: induction of LTD in slices from 50- to 65-day-old mice required a priming procedure consisting of multiple high frequency stimulus trains delivered in the presence of D-AP5 before the low-frequency stimulation. OFQ applied during the low-frequency stimulation, but not during the priming trains, blocked induction of primed LTD. (3) Depotentiation: high-frequency train-induced dentate LTP could be reversed by a subsequent low-frequency stimulation. This depotentiation was also attenuated by either OFQ or D-AP5 applied during low-frequency stimulation. These results, together with our previous findings, suggest that OFQ inhibits bidirectional changes in synaptic strength in the dentate; and its multiple actions on NMDA receptor-dependent, long-term synaptic plasticity might work in tandem to regulate hippocampus-dependent learning and memory.

Inhibitory effect of nociceptin on [3H]-5-HT release from rat cerebral cortex slices.

1. The effect of nociceptin (NC) on 5-hydroxytryptamine (5-HT) release was studied in rat cerebral cortex slices preincubated with [3H]-5-HT and electrically stimulated (3 Hz, for 2 min) at the 45th (St1) and the 75th (St2) min of superfusion. 2. NC (0.1 - 3 microM), present in the medium from the 70th min onward, concentration-dependently reduced electrically evoked [3H]-5-HT efflux (pEC50=6.54, Emax -54%). The inhibition was not antagonized by naloxone (1 microM) ruling out the involvement of opioid receptors. 3. Phe1psi(CH2-NH2)Gly2]NC(1-13)NH2, which acts as an opioid-like receptor (ORL1) antagonist at the peripheral level, behaved as a partial agonist in cerebral cortex slices i.e. it inhibited [3H]-5-HT efflux when added before St2, however, when present in the medium throughout the whole experiment, [Phe1psi(CH2-NH2)Gly2]NC(1-13)NH2 prevented the action of NC added at the 70th min. 4. The non-selective ORL1 receptor antagonist, naloxone benzoylhydrazone (3 microM), in the presence of 10 microM naloxone, did not modify the St2/St1 ratio but completely abolished the NC effect. 5. These findings demonstrate that NC inhibits 5-HT release from rat cerebral cortex slices via ORL1 receptors, suggesting its involvement in central processes mediated by 5-HT.

Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice.

The neuropeptide orphanin FQ (also known as nociceptin; OFQ/N) has been implicated in modulating stress-related behavior. OFQ/N was demonstrated to reverse stress-induced analgesia and possess anxiolytic-like activity after central administration. To further study physiological functions of OFQ/N, we have generated OFQ/N-deficient mice by targeted disruption of the OFQ/N gene. Homozygous mice display increased anxiety-like behavior when exposed to a novel and threatening environment. OFQ/N-null mice show elevated basal pain threshold but develop normal stress-induced analgesia. Interestingly, these mice show impaired adaptation to repeated stress when compared with wild-type mice, whereas their performance in spatial learning remained unaffected. Basal and poststress plasma corticosterone levels were found to be elevated in OFQ/N-deficient animals. Thus, OFQ/N appears to be crucially involved in the neurobiological regulation of stress-coping behavior and fear.

Orphanin FQ reduces morphine-induced dopamine release in the nucleus accumbens: a microdialysis study in rats

The effects induced by orphanin FQ (OFQ) on morphine-induced dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA) release in the nucleus accumbens were studied in rats by using microdialysis with electrochemical detection. Morphine administered intraperitoneally (i.p., 2, 5 and 10 mg/kg) dose-dependently increased DA and metabolites release in the nucleus accumbens. OFQ intracerebroventricularly (i.c.v.) administered at doses of 2, 5 and 10 nmol did not change DA and metabolites release in the nucleus accumbens. OFQ (10 nmol) administered i.c.v. 15 min before morphine (5 and 10 mg/kg, i.p.) significantly reduced morphine-induced DA and metabolites release in the nucleus accumbens. These effects suggest that OFQ may regulate the stimulant action linked to morphine-induced DA release in the nucleus accumbens.

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: Comparison of ORL1 receptor mRNA expression with125I-[14Tyr]-orphanin FQ binding

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor-like (ORL1) receptor, exhibit structural features suggestive of the micro, kappa, and delta opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with (125)I-[(14)Tyr]-OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes.

Pharmacology of [Tyr1]nociceptin analogs: receptor binding and bioassay studies.

Two series of nociceptin (NC)-related peptides with or without replacement of the N-terminal Phe by Tyr have been investigated in an attempt to obtain compounds that interact with the NC receptor (ORL1) and classic opioid receptors. When tested for their ability to displace [3H]NCNH2 ([3H]nociceptin amide; ORL1 sites) or the selective opioid receptor ligands [3H]DAMGO (mu), [3H]deltorphin II (delta) and [3H]U69593 (kappa) from their respective binding sites in guinea-pig brain membranes, [Tyr1]NCNH2 and [Tyr1]NC(1-13)NH2 showed high affinities (Ki 2nM and 5 nM, respectively) for ORL1 and approximately tenfold lower potency for mu (32nM and 44nM) and kappa sites (42 nM and 48 nM). They also interacted, but with low potency (Ki 410 nM and 310 nM) with delta sites. Shorter fragments as [Tyr1]NC(1-9)NH2 and [Tyr1]NC(1-5)NH2 were found to be inactive on ORL1, delta and kappa sites, and extremely weak (Ki 2224 nM and 4228 nM, respectively) on mu. Results of bioassays performed on the guinea-pig ileum (ORL1 and mu receptors), mouse vas deferens (ORL1 and delta receptors), and rabbit vas deferens (kappa receptor) confirmed (at least partially) the data of the binding by showing that [Tyr1]NC analogs interact with functional ORL1 as well as with classic opioid receptors. [Tyr1]NCNH2 and [Tyr1]NC(1-13)NH2 behaved as mixed ORL1/opioid receptor agonists showing similar affinities as the control NC sequence while [Tyr1]NC(1-9)NH2 and [Tyr1]NC(1-5)NH2 were inactive on ORL1 receptors but maintained some activities on opioid receptors: their effects were prevented by naloxone. The results of this study indicate that the replacement of Phe1 by Tyr in NC leads to compounds which bind both the ORL1 and mu/kappa receptors and may represent new promising agents for use in peripheral organs.