Inhibitory effect of nociceptin on somatostatin secretion of the isolated perfused rat stomach

The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) has recently been isolated from porcine and rat brain and identified as the endogenous ligand of the N/OFQ receptor (NOP). It shows structural similarity with opioid peptides. N/OFQ has also been demonstrated in the gastrointestinal tract, where it inhibits gastrointestinal motility. The effect of N/OFQ on gastric neuroendocrine function is unknown as yet.In the isolated perfused rat stomach, N/OFQ 10(-6) M shows a small, but not significant decrease of basal somatostatin (SRIF) secretion. At the doses of 10(-12) M, 10(-10) and 10(-8) M N/OFQ has neither an effect on basal SRIF nor on basal vasoactive intestinal polypeptide (VIP), gastrin, substance P or bombesin secretion, respectively. However, gastric inhibitory polypeptide (GIP) 10(-9) M prestimulated SRIF secretion is significantly inhibited by N/OFQ 10(-8) M (-45+/-11%; p<0.05 vs. GIP). During concomitant infusion of the specific competitive NOP receptor antagonist [Nphe(1)]nociceptin(1-13)NH(2) 10(-6) M, the effect of N/OFQ is abolished (6+/-11%; p<0.05 vs. GIP and N/OFQ) while the opiate receptor antagonist naloxone 10(-6) M has no significant effect (-32+/-9%; ns vs. GIP and N/OFQ). At the higher concentration of N/OFQ 10(-6) M, the inhibition of prestimulated SRIF secretion (-58+/-6%; p<0.05 vs. GIP) is not influenced by the NOP receptor antagonist at the concentration of 10(-6) M (-49+/-9%; ns vs. GIP and N/OFQ) and 10(-5) M (-69+/-10%; ns vs. GIP and N/OFQ), respectively. On the other hand, infusion of naloxone 10(-6) M attenuates the inhibitory effect of N/OFQ 10(-6) M significantly (-21+/-6%; p<0.05 vs. GIP and N/OFQ).Thus, N/OFQ is an inhibitor of gastric somatostatin secretion. At the lower dose, this effect is transmitted via NOP receptors, while at the higher dose of 10(-6) M, the effect is at least in part mediated via opiate receptors.     

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.     

Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) produces several biological actions by activating the N/OFQ peptide receptor (NOP). It has been previously shown that N/OFQ stimulates leukocyte chemotaxis both in vitro and in vivo. In the present study we investigated the ability of N/OFQ, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human neutrophil and monocyte chemotaxis and the release of lysozyme and superoxide anion (O2-) production from neutrophils. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC50 12.15) but not neutrophil chemotaxis. The production of O2- from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, Ro 64-6198, UFP-101 and the opioid antagonist naloxone were used for pharmacologically characterizing the receptor involved in the monocyte chemoattractant action of N/OFQ. [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg14, Lys15]N/OFQ (pEC50 13.22)>Ro 64-6198 (pEC50 12.96)>N/OFQ(1-13)NH2 (pEC50 12.67)>N/OFQ (pEC50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 microM while antagonized by UFP-101 10 microM (pA2 7.00). Thus, the order of potency of agonists and the antagonist selectivity demonstrated that N/OFQ stimulates human monocyte chemotaxis via NOP receptor activation.     

Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.     

The effects of [Arg14, Lys15] nociceptin/orphanin FQ, a highly potent agonist of the NOP receptor, on in vitro and in vivo gastrointestinal functions

Nociceptin/orphanin FQ (N/OFQ) administered into the lateral left cerebral ventricle of rats has been reported to inhibit in vivo gut motor and secretory functions. Recently, a novel N/OFQ analog, [Arg14, Lys15] N/OFQ, was synthesized and demonstrated to behave as a highly potent agonist at the human recombinant N/OFQ peptide (NOP) receptors and to produce long-lasting effects in vivo in mice compared with the natural ligand N/OFQ. In the present study, the pharmacological profile of [Arg14, Lys15] N/OFQ was further evaluated and compared with that of N/OFQ in vitro on guinea pig exocrine pancreas and in vivo on gastric emptying, colonic propulsion and gastric acid secretion in rats. [Arg14, Lys15] N/OFQ and N/OFQ significantly decreased the KCl-evoked amylase secretion from isolated pancreatic lobules of the guinea pig. In in vivo experiments, [Arg14, Lys15] N/OFQ mimicked the effects of N/OFQ, inducing, after intracerebroventricular injection, a delay (up to 70%) in the gastric emptying of a phenol red meal, an increase (about 40 times) of the mean bead colonic expulsion time and a decrease (up to 90%) of gastric acid secretion in water loaded rats after 90 min pylorus ligature. In all these assays, [Arg14, Lys15] N/OFQ was more effective than N/OFQ, and its effective doses were at least 10-fold lower than N/OFQ effective doses. The highly selective NOP receptor antagonist, UFP-101, decreased the efficacy of [Arg14, Lys15] N/OFQ in in vitro and in vivo assays above reported. These findings: (a) show that pancreatic NOP receptors mediate an in vitro inhibitory effect on stimulated guinea pig amylase secretion; (b) confirm that the stimulation of central NOP receptors exerts an inhibitory control on gastric emptying, colonic motility and gastric secretion in rats and (c) put in evidence that [Arg14, Lys15] N/OFQ, being more potent and effective than the natural ligand N/OFQ, represents a new pharmacological tool for the study of the physiological and pharmacological roles mediated by the N/OFQ-NOP receptor system.     

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH2 with aminophosphonate moiety

New series of N-modified analogues of the N/OFQ(1-13)NH(2) with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH(2) analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH(2), but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH(2) with aminophosphonate moiety was investigated for the first time.     

IL-12 receptor beta 2 (IL-12R beta 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites

Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.     

The gastric effects of UFP-112, a new nociceptin/orphanin receptor agonist, in physiological and pathological conditions

Nociceptin/orphanin FQ (N/OFQ), the endogenous NOP receptor ligand, centrally modulates gastric motor and secretory functions and prevents ethanol-induced gastric lesions in rats. A recently synthesized N/OFQ analog, [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), acts as a highly potent and selective peptide agonist for NOP receptors and produces longer-lasting in vitro and in vivo effects in mice than the natural ligand N/OFQ. In this study, we evaluated the effects of centrally (intracerebroventricularly/icv) and peripherally (intraperitoneally/ip) injected UFP-112 on gastric emptying and gastric acid secretion, and on the development of gastric mucosal lesions induced by 50% ethanol in the rat. When injected icv, it dose-dependently delayed gastric emptying of a phenol red meal (by up to 70%), decreased gastric secretion in water-loaded rats after 90 pylorus ligature, and reduced ethanol-induced gastric lesions (by up to 87%). In all three assays, UFP-112 was more effective than N/OFQ. The highly selective NOP receptor antagonist, UFP-101, decreased the efficacy of UFP-112, thus confirming that central NOP receptors mediate inhibitory control on these functional and pathological conditions in rats. Ip injected N/OFQ and UFP-112 induced non-dose-related gastric hypersecretory and antiulcer effects, which UFP-101 partially abolished. Ip N/OFQ appeared equiactive but about 30-100 times less potent than ip UFP-112 in stimulating gastric acid secretion and preventing lesion formation. When ip injected, both UFP-112 and N/OFQ left gastric emptying in rats unchanged, suggesting that peripheral NOP receptors have a role in mediating gastric hypersecretory and antiulcer effects but are not involved in regulating gastric motility. In addition, the inhibitory effects induced by this novel NOP receptor agonist lasted longer than those induced by N/OFQ. In conclusion, UFP-112 is a promising new pharmacological tool for studying the functional roles of the central and peripheral N/OFQ receptor system.     

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor

[(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC(50) 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1-100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.     

Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor referred to as N/OFQ peptide (NOP) receptor. NOP receptor activation by N/OFQ modulates several biological functions both at central and peripheral level. Structure activity relationship (SAR) studies demonstrated that the N/OFQ sequence can be divided into a N-terminal tetrapeptide 'message' crucial for receptor activation and a C-terminal 'address' important for receptor binding. On the basis of this message/address concept we synthesized some chimeric compounds in which we substituted the natural message domain with the nonselective nonpeptide NOP ligand (8-Naphthalen-1-yl-methyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4,5]dec-3-yl)-aceticacid methyl ester (NNC 63-0532) and used as address domain the peptide sequences Thr-NH2, N/OFQ(5-9)-NH2, N/OFQ(5-13)-NH2 and N/OFQ(5-17)-NH2. All the compounds were pharmacologically evaluated in the electrically stimulated guinea-pig ileum. NNC 63-0532 produced a concentration-dependent inhibition of the electrically induced twitches showing, in comparison with N/OFQ, lower potency and higher maximal effects. In addition, contrary to N/OFQ, the effects of NNC 63-0532 were insensitive to the NOP selective antagonist [Nphe1, Arg14, Lys15]N/OFQ-NH2 (UFP-101) while prevented by naloxone. Similar results were obtained with NNC 63-0532/Thr-NH2 and NNC 63-0532/N/OFQ(1-9)-NH2. On the contrary, the inhibitory effects of NNC 63-0532/N/OFQ(5-13)-NH2 and NNC 63-0532/N/OFQ(5-17)-NH2 were slightly antagonized by UFP-101 while naloxone prevented the effects of the high but not of the low concentrations of the two ligands. These data indicate that it is possible to functionalize with the N/OFQ address sequence a nonpeptide NOP ligand for increasing its binding to the NOP receptor. Moreover, these results corroborate the idea that the 5-13 sequence represents the crucial core of the N/OFQ address domain.     

Lack of involvement of dopamine and serotonin during the orphanin FQ/Nociceptin (OFQ/N)-induced prolactin secretory response

The purpose of these studies was to examine possible mechanisms of Orphanin FQ/Nociceptin (OFQ/N)-induced prolactin release. We investigated the involvement of the dopaminergic neurons by quantifying DOPAC:DA levels in the median eminence and neurointermediate lobe following central administration of OFQ/N to female Sprague-Dawley rats. To specifically determine the involvement of the tuberoinfundibular dopaminergic neurons, immunocytochemical studies were conducted to visualize c-fos protein expression in the arcuate nucleus following central administration of OFQ/N. In addition, the role of serotonergic activation was examined in dose response studies using the selective serotonin antagonist ritansarin and the nonselective antagonist metergoline. Finally, the pharmacological specificity of the prolactin response was examined by pretreating animals with [Nphe1] NC (1-13)NH2, a drug reported to antagonize OFQ/N effects. The results of these studies indicate that the increase in prolactin release following central administration of OFQ/N does not inhibit tuberoinfundibular, tuberohypophyseal or periventricular hypophysial dopaminergic neuronal activity at 10 min after drug administration, a time when prolactin levels were significantly elevated. Furthermore, serotonergic activation is not involved since pharmacological blockade of serotonergic receptors did not alter the prolactin secretory response to OFQ/N. NC (1-13)NH2 did not antagonize the stimulatory effects of OFQ/N on prolactin secretion. The neural effects of OFQ/N on dopaminergic neuronal activity may occur following a different time course than that of the prolactin increase.     

Novel potent agonist [(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and antagonist [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.     

UFP-101 antagonizes the spinal antinociceptive effects of nociceptin/orphanin FQ: behavioral and electrophysiological studies in mice

Nociceptin/orphanin FQ (N/OFQ) modulates various biological functions, including nociception, via selective stimulation of the N/OFQ peptide receptor (NOP). Here we used the NOP selective antagonist UFP-101 to characterize the receptor involved in the spinal antinociceptive effects of N/OFQ evaluated in the mouse tail withdrawal assay and to investigate the mechanism underlying this action by assessing excitatory postsynaptic currents (EPSC) in laminas I and II of the mouse spinal cord dorsal horn with patch-clamp techniques. Intrathecal (i.t.) injection of N/OFQ in the range of 0.1-10 nmol produced a dose dependent antinociceptive effect, which was prevented by UFP-101, but not by naloxone. In contrast the antinociceptive effect of the mu-opioid peptide receptor agonist endomorphin-1 was blocked by naloxone but not by UFP-101. Moreover, N/OFQ and endomorphin-1 induced a significant antinociceptive effect in wild type mice while in mice knockout for the NOP receptor gene only endomorphin-1 was found to be active. In mouse spinal cord slices 1 microM N/OFQ reduced EPSC to 60+/-4% of control values. This inhibitory effect was reversed in a concentration dependent manner by UFP-101 (pA2 value 6.44). The present results demonstrate that N/OFQ-induced spinal antinociception in vivo and inhibition of spinal excitatory transmission in vitro are mediated by receptors of the NOP type.     

Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells

AimThe nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr¹⁰]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of ¹²⁵I-[Tyr¹⁴]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr¹⁰]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr¹⁰]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP–MOP receptors. We also examined if [Tyr¹⁰]N/OFQ(1-11) distinguished heteromeric NOP–MOP receptors from homomeric NOP receptors.Main methodsThe NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors.Key findingsIn vlPAG neurons, [Tyr¹⁰]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC₅₀: 8.98 μM) but equi-efficacious as N/OFQ. [Tyr¹⁰]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr¹⁰]N/OFQ(1-11) displayed similar concentration–response curves in decreasing cAMP accumulation.Significance[Tyr¹⁰]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP–MOP and homomeric NOP receptors.     

Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1-13), (1-11) and (1-7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3-1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1-13) and (1-11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1-13) and 4.75 for N/OFQ (1-11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1-7) led to be less potent than N/OFQ and its fragments, (1-13) and (1-11). Antinociception induced by N/OFQ or N/OFQ (1-13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1-13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1-11) and (1-7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1-13) still possess antinociceptive activity through NOP receptors.     

Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg x kg(-1) x h(-1) iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 microm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 +/- 11 mmHg, 15 nmol/kg N/OFQ: 112 +/- 10 mmHg, P = 0.009), vasodilation (venules: 23.9 +/- 1.2 microm, 26.7 +/- 1.2 microm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 +/- 3.4, 123.5 +/- 11.8, P = 0.009), and leukocyte adhesion (2.0 +/- 0.9, 15.2 +/- 0.9/100 microm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 +/- 370 microm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 +/- 0.13 vs. 2.06 +/- 0.38, P < 0.05). The NOP antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101; 60 and 150 nmol/kg iv), H(1) and H(2)antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg x kg(-1) x min(-1)) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (approximately 200 microm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10(-5) M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 +/- 3.8%; N/OFQ: 32.6 +/- 8.4%; pyrilamine: 31.5 +/- 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.     

Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20 Hz, 320 mA, 1.5 ms, 10 s) was applied five times every 20 min. Contractions induced by EFS were abolished by either TTX (1 microM) or atropine (1 microM) and concentration-dependently (10 nM-1 microM) inhibited by N/OFQ (Emax, 11.5+/-1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7+/-2.9% inhibition, P<0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1 microM) while prevented by the selective NOP receptor antagonist UFP-101 (10 microM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 microM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents.     

Lymphoadenopathy in IL-2-deficient mice: further characterization and overexpression of the antiapoptotic molecule cellular FLIP

IL-2 was originally identified as a potent T cell growth factor. It was subsequently demonstrated that IL-2 also exerts proapoptotic effects under certain conditions. Inactivation of IL-2 by gene targeting in mice showed that whereas IL-2 is not essential for the generation, clonal expansion, or differentiation of lymphocytes to effector cells, it has a unique role in preventing the accumulation of activated lymphocytes. IL-2(-/-) mice show lymphoadenopathy and autoimmune reactions, suggesting that the proapoptotic effects of IL-2 may predominate in vivo. In this study, we confirm that lymph nodes (LNs) are enlarged in IL-2(-/-) animals, but surprisingly, we found that their spleens are almost normal in size. Subsequent to this observation, we compare lymphocytes from LNs and spleens of IL-2(-/-) and IL-2(+/-) animals to analyze molecular and cellular correlates of the immunopathological disorders found in IL-2-deficient mice. LN lymphocytes from IL-2(-/-) are selectively activated and show an enhanced survival capacity and an increased ability to proliferate in vitro when compared with LN cells from IL-2(+/-) mice and splenocytes from IL-2(-/-) and IL-2(+/-) mice. Because the apoptosis inhibitor FLIP has been shown in vitro to participate in the IL-2 control of activation-induced cell death, we analyze its expression in IL-2(-/-) mice. FLIP was found to be selectively overexpressed in the LNs of IL-2(-/-) mice, but no overexpression was found in spleen cells or thymocytes. These results suggest that FLIP, in conjunction with other IL-2-regulated genes previously characterized in our laboratory, is involved in controlling lymphoadenopathy in IL-2(-/-) mice.     

Possible involvement of endogenous nociceptin/orphanin FQ in the pain-related behavioral responses induced by its own metabolite, nociceptin/orphanin FQ(14-17)

Nociceptin/orphanin FQ(14-17) (N/OFQ(14-17)) is one of the major fragments that are released from N/OFQ, an endogenous ligand for the opioid receptor like-1 (ORL-1) receptor by endopeptidase 24.11. In the present study, we determined the pharmacological profiles of N/OFQ(14-17) on pain-related behavioral responses in the mouse. Intrathecal (i.t.) administration of N/OFQ(14-17) (5-160 pmol) evoked pain-related behaviors, and these behavioral responses were reduced by i.t. co-administration of an ORL-1 receptor antagonist, [Nphe(1)]N/OFQ(1-13)NH2 (4 pmol). However, in the ligand-binding receptor assay, N/OFQ(14-17) had no affinity for the ORL-1 receptor. Furthermore, i.t. pretreatment with an antiserum against N/OFQ (1:50) diminished the N/OFQ(14-17)-induced pain-related behaviors, suggesting that endogenous N/OFQ is involved in their expression. Therefore, N/OFQ(14-17)-induced pain-related behaviors may be mediated through the release of endogenous N/OFQ in the mouse spinal cord.     

Cyclic AMP analogs induce synthesis, processing, and secretion of prepro nociceptin/orphanin FQ-derived peptides by NS20Y neuroblastoma cells

Recent studies have shown that cAMP analogs can induce expression of prepro (pp) orphanin FA (OFQ)/nociceptin-related gene products in NS20Y mouse neuroblastoma cells (Saito et al. [1996]. J Biol Chem 271, 15615-15622). Additionally, exposure of NS20Y cells to cAMP analogs promoted neurite outgrowth and large dense-core vesicle formation. Even though an OFQ-like precursor (called 27K) was identified in NS20Y cell extracts, no secretion of OFQ-related peptides was detected. We have used reversed-phase high-performance liquid chromatography combined with a specific radioimmunoassay for OFQ(1-17) to determine if NS20Y cells secrete ppOFQ-derived peptides when stimulated by the cAMP analog ctp-cAMP. We found that NS20Y cells secreted abundant amounts of OFQ-derived products when stimulated by cAMP analogs. We also have determined that secretion of OFQ peptides was both time and concentration dependent and reversible on removal of cAMP analogs from the culture medium. In addition, the opioid agonist D-Pen2-D-Pen5-enkephalin inhibited forskolin-stimulated OFQ peptide secretion. Further, the synthetic glucocorticoid dexamethasone virtually abolished ctp-cAMP-stimulated OFQ peptide secretion. These results suggest that the biosynthesis, processing, and secretion of the OFQ neuropeptide transmitter system can be modulated through intracellular cAMP levels and that these functions are regulated by opioids and molecules involved in mediating the stress response. The NS20Y cell system will be extremely valuable for studying the regulation of OFQ-derived peptides by a variety of intra-cellular and extracellular signaling pathways.