Nociceptin/orphanin FQ and [Phe(1)psi(CH2-NH)Gly2]nociceptin(1-13)NH2 modulates the activity of hypothalamic paraventricular nucleus neurons in vitro

Nociceptin, also known as orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor-like(1) (ORL(1)) receptor, is moderately expressed in the hypothalamic paraventricular nucleus (PVN) involved in the integrative control of the function of the endocrine and autonomic nervous systems. Our previous study demonstrated that intracerebroventricular administration of N/OFQ elicits an inhibitory action on the function of the cardiovascular and sympathetic nervous systems in conscious rats. However, the effects of N/OFQ on PVN neurons have not been examined. We investigated the effects of N/OFQ on PVN neurons using a whole-cell patch-clamp recording technique in rat brain slices. N/OFQ (30-1000 nM) hyperpolarized membrane potentials in type 1 and type 2 neurons of the PVN classified by the electrophysiological property. [Phe(1)psi(CH2-NH)Gly2]nociceptin(1-13)NH2 (Phepsi) (1-9 microM), a presumed competitive antagonist of the ORL(1) receptor, also hyperpolarized membrane potential in both types of neurons. In voltage clamp studies, N/OFQ (3-3000 nM) activated a K+ current concentration-dependently in 69.7% of PVN neurons with an EC(50) of 72.4+/-12 nM. Phepsi (100-9000 nM) also activated a K+ current with an EC(50) of 818+/-162 nM in PVN neurons, and significantly reduced the amplitude of the N/OFQ-stimulated current. The N/OFQ-induced current was not antagonized by the classical opioid receptor antagonist naloxone and putative antagonist nocistatin. These findings suggest that N/OFQ may have a functional role in the PVN.     

Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) does not antagonize orphaninFQ/nociceptin-induced prolactin release

The specificity of the orphaninFQ (OFQ)/nociceptin (N)-induced prolactin increase was determined in male and female rats by pretreating animals with different doses of [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2), a compound originally reported to be a specific OFQ/N antagonist. In addition, the effect of naloxone pretreatment on OFQ/N-induced prolactin release was examined to determine if OFQ/N's effects were mediated by opiate receptors. Furthermore, dose response studies using [Phe(1)Psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) only were performed to determine potential agonist activity of this drug. Finally, growth hormone (GH) levels were determined as an index of specificity of the prolactin response. Our results confirm previous findings that OFQ/N potently stimulates prolactin release and that a gender difference exists in the magnitude of the response, with females showing a much greater response than male rats. The endocrine response is specific because OFQ/N potently stimulated prolactin, but not GH secretion. The prolactin response is not mediated by actions at opiate receptors because naloxone did not inhibit OFQ/N's effects on prolactin release. However, [Phe(1)Psi(CH(2)-NH) Gly2]NC(1-13) NH(2) did not antagonize OFQ/N's effects on prolactin release. Indeed, this drug acted as a potent agonist. Demonstrating pharmacological specificity of OFQ/N's effects on prolactin release awaits the development of more selective, specific antagonists.     

Nociceptin/orphanin FQ into the rat periaqueductal gray decreases the withdrawal latency to heat and loading, an effect reversed by (Nphe(1))nociceptin(1-13)NH(2).

The present study investigated the effect of intraperiaqueductal grey injection of nociceptin/orphanin FQ (N/OFQ) and an antagonist (Nphe(1))nociceptin(1-13)NH(2) on the hindpaw withdrawal response to thermal and mechanical stimulation in rats. N/OFQ (5 nmol) significantly decreased the nociceptive thresholds in both tests and 1, 5 and 10 nmol of (Nphe(1))nociceptin(1-13)NH(2) significantly reversed this effect in a dose dependent way. Our results demonstrate, that N/OFQ has a nociceptive action, possibly through inhibition of PAG neurons. This effect is blocked by the antagonist (Nphe(1))nociceptin(1-13)NH(2) probably via ORL1 receptors in the periaqueductal grey.     

Phe1phi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 acts as a partial agonist at ORL1 receptor endogenously expressed in mouse N1E-115 neuroblastoma cells.

The nociceptin derivative [Phe1phi(CH2-NH)Gly2]-nociceptin-(1-13)-NH2 (Phe(phi)noc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identified the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of Phe(phi)noc. Like nociceptin, Phe(phi)noc stimulated the binding of [35S]GTPgammaS (EC50 = 120 nM) and inhibited forskolin-stimulated [3H]cAMP formation (EC50 = 3.3 nM). However, Phe(phi)noc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (Ki = 0.9 nM). These data indicate that Phe(phi)noc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells.     

The putative OP(4) antagonist, [Nphe(1)]nociceptin(1-13)NH(2), prevents the effects of nociceptin in neuropathic rats.

Nociceptin or orphanin FQ (N/OFQ) is the natural ligand of the opioid receptor-like 1 receptor (ORL-1), which has been also classified as the fourth member of the opioid family of receptors and named OP(4). Elucidation of the biological role of N/OFQ has been hampered by the lack of compounds that selectively block the OP(4) receptor. Recently, a N/OFQ derivative, [Nphe(1)]N/OFQ(1-13)NH(2), has been found to possess OP(4) antagonistic properties both in vitro and in vivo models. We investigated its spinal effect in the chronic constriction injury of the sciatic nerve in the rat, a model relevant to neuropathic pain in humans. Intrathecal (i.t.) administration of N/OFQ (0.2--20 nmoles) dose-dependently reversed mechanical allodynic-like behavior, while [Nphe(1)]N/OFQ(1-13)NH(2) (20--120 nmoles, i.t.) was ineffective on its own. [Nphe(1)]N/OFQ(1-13)NH(2) (60--120 nmoles, i.t.) antagonized N/OFQ (about 80% of reduction) but did not modify the activity of morphine (20 nmoles, i.t.). These results further support, for the first time in a chronic model of pain, the specific antagonistic profile of [Nphe(1)]N/OFQ(1-13)NH(2)vs the OP(4) receptor. This pseudopeptide is an interesting pharmacological tool to better clarify the role of N/OFQ in pathophysiology.     

Pharmacological profile of nociceptin/orphanin FQ receptors regulating 5-hydroxytryptamine release in the mouse neocortex

A synaptosomal preparation was employed to pharmacologically characterize the role of presynaptic nociceptin/orphanin FQ (N/OFQ) receptors (NOP receptors) in the regulation of 5-hydroxytryptamine release in the Swiss mouse neocortex. In the present study, the NOP receptor ligands N/OFQ, Ac-RYYRWK-NH(2) and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) inhibited the K(+)-induced [(3)H]-5-HT overflow with similar maximal effects ( approximately -35%) but different potencies (pEC(50) of 8.56, 8.35 and 7.23, respectively). The novel agonist [Arg(14),Lys(15)]N/OFQ also inhibited [(3)H]-5-HT overflow, but the concentration-response curve was biphasic and the efficacy higher ( approximately -45%). Receptor selectivity of NOP receptor agonists was demonstrated by showing that synaptosomes from NOP receptor knockout mice were unresponsive to N/OFQ, [Arg(14),Lys(15)]N/OFQ and [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)-NH(2) but maintained full responsiveness to endomorphin-1. Moreover, the inhibitory effect of N/OFQ was prevented by peptide ([Nphe(1)]N/OFQ(1-13)-NH(2) and UFP-101) and nonpeptide (J-113397 and JTC-801) NOP receptor selective antagonists. Desensitization occurred under perfusion with high (3 and 10 microm) N/OFQ concentrations. This phenomenon was prevented by the protein kinase C inhibitor, bisindolylmaleimide. Moreover, N/OFQ-induced desensitization did not affect mu opioid receptor responsiveness. Finally, it was observed in a similar preparation of rat cerebrocortical synaptosomes, although it was induced by higher N/OFQ concentrations than that used in the mouse. Together, these findings indicate that presynaptic NOP receptors inhibit 5-hydroxytryptamine release in the mouse neocortex. Based on present and previous studies, we conclude that NOP receptors in the mouse are subtly different from the homologous receptor population in the rat, strengthening the view that there exist species differences in the pharmacology of central NOP receptors.     

Orphanin-FQ/nociceptin (OFQ/N) modulates the activity of suprachiasmatic nucleus neurons.

Neurons in the suprachiasmatic nucleus (SCN) constitute the principal circadian pacemaker of mammals. In situ hybridization studies revealed expression of orphanin-FQ/nociceptin (OFQ/N) receptor (NOR) mRNA in the SCN, whereas no expression of mRNA for preproOFQ/N (ppOFQ/N) was detected. The presence of OFQ/N peptide in the SCN was demonstrated by radioimmunoassay. SCN neurons (88%) responded dose-dependently to OFQ/N with an outward current (EC50 = 22.3 nM) that was reduced in amplitude by membrane hyperpolarization and reversed polarity near the theoretical potassium equilibrium potential. [Phe1psi(Ch2-NH)Gly2]OFQ/N(1-13)NH2 (3 microM), a putative NOR antagonist, activated a small outward current and significantly reduced the amplitude of the OFQ/N-stimulated current. OFQ/N reduced the NMDA receptor-mediated increase in intracellular Ca2+. When injected unilaterally into the SCN of Syrian hamsters housed in constant darkness, OFQ/N (1-50 pmol) failed to alter the timing of the hamsters' wheel-running activity. However, injection of OFQ/N (0.1-50 pmol) before a brief exposure to light during the midsubjective night significantly attenuated the light-induced phase advances of the activity rhythm. These data are consistent with the interpretation that OFQ/N acting at specific receptors modulates the activity of SCN neurons and, thereby, the response of the circadian clock to light.     

Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg(-1) protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO(hNOP)) cells. N/OFQ displaced [leucyl-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS binding with pEC(50) and E(max) values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO(hNOP), pEC(50) and E(max) values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(B) for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.     

Nociceptin/orphanin FQ into the rat periaqueductal gray decreases the withdrawal latency to heat and loading, an effect reversed by (Nphe)nociceptin(1–13)NH2

The present study investigated the effect of intraperiaqueductal grey injection of nociceptin/orphanin FQ (N/OFQ) and an antagonist (Nphe¹)nociceptin(1–13)NH₂ on the hindpaw withdrawal response to thermal and mechanical stimulation in rats. N/OFQ (5 nmol) significantly decreased the nociceptive thresholds in both tests and 1, 5 and 10 nmol of (Nphe¹)nociceptin(1–13)NH₂ significantly reversed this effect in a dose dependent way. Our results demonstrate, that N/OFQ has a nociceptive action, possibly through inhibition of PAG neurons. This effect is blocked by the antagonist (Nphe¹)nociceptin(1–13)NH₂ probably via ORL1 receptors in the periaqueductal grey.     

Acute desensitization of nociceptin/orphanin FQ inhibition of voltage-gated calcium channels in freshly dissociated hippocampal neurons

Nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for opioid receptor-like receptor, has been shown to inhibit high-voltage-gated calcium channels (VGCCs) in acutely dissociated rat hippocampal pyramidal cells [Knoflach, F., Reinscheid, R.K., Civelli, O. & Kemp, J.A. (1996), J. Neurosci., 16, 6657]. In this study, it was further demonstrated that N/OFQ inhibition of calcium channel current was blocked by its specific antagonist PGN, [Phe1-psi(CH2-NH)-Gly2]nociceptin (1-13)-NH2, and the EC50 of the N/OFQ inhibition was approximately 10 nM, indicating that this effect was really mediated via the opioid receptor-like receptor. The N/OFQ inhibition of the calcium channel current was significantly reduced, as the maximal inhibition decreased from 36 to 23%, by 1-min pretreatment of freshly dissociated hippocampal neurons with the same peptide. The inhibition completely recovered from this acute desensitization in less than 20 min. The N/OFQ inhibition was also greatly attenuated by pretreatment of the neurons with the GABAB (gamma-aminobutyric acid) agonist baclofen while the baclofen inhibition of the calcium channel current was significantly reduced by N/OFQ pretreatment, revealing the agonist-induced desensitization was heterologous in nature. This desensitization was blocked by pretreating the neurons with the sodium channel blocker, tetrodotoxin (TTX), or by removing the extracellular calcium, which indicates the necessity of membrane depolarization and extracellular calcium influx in the process. Furthermore, pretreatment of the neurons with the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), attenuated the N/OFQ inhibition of the calcium channel current whereas the cAMP-dependent kinase A activator, forskolin, showed no effect, suggesting the probable involvement of PKC in the N/OFQ-induced desensitization.     

Nociceptin/orphanin FQ and [Pheψ(CH2-NH)Gly]nociceptin(1–13)NH2 modulates the activity of hypothalamic paraventricular nucleus neurons in vitro

Nociceptin, also known as orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor-like₁ (ORL₁) receptor, is moderately expressed in the hypothalamic paraventricular nucleus (PVN) involved in the integrative control of the function of the endocrine and autonomic nervous systems. Our previous study demonstrated that intracerebroventricular administration of N/OFQ elicits an inhibitory action on the function of the cardiovascular and sympathetic nervous systems in conscious rats. However, the effects of N/OFQ on PVN neurons have not been examined. We investigated the effects of N/OFQ on PVN neurons using a whole-cell patch-clamp recording technique in rat brain slices. N/OFQ (30–1000 nM) hyperpolarized membrane potentials in type 1 and type 2 neurons of the PVN classified by the electrophysiological property. [Phe¹ψ(CH₂-NH)Gly²]nociceptin(1-13)NH₂ (Pheψ) (1–9 μM), a presumed competitive antagonist of the ORL₁ receptor, also hyperpolarized membrane potential in both types of neurons. In voltage clamp studies, N/OFQ (3–3000 nM) activated a K⁺ current concentration-dependently in 69.7% of PVN neurons with an EC₅₀ of 72.4±12 nM. Pheψ (100–9000 nM) also activated a K⁺ current with an EC₅₀ of 818±162 nM in PVN neurons, and significantly reduced the amplitude of the N/OFQ-stimulated current. The N/OFQ-induced current was not antagonized by the classical opioid receptor antagonist naloxone and putative antagonist nocistatin. These findings suggest that N/OFQ may have a functional role in the PVN.     

Role of nociceptin in the modulation of nociception in the arcuate nucleus of rats

Neuropeptide nociceptin/orphanin FQ is the endogenous ligand for the opioid-receptor-like receptor 1 (ORL1), mediating essential functions in the central and peripheral nervous systems. The present study was performed to investigate the role of nociceptin and ORL1 receptor in nociception and morphine-induced antinociception in the arcuate nucleus of hypothalamus in rats. Hindpaw withdrawal latencies (HWL) were measured by hot-plate and Randall Selitto tests. The HWL to both thermal and mechanical stimulation decreased significantly after intra-arcuate nucleus injection of nociceptin in a dose-dependent manner. The effect of nociceptin was blocked significantly by subsequent intra-arcuate nucleus administration of [Nphe(1)]nociceptin(1-13)-NH(2), an ORL1 receptor antagonist. Furthermore, an intra-arcuate nucleus injection of nociceptin dramatically attenuated the antinociceptive effect induced by morphine either injected in the same site or applied intraperitoneally. These results suggest that nociceptin in the arcuate nucleus induces a hyperalgesic effect by acting on ORL1 receptors. The present study also demonstrates an interaction between nociceptin and opioids in the arcuate nucleus of the hypothalamus.     

In vitro inhibitory effects of J-113397 on nociceptin/orphanin FQ-stimulated.

J-113397 (1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) is a recently developed antagonist of the opioid receptor-like 1 (ORL1) receptor. We compared the in vitro functional profile J-113397 on [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTPgammaS) binding to mouse brain with that of [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 and naloxone benzoylhydrazone (NalBzoH). J-113397 antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding to mouse brain with an IC50 value of 7.6 nM, but had no effect on basal [35S]GTPgammaS binding by itself. [Phe1psi(CH2-NH)Gly2]nociceptin(1-13)NH2 partially antagonized nociceptin/orphanin FQ-stimulated [35S]GTPgammaS binding but showed agonistic activity on ORL1 by itself. NalBzoH showed antagonistic activity on ORL1 receptor but had significant agonistic activity on other opioid receptors at lower doses. Schild plot analysis demonstrated competitive antagonism of J-113397 on ORL1 receptor in mouse brain. A [35S]GTPgammaS binding study using ORL1 receptor-deficient mice confirmed the selective antagonism of J-113397 on ORL1 receptor. These data indicate that J-113397 is the most potent and selective antagonist of ORL1 receptor in mouse brain that has yet been reported, and therefore will be a useful tool for characterization of ORL1 receptors in the brain.     

In vitro characterization of Ac-RYYRWK-NH(2), Ac-RYYRIK-NH(2) and [Phe1Psi(CH(2)-NH)Gly2] nociceptin(1-13)NH(2) at rat native and recombinant ORL(1) receptors

The pharmacology of ORL(1) compounds, [Phe1Psi(CH(2)-NH)Gly2]nociceptin(1-13)NH(2) (F/GNC13), Ac-RYYRIK-NH(2) and Ac-RYYRWK-NH(2) was evaluated at rat ORL(1) receptors in frontal cortex (CTX), transfected chinese hamster ovary (CHO) cells, vas deferens (VD) and anococcygeus (AC). Ranked affinities for the inhibition of [3H]nociceptin binding to CTX and CHO's were: Ac-RYYRWK-NH(2) identical withAc-RYYRIK-NH(2) identical withnociceptin>F/GNC13>Dynorphin A>naloxone.The full agonist, nociceptin stimulated [35S]GTPgammaS binding in CTX (E(max)=174%) and CHO's (E(max)=311%); all other ORL(1) peptides acted as partial agonists with the following rank order for E(max) values: Ac-RYYRWK-NH(2) (96% (CTX), 202% (CHO))>F/GNC13 (44% (CTX), 136% (CHO)) identical withAc-RYYRIK-NH(2) (44% (CTX), 115% (CHO)). Schild analysis generated pA(2) values in CTX of 8.59 (F/GNC13) and 9.13 (Ac-RYYRIK-NH(2)). cAMP production in CHO's was inhibited by 77% (nociceptin), 58% (Ac-RYYRWK-NH(2)), 55% (F/GNC13) and 49% (Ac-RYYRIK-NH(2)). Nociceptin inhibited electrically evoked contractions in isolated tissues by 95% (VD) and 98% (AC); partial inhibition was observed with Ac-RYYRWK-NH(2) (72% (VD), 66% (AC)) and Ac-RYYRIK-NH(2) (54% (VD); 37%(AC)).Ineffective in the VD, F/GNC13 caused a small inhibition in the AC that was reversed at higher concentrations. Schild analysis gave pA(2) affinities of 7.32(VD) and 7.34(AC) for F/GNC13 and 8.69(AC) for Ac-RYYRIK-NH(2).     

Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-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.     

Pro-nociceptin/orphanin FQ and NOP receptor mRNA levels in the forebrain of food deprived rats

Forebrain injections of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP opioid receptor, previously referred to as ORL1 or OP4 receptor, stimulate feeding in freely feeding rats, while the NOP receptor antagonist [Nphe(1)]N/OFQ(1-13)NH(2) inhibits food deprivation-induced feeding. To further evaluate whether the N/OFQ-NOP receptor system plays a physiological role in feeding control, the present study evaluated forebrain mRNA levels for the N/OFQ precursor (pro-N/OFQ), as well as for the NOP receptor in food deprived rats. The results obtained show that food deprived rats have lower mRNA levels for the NOP receptor in several forebrain regions; a significant reduction was found in the paraventricular and lateral hypothalamic nuclei and in the central nucleus of the amygdala. Food deprived rats also exhibited lower pro-N/OFQ mRNA levels in the central amygdala. These results suggest that the N/OFQ-NOP receptor system may have a physiological role in feeding control. The observation that food deprivation reduces gene expression of the N/OFQ-NOP receptor system is apparently not consistent with a direct hyperphagic action for N/OFQ. Taking into account that N/OFQ exerts inhibitory actions at cellular level, the present results may be in keeping with the hypothesis that N/OFQ stimulates feeding by inhibiting neurons inhibitory for food intake; under conditions of food deprivation, these neurons may be silent and the N/OFQ-NOP receptor system, which controls them, may also be regulated at a lower level. Consistently, in the present study N/OFQ stimulated food intake in freely feeding rats, but did not further increase feeding in food deprived rats.     

Involvement of nociceptin/orphanin FQ and its receptor in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain regulation. In the present study, the involvement of N/OFQ-NOP receptor system in electroacupuncture (EA)-produced anti-hyperalgesia was investigated in rats with peripheral inflammation. Intrathecal (i.t.) administration of N/OFQ (15 nmol) or EA at acupoints GB30 and GB34 could significantly attenuate hyperalgesia which was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into one hindpaw of rats, manifesting as decreased paw withdrawal latency (PWL) to the noxious thermal stimulus. The anti-nociceptive effect of N/OFQ or EA was significantly blocked by intrathecal injection of [Nphe(1)]nociceptin(1-13)NH(2) (20 nmol), a selective antagonist of the NOP receptor, indicating the NOP-receptor-mediated mechanism. Additionally, the combination of N/OFQ injection with EA treatment could enhance anti-hyperalgesia compared to that produced by each component alone. These findings suggested that the spinal N/OFQ-NOP system might be involved in EA analgesia, which may be one of the mechanisms underlying the anti-nociceptive effect of EA in rat's peripheral inflammatory pain.     

A combined pharmacological and genetic approach to investigate the role of orphanin FQ in learning and memory

Using a combination of the selective opioid receptor-like1 (ORL1) receptor agonist, Ro 64-6198, and orphanin FQ/nociceptin (OFQ/N) peptide knockout (KO) mice, the influence of OFQ/N on cognition has been studied in the rodent. In wild type, C57BL/6J mice, Ro 64-6198 (0.3-1 mg/kg i.p.) impaired the acquisition of spatial learning in the Morris water maze, although a mild neurological impairment was evident which complicated precise interpretation. In Lister hooded rats, Ro 64-6198 (6 mg/kg i.p.) produced delay dependent impairments in rats performing either a delayed matching or a delayed nonmatching to position task with only a modest (< 20%) effect on omissions - an effect consistent with a short-term memory impairment. Electrophysiological studies demonstrated an inhibitory effect of OFQ/N on LTP recorded from the CA1 region of wild type mice, but not in ORL1 receptor knockout mice. In contrast to the ORL1 agonist, mice deficient in the OFQ/N peptide showed some evidence of improved spatial learning, fear conditioning and passive avoidance retention. However, CA1 LTP was similar between OFQ/N peptide KO mice and wild type controls. Subsequent receptor radioautography studies demonstrated the presence of ORL1 receptors within various regions of the medial temporal lobe system: i.e. CA1, dentate gyrus molecular layer, subiculum, perirhinal cortex. Taken together, these results suggest a bi-directional effect of OFQ/N containing systems on aspects of cognitive behaviour, particularly those elements associated with hippocampal function. This is consistent with a likely modulatory role of OFQ/N on hippocampal and associated cortical circuitry.     

孤啡肽受体拮抗剂对机械性损伤神经元存活率的影响

目的既往研究表明,孤啡肽在脑损伤后的表达明显升高,本研究通过特异性阻断孤啡肽受体(opioid-receptor-like receptor,ORL-1),观察对受损神经元是否具有保护作用。方法建立神经元机械性损伤模型,用孤啡肽受体特异性阻断剂([Nphe1]-NC(1-13)-NH2,Nphe)阻断孤啡肽受体,通过四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法、乳酸脱氢酶(lactate dehydrogenase,LDH)活性,钙离子水平测定,研究Nphe对机械性损伤神经元存活率的影响。结果 MTT法测定神经元机械性损伤后12 h细胞存活率显示:单纯损伤组细胞存活率为46%±4%,与对照组相比显著下降(P<0.05),不同剂量Nphe(30、300、1 200 nM)干预组的细胞存活率分别为56%±5%、67%±7%、72%±8%,与单纯损伤组存活率46%±4%相比差异显著(P<0.05)。LDH活性检测提示损伤后12 h和48 h,Nphe干预组LDH活性与损伤组相比有显著差异(P<0.05)。神经元机械性损伤后12 h,Nphe能够降低损伤后细胞内钙离子水平(P<0.05)。结论 ORL-1的特异性拮抗剂Nphe能够减少机械性损伤后继发性神经元损害,对神经元具有一定的保护作用。     

The putative OP4 antagonist, [Nphe]nociceptin(1-13)NH2, prevents the effects of nociceptin in neuropathic rats

Nociceptin or orphanin FQ (N/OFQ) is the natural ligand of the opioid receptor-like 1 receptor (ORL-1), which has been also classified as the fourth member of the opioid family of receptors and named OP₄. Elucidation of the biological role of N/OFQ has been hampered by the lack of compounds that selectively block the OP₄ receptor. Recently, a N/OFQ derivative, [Nphe¹]N/OFQ(1-13)NH₂, has been found to possess OP₄ antagonistic properties both in vitro and in vivo models. We investigated its spinal effect in the chronic constriction injury of the sciatic nerve in the rat, a model relevant to neuropathic pain in humans. Intrathecal (i.t.) administration of N/OFQ (0.2–20 nmoles) dose-dependently reversed mechanical allodynic-like behavior, while [Nphe¹]N/OFQ(1-13)NH₂ (20–120 nmoles, i.t.) was ineffective on its own. [Nphe¹]N/OFQ(1-13)NH₂ (60–120 nmoles, i.t.) antagonized N/OFQ (about 80% of reduction) but did not modify the activity of morphine (20 nmoles, i.t.). These results further support, for the first time in a chronic model of pain, the specific antagonistic profile of [Nphe¹]N/OFQ(1-13)NH₂vs the OP₄ receptor. This pseudopeptide is an interesting pharmacological tool to better clarify the role of N/OFQ in pathophysiology.