Nociceptin/orphanin FQ Research Articles

A dual nociceptin and mu opioid receptor agonist exhibited robust antinociceptive effect with decreased side effects

The compelling demand of a consummate analgesic medication without addiction is rising due to the clinical mistreatment. Additionally, the series of severe untoward effects usually deterred the utilization while coping with serious pain. As a possible turning point, we revealed that compound 14 is a dual agonist of mu opioid receptor (MOR) and nociceptin-orphanin FQ opioid peptide (NOP) receptor in this study. More importantly, compound 14 achieves pain relieving at very small doses, meanwhile, reduces several unwanted side effects such as constipation, reward, tolerance and withdrawal effects. Here, we evaluated the antinociception and side effects of this novel compound from wild type and humanized mice to further develop a safer prescription analgesic drug.

Benzo[b]thiophene-2-carboxamides as novel opioid receptor agonists with potent analgesic effect and reduced constipation

Currently, there is a significant unmet need for novel analgesics with fewer side effects. In this study, we carried out structural modification of a hit compound previously identified in an artificial-intelligence (AI) virtual screening and discovered the potent analgesic, benzo[b]thiophene-2-carboxamide analog (compound 25) with new structural scaffold. We investigated the signaling pathways of opioid receptors mediated by compound 25, and found this racemic compound activated mu-opioid receptor through the cyclic adenosine monophosphate (cAMP) and β-arrestin-2-mediated pathways with strong potency and efficacy, and accompanying nociceptin-orphanin FQ opioid peptide and delta-opioid receptors through the cAMP pathway with weak potencies. Compound 25 elicited potent antinociception in thermal-stimulated pain (ED50 value of 127.1 ± 34.65 μg/kg) and inflammatory-induced allodynia models with less gastrointestinal transit inhibition and antinociceptive tolerance than morphine. Overall, this study revealed a novel analgesic with reduced risks of side effects.

Buprenorphine Analogs to Treat Relapse

Substance use disorders (SUDs) – the overuse and dependence on at least one substance – have a lifetime prevalence of ~10% in the U.S. While buprenorphine (BUP) treatment reduces relapse rates, it is only marginally effective, as 70% of SUD patients relapse within a year. Due to BUP’s marginal effectiveness and abuse potential, there is a need to improve SUD therapies that prevent relapse to multiple drugs of abuse after exposure to stress, drug‐associated context and drug priming. Here we evaluate BUP and two analogs for their ability to inhibit cocaine‐ and stress‐induced relapse using a conditioned place preference (CPP) paradigm. Pharmacologically, BUP is a potent (sub nM) partial agonist at the m‐opioid receptor (MOR) with potent antagonist activity at the k‐(KOR), and d‐opioid receptors (DOR). In vitro BUP can act as a weak (mM) partial agonist at the nociceptin/OrphaninFQ (NOP) receptor with uncertain in vivo consequences. We measured in vitro potency and efficacy using [35S]GTPgS functional assays in CHO cells separately expressing DOR, KOR, MOR or NOP. Since NOP receptor agonists reduce relapse in animal models, we compared BU10119 with NOP agonist activity (EC50 = 120 nM ± 20; EMAX = 44% ± 5%) or BU12004 with NOP antagonist activity (KB = 190 nM ±30) to BUP (EC50 = 1300 nM ± 320; EMAX = 39% ± 7%). Furthermore, BU12004 and BU10119 were MOR antagonists (KB = 0.42 nM ± 0.08 and KB = 1.1 nM ± 0.2, respectively) with less abuse liability than the MOR partial agonist BUP (EC50 = 0.79 nM ± 0.31; EMAX = 34% ± 6%). BUP, BU10119, and BU12004 were all potent antagonists at DOR and KOR. BU10119 (1.0 mg/kg) attenuated cocaine‐primed reinstatement in CPP, but 10 mg/kg BU12004 did not. BUP trended toward reduced drug‐primed reinstatement. The in vivo rank order of BU10119 >BUP >BU12004 matches the rank order for NOP agonism in vitro. Additionally, 1.0 mg/kg of either BU10119 or BU12004 blocked swim stress‐primed reinstatement, ostensibly due to their KOR antagonist activity. Taken together, BU10119 provides a promising lead to treat SUDs with better effectiveness and reduced abuse potential compared to BUP.Support or Funding InformationSupported by NIDA R01 DA07315 and 035316.

BPR1M97, a dual mu opioid receptor/nociceptin-orphanin FQ peptide receptor agonist, produces potent antinociceptive effects with safer properties than morphine

There is unmet need to design an analgesic with fewer side effects for severe pain management. Although traditional opioids are the most effective painkillers, they are accompanied by severe adverse responses, such as respiratory depression, constipation symptoms, tolerance, withdrawal, and addiction. We indicated BPR1M97 as a dual mu opioid receptor (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor full agonist and investigated the pharmacology of BPR1M97 in multiple animal models. In vitro studies on BPR1M97 were assessed using cyclic-adenosine monophosphate production, β-arrestin, internalization, and membrane potential assays. In vivo studies were characterized using the tail-flick, tail-clip, lung functional, heart functional, acetone drop, von Frey hair, charcoal meal, glass bead, locomotor activity, conditioned place preference (CPP) and naloxone precipitation tests. BPR1M97 elicited full agonist properties for all cell-based assays tested in MOP-expressing cells. However, it acted as a G protein-biased agonist for NOP. BPR1M97 initiated faster antinociceptive effects at 10 min after subcutaneous injection and elicited better analgesia in cancer-induced pain than morphine. Unlike morphine, BPR1M97 caused less respiratory, cardiovascular, and gastrointestinal dysfunction. In addition, BPR1M97 decreased global activity and induced less withdrawal jumping precipitated by naloxone. Thus, BPR1M97 could serve as a novel small molecule dual receptor agonist for antinociception with fewer side effects than morphine.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Concomitant in Vivo Voltammetric and Electrophysiological Analysis Indicate that Nociceptin/Orphanin FQ Affects Dopamine and then Serotonin Activities in Brain Substancia Nigra.

Nociceptin/orphanin-FQ (NOCI) together with its receptor NOP are widely expressed in cortical and subcortical motor areas and it is known that NOCI acts as an anxiolytic attenuating the behavioral inhibition of animals acutely exposed to stressful/anxiogenic conditions. Influence of NOCI upon the dopaminergic system has been observed in the ventral tegmental area and in the nucleus accumbens as well as an inhibitory action of NOCI is described upon serotoninergic mechanisms at cells and terminal levels. In particular, it is known that serotoninergic fibers from the raphe system project to the substancia nigra (SN) and that this modulation is behaviourally relevant. In the present work, the effect of exogenous NOCI injected into the SN upon DA and 5-HT levels have been analyzed by means of differential pulse voltammetry and nafion-carbon fiber microelectrodes. Electrophysiological monitoring of multicell activity was concomitantly performed with the same microsensor. It appeared that both levels of these biogenic amines were specularly altered, with possibly a driving influence of the DA activity upon the serotoninergic function(s).

Distinct functions of opioid-related peptides and gastrin-releasing peptide in regulating itch and pain in the spinal cord of primates.

How neuropeptides in the primate spinal cord regulate itch and pain is largely unknown. Here we elucidate the sensory functions of spinal opioid-related peptides and gastrin-releasing peptide (GRP) in awake, behaving monkeys. Following intrathecal administration, β-endorphin (10–100 nmol) and GRP (1–10 nmol) dose-dependently elicit the same degree of robust itch scratching, which can be inhibited by mu-opioid peptide (MOP) receptor and GRP receptor (BB2) antagonists, respectively. Unlike β-endorphin, which produces itch and attenuates inflammatory pain, GRP only elicits itch without affecting pain. In contrast, enkephalins (100–1000 nmol) and nociceptin-orphanin FQ (3–30 nmol) only inhibit pain without eliciting itch. More intriguingly, dynorphin A(1–17) (10–100 nmol) dose-dependently attenuates both β-endorphin- and GRP-elicited robust scratching without affecting pain processing. The anti-itch effects of dynorphin A can be reversed by a kappa-opioid peptide (KOP) receptor antagonist nor-binaltorphimine. These nonhuman primate behavioral models with spinal delivery of ligands advance our understanding of distinct functions of neuropeptides for modulating itch and pain. In particular, we demonstrate causal links for itch-eliciting effects by β-endorphin-MOP receptor and GRP-BB2 receptor systems and itch-inhibiting effects by the dynorphin A-KOP receptor system. These studies will facilitate transforming discoveries of novel ligand-receptor systems into future therapies as antipruritics and/or analgesics in humans.

A novel, potent, orally‐bioavailable nociceptin‐1 receptor antagonist reduces ethanol self‐administration and ethanol‐seeking in animal models of alcohol addiction (656.2)

Background: The receptor for nociceptin/orphanin‐FQ (NOP) is localized in the mesolimbic reward pathway and has been suggested to play a role in addiction and motivated behavior. We characterize here a novel, potent, orally‐bioavailable NOP antagonist in several ethanol self‐administration models.Methods: Compound 1 was evaluated in homecage ethanol self‐administration models, progressive ratio operant self‐administration, stress‐induced reinstatement to ethanol‐seeking, and in vivo microdialysis.Results: Compound 1 dose‐dependently reduced homecage ethanol self‐administration in both P and msP rats, without affecting food or water intake. Compound 1 also attenuated progressive ratio operant responding and breakpoints in P rats. Stress‐induced reinstatement of ethanol‐seeking in msP rats was completely blocked by compound 1. Furthermore, compound 1 blocked ethanol‐induced dopamine release in the nucleus accumbens.Conclusions: Compound 1 demonstrates efficacy in attenuating ethanol self‐administration and stress‐induced ethanol‐seeking in two different lines of rats that exhibit excessive ethanol consumption. Compound 1 also blocks the motivation to consume ethanol and ethanol‐stimulated increases in extracellular dopamine levels in the nucleus accumbens, suggesting potential therapeutic utility of NOP receptor antagonists in treating alcohol addiction.

Opioid Receptor Gene Expression in Human Neuroblastoma SH-SY5Y Cells Following Tapentadol Exposure

Recent studies showed that combination of mu opioid receptor (MOP) agonism and monoamine reuptake inhibition may improve the therapeutic effect of opioids by reducing requirement for MOP activation. Tapentadol, showing such a combined mechanism of action, exhibits delayed analgesic tolerance development compared to pure MOP agonists. Here we investigated how opioid receptors are regulated following different schedules (two ranges of concentrations for 24 and 48 h) of tapentadol exposure in vitro in SH-SY5Y cells. MOP and nociceptin/orphaninFQ (NOP) receptor gene expressions were quantified using qReal-Time PCR. Moreover, studies were performed in U2 cells to assess tapentadol effect on MOP internalization compared with morphine and DAMGO. Ten and 100 nM tapentadol for 48 h induced a significant increase of MOP gene expression; cells exposed to 100 μM tapentadol for 24 and 48 h showed a significant increase of MOP mRNA levels. NOP gene expression showed a significant decrease following tapentadol at all low concentrations used after 24 h and at high concentrations (45 and 60 μM) after 24 h and (60 μM) after 48 h. Differently from DAMGO, tapentadol or morphine showed no effects on MOP internalization. This study suggests that tapentadol affects MOP and NOP gene expression and MOP internalization showing a pattern distinct from classical MOP agonists. Whether these differences can explain the improved therapeutic profile of tapentadol remains to be investigated.

S18 Activation of nociceptin orphanin FQ (N/OFQ) – N/OFQ peptide (NOP) receptor system plays a key immunomodulatory role in asthma

Asthma is a complex heterogeneous disease characterised by variable airflow obstruction, bronchial hyper-responsiveness, airway inflammation and remodelling. The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide (NOP) receptor, a non-opioid member of the opioid receptor family. The role of N/OFQ-NOP system in asthma is uncertain. We sought to evaluate N/OFQ-NOP expression in healthy and asthmatic human airway tissues and relate this to an established animal model of asthma. NOP expression in human airway cells was investigated predominantly by qRT-PCR. The functional role of N/OFQ on human airway structural and immune cells was then interrogated using a range of functional assays including proliferation,migration,collagen gel contraction and wound healing. We further investigated the functional role of N/OFQ in vivo using ovalbumin-sensitised mice. NOP expression was detected in human airway smooth muscle cells (HASM; mean?C? = 11 ± 0.7,n = 13), bronchial epithelial cells (HBEC;mean?C? = 10 ± 0.49,n = 12), lung mast cells (mean?C? = 7 ± 0.64,n = 5) and peripheral blood eosinophils (mean?C? = 10.4 ± 1.2,n = 16). N/OFQ inhibited chemoattractant-induced migration of mast cells and eosinophils (see Figure). N/OFQ stimulated significant HBEC wound closure with 49.62 ± 3.58% (p < 0.001, n = 8) of the wound area healed relative to the control (30.88 ± 4.13%, n = 8)18 h post-wound. Similarly N/OFQ significantly promoted HASM wound closure (p < 0.01). Our findings showed that SCF-stimulated IL-8 release (4.43 ± 0.69 fold over control, p < 0.01,n = 7) was significantly inhibited by N/OFQ (3.32 ± 0.56 fold over control, p < 0.01, n = 7). Ex vivo human studies demonstrate significantly (p < 0.01) increased endogenous N/OFQ in asthmatic airways relative (sputum N/OFQ:59.02 ± 2.57 pg/ml,n = 26) to healthy airways (sputum N/OFQ:44.69 ± 0.43,n = 10) and identifies eosinophils as a potential source for these. Pre-treatment with N/OFQ was shown to significantly reduce agonist-induced bronchial hyper-responsiveness using in vitro (p < 0.01) and in vivo models (p < 0.001). Ex vivo animal studies show that N/OFQ significantly inhibits release of inflammatory mediators including IL4, IL5, IL12, IL13 and inflammatory cell recruitment including mast cells and eosinophils within the airways. Further mucus hyper-secretion was also reduced following N/OFQ pre-treatment in these models. ![Abstract S18 Figure 1.][1] Abstract S18 Figure 1. N/OFQ inhibits chemoattractant-induced migration of lung mast cells and eosinophils. Migration of HMC-l cells (through an 8- µm pore-size transwell membrane) was enhanced following stimulation With SCF (p<0.01, n = 5), CXCL10 (p<0.001, n = 5) and TNF-α stimulated ASM s/n (p<0.001, n = 8) and this was significantly inhibited by N/OFQ (p<0.01). HLMC migration induced by SCF (p<0.001, n = 7) was significantly attenuated by N/OFQ (p<0.001, n = 7). Migration of peripheral blood eosinophils (n = 6) stimulated by epithelial conditioned media (p<0.01) was significantly inhibited by N/OFQ (p<0.01). Cell counts were performed by a blinded observer and data was represented as fold change over control in log (2) scale (mean ± SEM). Statistical analysis was performed by one-Way ANOVA With Bonferroni’s multiple comparison test. (*: p value vs control; #: p value vs different relevant stimulation). This is the first study to perform a comprehensive and complementary in vivo and in vitro study of the expression and actions of the N/OFQ-NOP system in the airways and provide evidence for a role of NOP activation in the management of asthma. [1]: pending:yes

The nociceptin orphanin FQ peptide receptor agonist, Ro64-6198, impairs recognition memory formation through interaction with glutamatergic but not cholinergic receptor antagonists

We previously reported that the selective nociceptin orphanin peptide (NOP) receptor agonist, Ro64-6198, impairs mnemonic function through glutamatergic-dependent mechanisms. The aim of the current study was to determine whether the amnesic effects of Ro64-6198 involve a cholinergic component. The effects of systemic administration of Ro64-6198 (0.3 and 1mg/kg, i.p.), the cholinergic nicotinic receptor antagonist, mecamylamine (0.1 and 1mg/kg, s.c.), the cholinergic muscarinic receptor antagonist, scopolamine (0.1 and 0.3mg/kg, s.c.), and the glutamatergic NMDA receptor antagonist, MK-801 (0.03 and 0.1mg/kg, s.c.), were studied in the mouse object recognition task. All compounds tested were effective in disrupting formation of long-term (24-h delay) recognition memory. Drug interaction studies were then conducted to reveal the existence of functional interactions between NOP receptors and cholinergic and/or NMDA receptors. Co-administration of silent doses of Ro64-6198 (0.3mg/kg) and MK-801 (0.01mg/kg) produced clear-cut memory impairment. Similar synergistic effects were observed with the combination of mecamylamine (0.03mg/kg) and scopolamine (0.1mg/kg). In contrast, co-administration of Ro64-6198 (0.3mg/kg) with either mecamylamine (0.03 and 0.1mg/kg) or scopolamine (0.1mg/kg) was without any effect on recognition memory. These findings suggest that NOP receptor may modulate memory formation through a functional interaction with glutamatergic but not cholinergic receptors.

Orphanin FQ/Nociceptin Activates Nuclear Factor Kappa B

Endogenous neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, nociceptin orphanin FQ peptide receptor (NOPr), play a modulatory role throughout the body including nociceptive sensitivity, motor function, spatial learning, and the immune system. NOPr is an inhibitory G protein coupled receptor (GPCR) that modulates expression and release of inflammatory mediators from immune cells and in the CNS. Inhibitory GPCRs have been shown to activate the immune and central nervous system regulator, nuclear factor kappa B (NFκB), whose family consists of several subunits. When activated, NFκB translocates to the nucleus and can modify transcription. To determine if OFQ/N modulates NFκB activity, SH-SY5Y human neuroblastoma cells were treated with OFQ/N and assessed for changes in nuclear accumulation, DNA binding, and transcriptional activation. For the first time, we show that OFQ/N increases the nuclear accumulation (1.9-2.8-fold) and the DNA binding of NFκB (2.9-fold) by 2h as determined by immunoblotting and electromobility shift assay, respectively. OFQ/N induction of NFκB binding to DNA is protein kinase C-dependent and NOPr-specific. OFQ/N stimulated binding of both NFκB p50 and p65 subunits to their consensus binding site on DNA. OFQ/N also induces transcriptional activation of an NFκB reporter gene 2.2-fold by 2h with an EC(50) of 6.3nM. This activation of NFκB by OFQ/N suggests a likely mechanism for its modulation of the central nervous and immune systems.

Pharmacological blockade or genetic knockout of the NOP receptor potentiates the rewarding effect of morphine in rats

The Nociceptin/OrphaninFQ (NOP) system is believed to be involved in drug abuse and addiction. We have recently demonstrated that activation of the NOP receptor, by systemic administration of the NOP receptor agonist Ro65-6570, attenuated the rewarding effect of various opioids in conditioned place preference (CPP) in rats and this attenuating effect was reversed by the NOP receptor antagonist J-113397. The present study demonstrates that co-administration of J-113397 (4.64 mg/kg, i.p.) during conditioning, facilitates morphine-induced CPP. Moreover, we found that NOP receptor knockout rats (oprl1 −/−) are more sensitive to the rewarding effect of morphine than wildtype control rats. Thus, pharmacological or genetic inactivation of the NOP system rendered rats more susceptible to the rewarding effect of morphine. These findings support the suggestion that the endogenous NOP system attenuates the rewarding effect of opioids and therefore offers a therapeutic target for the treatment of drug abuse and addiction.

Nociceptin receptor antagonist JTC-801 inhibits nitrous oxide-induced analgesia in mice

The mechanism of the analgesic effect of nitrous oxide (N(2)O) has not been completely clarified. Although we have reported that the analgesic effect of N(2)O was significantly decreased in nociceptin-orphanin FQ (N/OFQ) receptor (NOP)-deficient mice, the effect of nociceptin receptor antagonists on N(2)O-induced analgesia has not been reported. In this investigation, we examined the effect of the NOP antagonist JTC-801 on N(2)O-induced analgesia in 129Sv mice by the writhing test and tail flick test, and demonstrated that the analgesic effect of N(2)O was suppressed by the intraperitoneal administration of JTC-801.

A combined marble burying–locomotor activity test in mice: A practical screening test with sensitivity to different classes of anxiolytics and antidepressants

Over the last decades, the inhibition of spontaneous burying of glass marbles by mice has been used as an index of anxiolytic drug action in the so-called marble burying test. Indeed, acute administration of rapid-onset (e.g. diazepam) and slow-onset (e.g. fluoxetine) anxiolytics inhibit marble burying. However, non-anxiolytic compounds such as classical antipsychotics also reduce marble burying thus suggesting that the predictive validity of this procedure for anxiety may be limited. In the present study, after having selected a strain of mice (C57BL/6J) that showed spontaneous avoidance of glass marbles, we tried to improve the predictive validity of the marble burying test for anxiety by measuring locomotor activity during the marble burying test and – if needed – in control experiments by using a videotracking system. Twenty-four reference compounds were tested including anxiolytics, anxiogenics, antidepressants, antipsychotics and other classes. By comparing marble burying scores with locomotor measures, we found that, based on our criteria, most of the anxiolytics and antidepressants selectively inhibited marble burying in contrast to most of the other compounds (e.g. haloperidol, morphine). Two putative anxiolytics, i.e. the nociceptin orphanin FQ peptide receptor agonist Ro 64-6198 and the metabotropic glutamate 5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, also showed a selective profile. We propose this modified procedure, requiring only a limited number of animals, as a valuable screening test for the detection of compounds having anxiolytic effects.

Regulation of the prepronociceptin gene and its effect on neuronal differentiation

Nociceptin/orphanin FQ (NOP/OFQ) is the endogenous ligand for the NOP receptor and is processed from a precursor protein in the family of opioid peptides. Prepronociceptin (ppN/OFQ) mRNA has been shown to be upregulated by an increase in cAMP, a treatment that leads to differentiation of NS20Y neuroblastoma cells. Although a large increase in endogenous ppN/OFQ mRNA upon cAMP stimulation can be shown in cellular systems, a similar increase cannot be expressed in pGL3 luciferase vector containing 1.3 kb proximal promoter, suggesting that a larger portion of the sequence or a different chromatin structure is necessary for a fully functional promoter. The induction of ppN/OFQ mRNA by cAMP appears to be mediated by a cAMP-response element. Chromatin immunoprecipitation (ChIP) assays show that CREB is recruited to the promoter region upon treatment of NS20Y cells with dibutyryl cAMP. In addition, the production of ppN/OFQ mRNA is regulated by histone acetylation, also through CREB, as the histone deacetylase (HDAC) inhibitor trichostatin A increases both CREB binding to the promoter and ppN/OFQ mRNA expression. In rat progenitor and mouse neuroblastoma cell lines, agents that increase ppN/OFQ mRNA expression also induce neurite outgrowth, suggesting a close relationship between ppN/OFQ and cellular differentiation.

NOC/oFQ activates ERK and JNK but not p38 MAPK to impair prostaglandin cerebrovasodilation after brain injury

Fluid percussion brain injury (FPI) elevates the CSF concentration of the opioid nociceptin/orphanin FQ (NOC/oFQ), which contributes to impairment of pial artery dilation to the prostaglandins (PG) PGE 2 and PGI 2. This study investigated the role of the ERK, p38, and JNK isoforms of mitogen-activated protein kinase (MAPK) in impaired PG cerebrovasodilation after FPI, and the relationship of brain injury induced release of NOC/oFQ to MAPK in such vascular impairment in newborn pigs equipped with a closed cranial window. FPI blunted PGE 2 pial artery dilation, but U 0126 and SP 600125 (10 −6 M) (ERK and JNK MAPK inhibitors, respectively) partially prevented such impairment (7 ± 1, 12 ± 1, and 17 ± 1 vs. 2 ± 1, 3 ± 1, and 5 ± 1 vs. 4 ± 1, 7 ± 1, and 12 ± 1% for 1, 10, and 100 ng/ml PGE 2 in control, FPI, and FPI + U 0126 pretreated animals, respectively). In contrast, administration of SB 203580 (10 −5 M) (p38 MAPK inhibitor) did not prevent FPI impairment of PGE 2 dilation. Co-administration of NOC/oFQ at the dose of 10 −10 M, the cerebrospinal fluid concentration observed after FPI, with PGE 2 under non-brain injury conditions blunted PG dilation, but U 0126 or SP 600125 partially prevented such impairment (7 ± 1, 11 ± 1, and 16 ± 2 vs. 0 ± 1, 1 ± 1, and 2 ± 1, vs. 5 ± 1, 9 ± 1, and 13 ± 2 for responses to PGE 2 in control, NOC/oFQ, and NOC/oFQ + U 0126 treated animals, respectively). Administration of SB 203580 did not prevent impairment of PG pial artery dilation by NOC/oFQ. These data show that activation of ERK and JNK but not p38 MAPK contributes to impairment of PG cerebrovasodilation after FPI. These data suggest that NOC/oFQ induced ERK and JNK but not p38 MAPK activation contributes to impaired cerebrovasodilation to PG after FPI.

NMDA and age dependent cerebral hemodynamics after traumatic brain injury

Activation of N-methyl- D-aspartate (NMDA) glutamatergic receptors elicits cerebrovascular dilation, may couple local cerebral metabolism to blood flow but contribute to excitotoxic neuronal cell death. While cerebral hemodynamics following traumatic brain injury may correlate with neurologic status, the role of NMDA vascular activity is uncertain in the sequelae of brain injury. NMDA dilation was impaired following fluid percussion brain injury (FPI) in an age dependent manner in the pig and the newly described opioid nociceptin/orphanin FQ (NOC/oFQ) contributes to such impairment via the cyclooxygenase dependent generation of superoxide. Further, hypotensive pial artery dilation (PAD) was blunted after FPI but partially protected by pretreatment with the NMDA antagonist MK801. Cerebral blood flow (CBF) was reduced during normotension by FPI, further reduced by hypotension, but both were partially protected by MK801 in the newborn. In contrast, blunted hypotensive PAD was protected significantly less by MK801 in the juvenile pig. Similarly, MK801 had less protective effect on normotensive and hypotensive CBF values post FPI in the juvenile. These data indicate that NMDA receptor activation contributes to impaired hypotensive cerebral hemodynamics following FPI in an age dependent manner. Further, these data suggest that NMDA receptor activation, NOC/oFQ, and prostaglandins dynamically interact to impair cerebral hemodynamics following FPI.

Nociceptin/orphanin FQ alters prostaglandin cerebrovascular action following brain injury.

Previous studies have observed that fluid percussion brain injury (FPI) elevated the CSF concentration of the opioid nociceptin/orphanin FQ (NOC/oFQ). In separate studies, FPI impaired pial artery dilation to the prostaglandins PGI2 and PGE2. This study was designed to investigate the following: (1) role of NOC/oFQ in impaired dilation to PGI2 and PGE2, (2) the effects of FPI on vasoconstriction to the TXA2 mimic U46619 and PGF2alpha, and (3) the role of NOC/oFQ in such FPI induced effects on U46619 and PGF(2alpha). Lateral FPI was induced in newborn pigs equipped with a closed cranial window. PGI2 (1, 10, 100 ng/ml) vasodilation was blunted by FPI and fully restored by the NOC/oFQ antagonist, [F/G] NOC/oFQ (1-13) NH2 (10(-6)M) (9 +/- 1, 13 +/- 1, and 19 +/- 1 vs. 2 +/- 1, 4 +/- 1, and 5 + 1 vs 7 +/- 1, 12 +/- 2, and 17 +/- 3% for control, FPI, and FPI + [F/G] NOC/oFQ (1-13) NH2, respectively). Similar effects were observed for PGE2. In contrast, U46619 (1, 10 ng/ml) induced vasoconstriction was potentiated by FPI but returned to the response observed prior to FPI by [F/G] NOC/oFQ (1-13) NH2 ( -8 +/- 1 and -14 +/- 1 vs. -15 +/- 1 and -25 +/- 1 vs. -7 +/- 1 and -12 +/- 2% for control, FPI, and FPI + [F/G] NOC/oFQ (1-13) NH2, respectively). Similar effects were observed for PGF(2alpha). Coadministration of NOC/oFQ (10(-10)M), the CSF concentration observed after FPI, with agonists under nonbrain injury conditions blunted PGI2 and PGE2 vasodilation, but potentiated U46619 and PGF2alpha vasoconstriction similarly to that observed after FPI. These data show that FPI blunted PGI2 and PGE2 vasodilation but potentiated U46619 and PGF2alpha vasoconstriction. Additionally, these data show that administration of a NOC/oFQ receptor antagonist prevented such FPI associated events. NOC/oFQ administrated in a concentration observed after FPI produced blunted dilator prostaglandin and potentiated vasoconstriction prostaglandin vascular responses under nonbrain injury conditions. Finally, these data suggest that NOC/oFQ alters prostaglandin cerebrovascular action following brain injury.

PTK, MAPK, and NOC/oFQ impair hypercapnic cerebrovasodilation after hypoxia/ischemia.

This study characterized the contributions of protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) in nociceptin/orphanin FQ (NOC/oFQ)-induced impairment of hypercapnic pial artery dilation (PAD) after hypoxia/ischemia (H/I) in piglets equipped with a closed cranial window. NOC/oFQ (10(-10) M cerebrospinal fluid H/I concentration) impaired hypercapnic PAD (21 +/- 2% vs. 13 +/- 1%). Coadministration of either of the PTK inhibitors genistein or tyrphostin A23 or the MAPK inhibitors U-0126 or PD-98059 with NOC/oFQ (10(-10) M) partially prevented the inhibition of hypercapnic PAD compared with that observed in their absence (21 +/- 2% vs. 17 +/- 1% for genistein). After exposure to H/I, PAD in response to hypercapnia was impaired, but pretreatment with either genistein, tyrphostin A23, U-0126, or PD-98059 partially protected such impairment (17 +/- 1% vs. 4 +/- 1% vs. 9 +/- 1% for sham control, H/I, and H/I + genistein pretreatment, respectively). These data show that PTK and MAPK activation contribute to NOC/oFQ-induced impairment of hypercapnic PAD. These data suggest that activation of PTK and MAPK is also involved in the mechanism by which NOC/oFQ impairs hypercapnic PAD after H/I.

Characterization of nociceptin/orphanin FQ-induced pain responses by the novel receptor antagonist N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride.

At the spinal level, nociceptin/orphanin FQ (Noc/OFQ) produces pronociceptive and allodynic effects at low doses (picogram range), while causing antinociceptive effects at high doses (microgram range). The discrepancy of pain modulation by Noc/OFQ at low and high doses raised a question whether Noc/OFQ exerted actions through the same Noc/OFQ receptor. In the present study, we examined the involvement of the Noc/OFQ receptor in pain responses with the novel nonpeptide antagonist N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride (JTC-801). Allodynia and hyperalgesia evoked by intrathecal administration of Noc/OFQ (50 pg/mouse) were dose dependently blocked by simultaneous administration of JTC-801 with IC(50) values of 32.2 and 363 pg, respectively. JTC-801 did not induce allodynia by itself. Subcutaneous injection of formalin into a hindpaw evoked biphasic pain behaviors such as flinching and biting in mice. Noc/OFQ at 10 pg increased the second-phase pain behaviors evoked by 1% formalin, whereas it strongly inhibited both the first-phase and second-phase pain evoked by 2% formalin at 1 microg. Although the pronociceptive effect by 10 pg of Noc/OFQ was dose dependently blocked by JTC-801 with an IC(50) value of 4.58 pg, the antinociceptive effects by 1 microg of Noc/OFQ were not antagonized by JTC-801. Furthermore, both phases of 2% formalin-induced pain behaviors were relieved by JTC-801. These results demonstrate that pronociceptive responses induced by a low dose of Noc/OFQ may be mediated through the Noc/OFQ receptor in the spinal cord and that JTC-801 can be a useful antagonist to examine the involvement of endogenous Noc/OFQ and mediation of the Noc/OFQ receptor under physiological and pathophysiological conditions including pain.